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Assessing effects of antimetastatic treatment

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Combining Kinetic Ligand Binding and 3D Tumor Invasion Technologies to Assess Drug Residence Time and Anti-metastatic Effects of CXCR4 Inhibitors

Application Note 3D Cell Culture, ADME/Tox, Cell Imaging, Cell-Based Assays
BioTek Instruments, Inc. P.O. Box 998, Highland Park, Winooski, Vermont 05404-0998
Brad Larson and Leonie Rieger, BioTek Instruments, Inc., Winooski, VT
Nicolas Pierre, Cisbio US, Inc., Bedford, MA
Hilary Sherman, Corning Incorporated, Life Sciences, Kennebunk, ME

http://vertassets.blob.core.windows.net/download/ba9da411/ba9da411-a56c-42d3-a1a0-8c128224947f/cisbio_residence_time_app_note_final.pdf

 

Metastasis, the spread of cancer cells from the original tumor to secondary locations within the body, is linked to approximately 90% of cancer deaths1 . The expression of chemokine receptors, such as CXCR4 and CCR7, is tightly correlated with the metastatic properties of breast cancer cells. In vivo, neutralizing the interaction of CXCR4 and its known ligand, SDF1-α (CXCL12), significantly impaired the metastasis of breast cancer cells and cell migration2 . Traditionally, the discovery of novel agents has been guided by the affinity of the ligand for the receptor under equilibrium conditions, largely ignoring the kinetic aspects of the ligandreceptor interaction. However, awareness of the importance of binding kinetics has started to increase due to accumulating evidence3, 4, 5, 6 suggesting that the in vivo effectiveness of ligands may be attributed to the time a particular ligand binds to its receptor (drug-target residence time).

Similarly, appropriate in vitro cell models have also been lacking to accurately assess the ability of novel therapies to inhibit tumor invasion. Tumors in vivo exist as a three-dimensional (3D) mass of multiple cell types, including cancer and stromal cells7 . Therefore, incorporating a 3D spheroid-type cellular structure that includes co-cultured cell types forming a tumoroid, provides a more predictive model than the use of individual cancer cells cultured on the bottom of a well in traditional two-dimensional (2D) format.

Here we examine the drug-target residence time of various CXCR4 inhibitors using a direct, homogeneous ligand binding assay and CXCR4 expressing cell line in a kinetic format. This inhibitor panel was further tested in a 3D tumor invasion assay to determine whether there is a correlation between the molecule’s CXCR4 residence time and inhibition of the phenotypic effect of tumor invasion. MDA-MB-231 breast adenocarcinoma cells, known to be invasive, and metastasize to lung from primary mammary fat pad tumors8 , were included, in addition to primary human dermal fibroblasts. Cellular analysis algorithms provided accurate quantification of changes to the original tumoroid structure, as well as invadopodia development. The combination presents an accurate, yet easy-to-use method to assess target-based and phenotypic effects of new, potential anti-metastatic drugs.

……

Cytation™ 5 Cell Imaging Multi-Mode Reader Cytation 5 is a modular multi-mode microplate reader that combines automated digital microscopy and microplate detection. Cytation 5 includes filter- and monochromator-based microplate reading; the microscopy module provides high resolution microscopy in fluorescence, brightfield, color brightfield and phase contrast. With special emphasis on live-cell assays, Cytation 5 features temperature control to 65 °C, CO2 / O2 gas control and dual injectors for kinetic assays. Shaking and Gen5 software are also standard. The instrument was used to image spheroids, as well as individual cell invasion through the Matrigel matrix.

Tag-lite® Receptor Ligand Binding Assay

Figure 1. Tag-lite® Receptor Ligand Binding Assay Procedure. The Tag-lite CXCR4 assay relies on a fully functional SNAP-tag fused CXCR4 receptor and fluorescently labeled ligand SDF1-α. Being homogeneous, the binding assay allows for binding events to be precisely recorded in time. The assay can be used to derive the kinetic binding parameters of unlabeled compounds by application of the Motulsky and Mahan equations.

……

Results and Discussion

Drug-Target Residence Time

Determination Association Kinetics of SDF1-α-d2 Labeled Ligand

The final Drug-Target Residence Time value takes into account the observed on and off rates of the unlabeled inhibitors as well as the labeled SDF1-α-d2 ligand, and is computed by incorporation of the Motulsky and Mahan equation9 . The first step to calculate the final value was to perform an associative binding experiment using a concentration range of 0-100 nM of the d2 acceptor fluor labeled ligand. Binding was monitored kinetically over a period of 40 minutes.

Figure 2. Association binding graph of SDF1-α-d2. Observed associative binding curves calculated from HTRF ratios of wells containing SDF1-α-d2 ligand concentrations ranging from 0-100 nM. Non-specific binding values subtracted from total ratios to determine observed specific binding.

Binding increases over time until it plateaus after several minutes (Figure 2). The plateau in an association experiment depends on the concentration of labeled SDF1-α used. Higher plateaus will be obtained with higher concentrations. Fitting of the curves with Graph Pad Prism yields the observed association rate values for all concentrations tested or kobs.

The Kd value of the labeled ligand was also determined by plotting the HTRF ratios generated after a binding equilibrium was reached with the different concentrations of ligand tested.

Figure 3. SDF1-α-d2 saturation binding curve. HTRF ratios generated upon the achievement of binding equilibrium of tested [SDF1-α-d2].

In a saturation binding experiment, increasing concentrations of labeled SDF1-α result in increased binding. Saturation is obtained when no further binding can be recorded. The ligand concentration that binds to half the receptor sites at equilibrium or Kd was 29 nM.

An assessment of whether the labeled SDF1-α ligand follows the Law of Mass action can also be carried out. If the system does follow the Law of Mass action then kobs increases linearly with increasing concentrations of SDF1-α.

Due to the linear shape of the curve, and an R2 value >0.9, Law of Mass Action was proven for the labeled SDF1-α ligand. This allowed for the use of Graph Pad Prism software to derive association and dissociation rate constants from the linear regression line. The rate constant values experimentally found or mathematically derived are summarized in Table 1. kon,SDF1-α-d2 and koff ,SDF1-α-d2 were 0.001 nM-1.s-1 and 0.04 s-1, respectively

Table   SDF1-α-d2 Kinetic Binding Characterization

Association Kinetics of SDF1-α-d2 Labeled Ligand In the theory developed by Motulsky and Mahan, an unlabeled competitor is co-incubated with a labeled ligand during a kinetic association experiment. Here, a single concentration of the SDF1-α-d2 ligand, 25 nM, was co-incubated with multiple concentrations of the unlabeled SDF1-α competitors in the presence of the CXCR4 expressing cells. Kinetic binding of the labeled ligand was then monitored over time.

Figure 5. Kinetics of Competitive Binding. Plot of specific binding HTRF ratios over time for the SDF1-α-d2 ligand when in the presence of 100, 10, or 1 nM concentrations of (A.) AMD 3100, (B.) AMD 3465, or (C.) IT1t.

From the curve fitting of the observed SDF1-α-d2 kinetic binding, and incorporation of the Law of Mass Action linear regression line, k(off) (Min-1) values were then calculated. Final residence time (R) values could then be determined using the following formula:

R = 1/k(off)

Therefore, molecules having a lower k(off) rate reside at the target receptor for longer periods of time.

Table 2. SDF1-α Competitor Dissociation Rate and Residence Time Values.

 

From the shape of the curves in Figure 5, and a comparison of the residence time values generated for the labeled ligand and unlabeled competitors (Table 2), qualitative and quantitative assumptions regarding the various competitors can then be made. First, if the competitor dissociates faster from its target than the ligand (smaller R value), such as is seen with AMD 3100 (Figure 5A), the specific binding of the ligand will slowly and monotonically approach its equilibrium in time. However, when the competitor dissociates slower (larger R value), the association curve of the ligand consists of two phases, starting with a typical “overshoot” and then a decline until a new equilibrium is reached. Competitors whose residence times are greater than that of the SDF1-α-d2 ligand, such as AMD 3465 and IT1t (Figure 5B and C), may then exhibit a stronger inhibitory response when used in the confirmatory phenotypic 3D tumor invasion assay.

Interruption of Invasion via SDF1-α Ligand Binding Inhibition As stated previously, interruption of the interaction between CXCR4 and its known ligand, SDF1-α, impairs metastasis of breast cancer and cell migration2 . Therefore, a phenotypic assessment of the CXCR4 inhibitor panel was then performed to determine whether changes in the level of tumor migration could be detected, and more importantly, if compounds exhibiting longer residence times compared to SDF1-α-d2 exhibited a higher inhibitory effect on migration through the 3D matrix. MDA-MB-231 breast adenocarcinoma cells, co-cultured with human dermal fibroblasts, were used as the in vitro tumor model. This breast cancer cell line has been previously shown to express the CXCR4 receptor10.

 

Figure 6. Image-based Monitoring of MDA-MB-231/Fibroblast Tumor Invasion. Overlaid brightfield and fluorescent images captured using a 4x objective, after a 0 and 5 day incubation period with AMD 3465, IT1t, and CTCE 9908. Imaging channel representation: Brightfield – Total cells and invadopodia; GFP – MDA-MB-231 cells; RFP – Fibroblasts.

Figure 7. Quantification of Invasive Tumor Area. 4x overlaid images captured following 5 day (A.) 100 and (B.) 0 μM IT1t incubation with tumoroids. Object masks automatically drawn by Gen5 using the following criteria: Threshold: 5000 RFU; Min. Object Size: 400 μm; Max. Object Size: 1500 μm; Image Smoothing Strength: 0; Background Flattening Size: Auto.

 

Cellular analysis is performed with the Cytation 5 using the brightfield signal to quantify the extent of invasion. Minimum and maximum object sizes, as well as brightfield threshold values are set such that a precise object mask is automatically drawn around each tumoroid in its entirety (Figure 7A and B). The same criteria are used for all images evaluated during the experiment. This allows for a quantitative comparison of the area covered within each object mask to be completed.

Figure 8. Tumor Invasion Inhibition Determination. Graphs of individual tumoroid areas on day 0, and subsequent to five day invasion period in the presence of inhibitor concentrations.

 

The 4x images displayed (Figure 6), as well as the graphs in Figure 8, demonstrating total tumoroid area coverage before and after the incubation period illustrate the ability of CXCR4 inhibitors to interrupt tumor invasion consistent with the previously determined residence time. AMD 3465 and IT1t, which exhibit a residence time longer than SDF1-α-d2, effectively minimize tumor invasion in a dose dependent manner. The decrease in MDAMB-231 GFP and fibroblast RFP expression exhibited after a 5 day 100 μM IT1t incubation, also seen after a 7 day AMD 3465 incubation of the same concentration (data not shown), may also indicate the chronic cytotoxic effects that elevated dosing of these compounds can have on both cancer and stromal cells. All other compounds show little to no effect on the ability of the tumoroid to migrate through the 3D matrix. While AMD 3465 and ITt1 display the same sub-nanomolar potency, AMD3465 prevails as a CXCR4 inhibitor due to its greater residence time.

 

Conclusions The Tag-lite CXCR4 ligand binding assay provides a simple, yet robust cell-based approach to determine kinetic binding of known receptor ligands, as well as competitive binding of test molecules. The simultaneous dual emission capture and injection capabilities of the Synergy Neo allow accurate calculations of kinetic association and dissociation rates to be made when used in conjunction with the Tag-lite® assay. Corning Spheroid Microplates then provide an easy-to-use, consistent method to perform spheroid aggregation and confirmatory 3D tumor invasion assays. Imaging of spheroid formation, as well as invading structures can be performed by the Cytation™ 5 using brightfield or fluorescent channels to easily track tumoroid invasion. The flexible cellular analysis capacity of the Gen5™ Data Analysis Software also allows for accurate assessment of 3D tumor invasion during the entire incubation period. The combination of assay chemistry, cell model, kinetic microplate and image-based monitoring, in addition to cellular analysis provide an ideal method to better understand the target-based and phenotypic effects of potential inhibitors of tumor invasion and metastasis.

References

1. Saxe, Charles. ‘Unlocking The Mysteries Of Metastasis’. ExpertVoices 2013. http://www.cancer.org/ cancer/news/expertvoices/post/2013/01/23/unlockingthe-mysteries-of-metastasis.aspx. Accessed 16 Mar. 2015.

2. Müller, A., Homey, B., Soto, H., Ge, N., Catron, D., Buchanan, M., McClanahan, T., Mruphy, E., Yuan, W., Wagner, S., Barrera, J., Mohar, A., Verástegui, E., Zlotnik, A. Involvement of chemokine receptors in breast cancer metastasis. Nature. 2001, 410, 50-56.

3. Swinney, D. Biochemical mechanisms of drug action: what does it take for success? Nat Rev Drug Discov. 2004, 3, 801-808.

4. Copeland, R., Pompliano, D., Meek, T. Drugtarget residence time and its implications for lead optimization. Nat Rev Drug Discov. 2006,5, 730-739.

5. Tummino, P., Copeland, R. Residence time of receptor-ligand complexes and its effect on biological function. Biochemistry. 2008, 47, 5481-5492.

6. Zhang, R., Monsma, F. The importance of drug-target residence time. Curr Opin Drug Discov Devel. 2009, 12, 488-496.

7. Mao, Y., Keller, E., Garfield, D., Shen, K., Wang, J. Stromal cells in tumor microenvironment and breast cancer. Cancer Metast Rev. 2013, 32, 303-315.

8. Kamath, L., Meydani, A., Foss, F., Kuliopulos, A. Signaling from protease-activated receptor-1 inhibits migration and invasion of breast cancer cells. Cancer Res. 2001, 61, 5933-5940.

9. Motulsky, H., Mahan, L. The kinetics of competitive radioligand binding predicted by the law of mass action. Mol Pharmacol. 1984, 25, 1-9.

10. Sun, Y., Mao, X, Fan, C, Liu, C., Guo, A., Guan, S., Jin, Q., Li, B., Yao, F., Jin, F. CXCL12-CXCR4 axis promotes the natural selection of breast cancer cell metastasis. Tumor Biol. 2014, 35, 7765-7773.

 

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New glucokinase activator

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

RO-28-1675 for Type 2 Diabetes

by DR ANTHONY MELVIN CRASTO Ph.D

http://www.medchemexpress.com/product_pic/hy-10595.gif

 

RO-28-1675

  • (2R)-3-Cyclopentyl-2-[4-(methanesulfonyl)phenyl]-N-(thiazol-2-yl)propionamide
  • Ro 028-1675
  • Ro 0281675
  • Ro 28-1675

3-Cyclopentyl-2(R)-[4-(methylsulfonyl)phenyl]-N-(2-thiazolyl)propionamide

MW 378.51 .-70.4 °Conc 0.027 g/100mL; chloroform, 589 nm;  23 °C

 

Formula C18H22N2O3S2
CAS No 300353-13-3

Glucokinase Activators

Ro 28-1675 (Ro 0281675) is a potent allosteric GK activator with a SC1.5 value of 0.24± 0.0019 uM.

Roche (Innovator)

Hoffmann La Roche

PHASE 1    Type 2  DIABETES,
IC50 value: 0.24± 0.0019 uM (SC1.5) [1]
Target: Glucokinase activator
The R stereoisomer Ro 28-1675 activated GK with a SC1.5 of 0.24 uM, while the S isomer did not activated GK up to 10 uM. Oral administration of Ro 28-1675 (50 mg/Kg) to male C57B1/6J mice caused a statistically significant reduction in fasting glucose levels and improvement in glucose tolerance relative to the vehicle treated animals [1].
Comparison of rat PK parameters indicated that Ro 28-1675 displayed lower clearance and higher oral bioavailability compared to 9a.

Following a single oral dose, Ro 28-1675 reduced fasting and postprandial glucose levels following an OGTT, was well tolerated, and displayed no adverse effects related to drug administration other than hypoglycemia at the maximum dose (400 mg).

RO-28-1675 as glucokinase activator.

Joseph Grimsby et al., of Roche have recently discovered activators of glucokinase that increase kcat and decrease the S0.5 for glucose, and these may offer a treatment for type II diabetes. Glucokinase (GK) plays a key role in whole-body glucose homeostasis by catalyzing the phosphorylation of glucose in cells that express this enzyme, such as pancreatic β cells and hepatocytes.

By screening of a library of 120,000 structurally diverse synthetic compounds, they found one small molecule that increased the enzymatic activity of GK. Chemical optimization of this initial molecule led to the synthesis of RO-28-0450 as a lead GK activator which is a class of antidiabetic agents that act as nonessential, mixed-type GK activators (GKAs) that increase the glucose affinity and maximum velocity (Vmax) of GK. RO-28-0450 is a racemic compound.

Activation of GK was exquisitely sensitive to the chirality of the molecule: The R enantiomer, RO-28-1675, was found to be a potent GKA, whereas the S enantiomer, RO-28-1674, was inactive. RO-28-1675 also reversed the inhibitory action of the human glucokinase regulatory protein (GKRP). The activators binding in a glucokinase regulatory site originally was discovered in patients with persistent hyperinsulinemic hypoglycemi.

The result of RO-28-1675 as a potent small molecule GKA may shed light to the chemical biologists to devise strategy for developing activators. Thus for a success to this end we must focus on highly regulated enzymes, or cooperative enzymes such as glucokinase, where nature has provided binding sites that are designed to modulate catalysis.

 

SYNTHESIS

Paper

J. Med. Chem., 2010, 53 (9), pp 3618–3625
DOI: 10.1021/jm100039a
Abstract Image

Glucokinase (GK) is a glucose sensor that couples glucose metabolism to insulin release. The important role of GK in maintaining glucose homeostasis is illustrated in patients with GK mutations. In this publication, identification of the hit molecule 1 and its SAR development, which led to the discovery of potent allosteric GK activators 9a and21a, is described. Compound 21a (RO0281675) was used to validate the clinical relevance of targeting GK to treat type 2 diabetes.

Flash chromatography (Merck Silica gel 60, 70-230 mesh, 9/1, 3/1, and then 11/9 hexanes/ethyl acetate) afforded (2R)-3-cyclopentyl-2-(4-methanesulfonylphenyl)-N-thiazol-2-yl-propionamide (2.10 g, 74%) as a white foam.   ….

PATENT

WO 2000058293

http://www.google.com/patents/WO2000058293A2?cl=en

 

Discovery, Structure−Activity Relationships, Pharmacokinetics, and Efficacy of Glucokinase Activator (2R)-3-Cyclopentyl-2-(4-methanesulfonylphenyl)-N-thiazol-2-yl-propionamide (RO0281675)

J. Med. Chem., 2010, 53 (9), pp 3618–3625   DOI:http://dx.doi.org:/10.1021/jm100039a
Abstract Image
Glucokinase (GK) is a glucose sensor that couples glucose metabolism to insulin release. The important role of GK in maintaining glucose homeostasis is illustrated in patients with GK mutations. In this publication, identification of the hit molecule 1 and its SAR development, which led to the discovery of potent allosteric GK activators 9a and 21a, is described. Compound 21a (RO0281675) was used to validate the clinical relevance of targeting GK to treat type 2 diabetes.

REFERENCES

[1]. Haynes NE, et al. Discovery, structure-activity relationships, pharmacokinetics, and efficacy of glucokinase activator (2R)-3-cyclopentyl-2-(4-methanesulfonylphenyl)-N-thiazol-2-yl-propionamide (RO0281675).

Glucokinase (GK) is a glucose sensor that couples glucose metabolism to insulin release. The important role of GK in maintaining glucose homeostasis is illustrated in patients with GK mutations. In this publication, identification of the hit molecule 1 and its SAR development, which led to the discovery of potent allosteric GK activators 9a and 21a, is described. Compound 21a (RO0281675) was used to validate the clinical relevance of targeting GK to treat type 2 diabetes.

http://www.nature.com/nrd/journal/v8/n5/fig_tab/nrd2850_T2.html

NMR…..http://www.medchemexpress.com/product_pdf/HY-10595/Ro%2028-1675-NMR-HY-10595-13569-2014.pdf

http://www.medchemexpress.com/product_pdf/HY-10595/Ro%2028-1675-Lcms_Ms-HY-10595-13569-2014.pdf

J Grimsby et al. Allosteric Activators of Glucokinase: Potential Role in Diabetes Therapy. Science Signaling 2003, 301(5631), 370-373.
T Kietzmann and GK Ganjam. Glucokinase: old enzyme, new target. Exp. Opin. Ther. Patents. 2005, 15(6), 705-713.

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P13K delta-gamma anticancer agent

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

RP 6350, Rhizen Pharmaceuticals S.A. and Novartis tieup for Rhizen’s inhaled dual Pl3K-delta gamma inhibitor

by DR ANTHONY MELVIN CRASTO Ph.D

 

(A)           and                         (Al)                  and                (A2)

(S)-2-(l-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-one (Compound A1 is RP 6350).

 

str1

 

RP 6350, RP6350, RP-6350

(S)-2-(l-(9H-purin-6-ylamino)propyl)-3-(3-fluorophenyl)-4H-chromen-4-one

mw 415

Rhizen Pharmaceuticals is developing RP-6530, a PI3K delta and gamma dual inhibitor, for the potential oral treatment of cancer and inflammation  In November 2013, a phase I trial in patients with hematologic malignancies was initiated in Italy ]\. In September 2015, a phase I/Ib study was initiated in the US, in patients with relapsed and refractory T-cell lymphoma. At that time, the study was expected to complete in December 2016

PATENTS……..WO 11/055215 ,  WO 12/151525.

  • Antineoplastics; Small molecules
  • Mechanism of Action Phosphatidylinositol 3 kinase delta inhibitors; Phosphatidylinositol 3 kinase gamma inhibitors
  • Phase I Haematological malignancies
  • Preclinical Multiple myeloma

 

Swaroop K. V. S. Vakkalanka,
COMPANY Rhizen Pharmaceuticals Sa

https://clinicaltrials.gov/ct2/show/NCT02017613

 

PI3K delta/gamma inhibitor RP6530 An orally active, highly selective, small molecule inhibitor of the delta and gamma isoforms of phosphoinositide-3 kinase (PI3K) with potential immunomodulating and antineoplastic activities. Upon administration, PI3K delta/gamma inhibitor RP6530 inhibits the PI3K delta and gamma isoforms and prevents the activation of the PI3K/AKT-mediated signaling pathway. This may lead to a reduction in cellular proliferation in PI3K delta/gamma-expressing tumor cells. In addition, this agent modulates inflammatory responses through various mechanisms, including the inhibition of both the release of reactive oxygen species (ROS) from neutrophils and tumor necrosis factor (TNF)-alpha activity. Unlike other isoforms of PI3K, the delta and gamma isoforms are overexpressed primarily in hematologic malignancies and in inflammatory and autoimmune diseases. By selectively targeting these isoforms, PI3K signaling in normal, non-neoplastic cells is minimally impacted or not affected at all, which minimizes the side effect profile for this agent. Check for active clinical trials using this agent. (NCI Thesaurus)

Company Rhizen Pharmaceuticals S.A.
Description Dual phosphoinositide 3-kinase (PI3K) delta and gamma inhibitor
Molecular Target Phosphoinositide 3-kinase (PI3K) delta ; Phosphoinositide 3-kinase (PI3K) gamma
Mechanism of Action Phosphoinositide 3-kinase (PI3K) delta inhibitor; Phosphoinositide 3-kinase (PI3K) gamma inhibitor
Therapeutic Modality Small molecule

 

Dual PI3Kδ/γ Inhibition By RP6530 Induces Apoptosis and Cytotoxicity In B-Lymphoma Cells
 Swaroop Vakkalanka, PhD*,1, Srikant Viswanadha, Ph.D.*,2, Eugenio Gaudio, PhD*,3, Emanuele Zucca, MD4, Francesco Bertoni, MD5, Elena Bernasconi, B.Sc.*,3, Davide Rossi, MD, Ph.D.*,6, and Anastasios Stathis, MD*,7
 1Rhizen Pharmaceuticals S A, La Chaux-de-Fonds, Switzerland, 2Incozen Therapeutics Pvt. Ltd., Hyderabad, India, 3Lymphoma & Genomics Research Program, IOR-Institute of Oncology Research, Bellinzona, Switzerland, 4IOSI Oncology Institute of Southern Switzerland, Bellinzona, Switzerland, 5Lymphoma Unit, IOSI-Oncology Institute of Southern Switzerland, Bellinzona, Switzerland, 6Italian Multiple Myeloma Network, GIMEMA, Italy, 7Oncology Institute of Southern Switzerland, Bellinzona, Switzerland

RP6530 is a potent and selective dual PI3Kδ/γ inhibitor that inhibited growth of B-cell lymphoma cell lines with a concomitant reduction in the downstream biomarker, pAKT. Additionally, the compound showed cytotoxicity in a panel of lymphoma primary cells. Findings provide a rationale for future clinical trials in B-cell malignancies.

POSTER SESSIONS
Blood 2013 122:4411; published ahead of print December 6, 2013
Swaroop Vakkalanka, Srikant Viswanadha, Eugenio Gaudio, Emanuele Zucca, Francesco Bertoni, Elena Bernasconi, Davide Rossi, Anastasios Stathis
  • Dual PI3K delta/gamma Inhibition By RP6530 Induces Apoptosis and Cytotoxicity
  • RP6530, a novel, small molecule PI3K delta/gamma
  • Activity and selectivity of RP6530 for PI3K delta and gamma isoforms

Introduction Activation of the PI3K pathway triggers multiple events including cell growth, cell cycle entry, cell survival and motility. While α and β isoforms are ubiquitous in their distribution, expression of δ and γ is restricted to cells of the hematopoietic system. Because these isoforms contribute to the development, maintenance, transformation, and proliferation of immune cells, dual targeting of PI3Kδ and γ represents a promising approach in the treatment of lymphomas. The objective of the experiments was to explore the therapeutic potential of RP6530, a novel, small molecule PI3Kδ/γ inhibitor, in B-cell lymphomas.

Methods Activity and selectivity of RP6530 for PI3Kδ and γ isoforms and subsequent downstream activity was determined in enzyme and cell-based assays. Additionally, RP6530 was tested for potency in viability, apoptosis, and Akt phosphorylation assays using a range of immortalized B-cell lymphoma cell lines (Raji, TOLEDO, KG-1, JEKO, OCI-LY-1, OCI-LY-10, MAVER, and REC-1). Viability was assessed using the colorimetric MTT reagent after incubation of cells for 72 h. Inhibition of pAKT was estimated by Western Blotting and bands were quantified using ImageJ after normalization with Actin. Primary cells from lymphoid tumors [1 chronic lymphocytic leukemia (CLL), 2 diffuse large B-cell lymphomas (DLBCL), 2 mantle cell lymphoma (MCL), 1 splenic marginal zone lymphoma (SMZL), and 1 extranodal MZL (EMZL)] were isolated, incubated with 4 µM RP6530, and analyzed for apoptosis or cytotoxicity by Annexin V/PI staining.

Results RP6530 demonstrated high potency against PI3Kδ (IC50=24.5 nM) and γ (IC50=33.2 nM) enzymes with selectivity over α (>300-fold) and β (>100-fold) isoforms. Cellular potency was confirmed in target-specific assays, namely anti-FcεR1-(EC50=37.8 nM) or fMLP (EC50=39.0 nM) induced CD63 expression in human whole blood basophils, LPS induced CD19+ cell proliferation in human whole blood (EC50=250 nM), and LPS induced CD45R+ cell proliferation in mouse whole blood (EC50=101 nM). RP6530 caused a dose-dependent inhibition (>50% @ 2-7 μM) in growth of immortalized (Raji, TOLEDO, KG-1, JEKO, REC-1) B-cell lymphoma cells. Effect was more pronounced in the DLBCL cell lines, OCI-LY-1 and OCI-LY-10 (>50% inhibition @ 0.1-0.7 μM), and the reduction in viability was accompanied by corresponding inhibition of pAKT with EC50 of 6 & 70 nM respectively. Treatment of patient-derived primary cells with 4 µM RP6530 caused an increase in cell death. Fold-increase in cytotoxicity as evident from PI+ staining was 1.6 for CLL, 1.1 for DLBCL, 1.2 for MCL, 2.2 for SMZL, and 2.3 for EMZL. Cells in early apotosis (Annexin V+/PI-) were not different between the DMSO blank and RP6530 samples.

Conclusions RP6530 is a potent and selective dual PI3Kδ/γ inhibitor that inhibited growth of B-cell lymphoma cell lines with a concomitant reduction in the downstream biomarker, pAKT. Additionally, the compound showed cytotoxicity in a panel of lymphoma primary cells. Findings provide a rationale for future clinical trials in B-cell malignancies.

Disclosures:Vakkalanka:Rhizen Pharmaceuticals, S.A.: Employment, Equity Ownership; Incozen Therapeutics Pvt. Ltd.: Employment, Equity Ownership.Viswanadha:Incozen Therapeutics Pvt. Ltd.: Employment. Bertoni:Rhizen Pharmaceuticals SA: Research Funding.

 

PI3K Dual Inhibitor (RP-6530)


Therapeutic Area Respiratory , Oncology – Liquid Tumors , Rheumatology Molecule Type Small Molecule
Indication Peripheral T-cell lymphoma (PTCL) , Non-Hodgkins Lymphoma , Asthma , Chronic Obstructive Pulmonary Disease (COPD) , Rheumatoid Arthritis
Development Phase Phase I Rt. of Administration Oral

Description

Rhizen is developing dual PI3K gamma/delta inhibitors for liquid tumors and inflammatory conditions.

Situation Overview

Dual Pl3K inhibition is strongly implicated as an intervention treatment in allergic and non-allergic inflammation of the airways and autoimmune diseases manifested by a reduction in neutrophilia and TNF in response to LPS. Scientific evidence for PI3-kinase involvement in various cellular processes underlying asthma and COPD stems from inhibitor studies and gene-targeting approaches, which makes it a potential target for treatment of respiratory disease. Resistance to conventional therapies such as corticosteroids in several patients has been attributed to an up-regulation of the PI3K pathway; thus, disruption of PI3K signaling provides a novel strategy aimed at counteracting the immuno-inflammatory response. Given the established criticality of these isoforms in immune surveillance, inhibitors specifically targeting the ? and ? isoforms would be expected to attenuate the progression of immune response encountered in most variations of airway inflammation and arthritis.

Mechanism of Action

While alpha and beta isoforms are ubiquitous in their distribution, expression of delta and gamma is restricted to circulating hematogenous cells and endothelial cells. Unlike PI3K-alpha or beta, mice lacking expression of gamma or delta do not show any adverse phenotype indicating that targeting of these specific isoforms would not result in overt toxicity. Dual delta/gamma inhibition is strongly implicated as an intervention strategy in allergic and non-allergic inflammation of the airways and other autoimmune diseases. Scientific evidence for PI3K-delta and gamma involvement in various cellular processes underlying asthma and COPD stems from inhibitor studies and gene-targeting approaches. Also, resistance to conventional therapies such as corticosteroids in several COPD patients has been attributed to an up-regulation of the PI3K delta/gamma pathway. Disruption of PI3K-delta/gamma signalling therefore provides a novel strategy aimed at counteracting the immuno-inflammatory response. Due to the pivotal role played by PI3K-delta and gamma in mediating inflammatory cell functionality such as leukocyte migration and activation, and mast cell degranulation, blocking these isoforms may also be an effective strategy for the treatment of rheumatoid arthritis as well.

Given the established criticality of these isoforms in immune surveillance, inhibitors specifically targeting the delta and gamma isoforms would be expected to attenuate the progression of immune response encountered in airway inflammation and rheumatoid arthritis.

 

http://www.rhizen.com/images/backgrounds/pi3k%20delta%20gamma%20ii.png

http://www.rhizen.com/images/backgrounds/pi3k%20delta%20gamma%20ii.pngtps:/

Clinical Trials

Rhizen has identified an orally active Lead Molecule, RP-6530, that has an excellent pre-clinical profile. RP-6530 is currently in non-GLP Tox studies and is expected to enter Clinical Development in H2 2013.

In December 2013, Rhizen announced the start of a Phase I clinical trial. The study entitled A Phase-I, Dose Escalation Study to Evaluate Safety and Efficacy of RP6530, a dual PI3K delta /gamma inhibitor, in patients with Relapsed or Refractory Hematologic Malignancies is designed primarily to establish the safety and tolerability of RP6530. Secondary objectives include clinical efficacy assessment and biomarker response to allow dose determination and potential patient stratification in subsequent expansion studies.

 

Partners by Region

Rhizen’s pipeline consists of internally discovered (with 100% IP ownership) novel small molecule programs aimed at high value markets of Oncology, Immuno-inflammtion and Metabolic Disorders. Rhizen has been successful in securing critical IP space in these areas and efforts are on for further expansion in to several indications. Rhizen seeks partnerships to unlock the potential of these valuable assets for further development from global pharmaceutical partners. At present global rights on all programs are available and Rhizen is flexible to consider suitable business models for licensing/collaboration.

In 2012, Rhizen announced a joint venture collaboration with TG Therapeutics for global development and commercialization of Rhizen’s Novel Selective PI3K Kinase Inhibitors. The selected lead RP5264 (hereafter, to be developed as TGR-1202) is an orally available, small molecule, PI3K specific inhibitor currently being positioned for the treatment of hematological malignancies.

PATENT
WO2014195888, DUAL SELECTIVE PI3 DELTA AND GAMMA KINASE INHIBITORS

This scheme provides a synthetic route for the preparation of compound of formula wherein all the variables are as described herein in above

Figure imgf000094_0001

15 14 10 12 12a

REFERENCES
April 2015, preclinical data were presented at the 106th AACR Meeting in Philadelphia, PA. RP-6530 had GI50 values of 17,028 and 22,014 nM, respectively
December 2014, data were presented at the 56th ASH Meeting in San Francisco, CA.
December 2013, preclinical data were presented at the 55th ASH Meeting in New Orleans, LA.
June 2013, preclinical data were presented at the 18th Annual EHA Congress in Stockholm, Sweden. RP-6530 inhibited PI3K delta and gamma isoforms with IC50 values of 24.5 and 33.2 nM, respectively.
  • 01 Sep 2015 Phase-I clinical trials in Hematological malignancies (Second-line therapy or greater) in USA (PO) (NCT02567656)
  • 18 Nov 2014 Preclinical trials in Multiple myeloma in Switzerland (PO) prior to November 2014
  • 18 Nov 2014 Early research in Multiple myeloma in Switzerland (PO) prior to November 2014

 

WO2011055215A2 Nov 3, 2010 May 12, 2011 Incozen Therapeutics Pvt. Ltd. Novel kinase modulators
WO2012151525A1 May 4, 2012 Nov 8, 2012 Rhizen Pharmaceuticals Sa Novel compounds as modulators of protein kinases
WO2013164801A1 May 3, 2013 Nov 7, 2013 Rhizen Pharmaceuticals Sa Process for preparation of optically pure and optionally substituted 2- (1 -hydroxy- alkyl) – chromen – 4 – one derivatives and their use in preparing pharmaceuticals
US20110118257 May 19, 2011 Rhizen Pharmaceuticals Sa Novel kinase modulators
US20120289496 May 4, 2012 Nov 15, 2012 Rhizen Pharmaceuticals Sa Novel compounds as modulators of protein kinases
WO 2011055215

 

 

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New Beta Lactamase Inhibitors

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Tazobactam

DR ANTHONY MELVIN CRASTO

Tazobactam.png

 

Tazobactam; Tazobactam acid; 89786-04-9; Tazobactamum; CHEMBL404; YTR-830H;

(2S,3S,5R)-3-methyl-4,4,7-trioxo-3-(triazol-1-ylmethyl)-4$l^{6}-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid

MOLECULAR FORMULA: C10H12N4O5S
MOLECULAR WEIGHT: 300.29108 g/mol

Tazobactam is a beta Lactamase Inhibitor. The mechanism of action of tazobactam is as a beta Lactamase Inhibitor.

Tazobactam is a penicillanic acid sulfone derivative and beta-lactamase inhibitor with antibacterial activity. Tazobactam contains a beta-lactam ring and irreversibly binds to beta-lactamase at or near its active site. This protects other beta-lactam antibiotics from beta-lactamase catalysis. This drug is used in conjunction with beta-lactamase susceptible penicillins to treat infections caused by beta-lactamase producing organisms.

Tazobactam is a pharmaceutical drug that inhibits the action of bacterial β-lactamases, especially those belonging to the SHV-1 and TEM groups. It is commonly used as its sodium salt, tazobactam sodium.

Tazobactam is combined with the extended spectrum β-lactam antibioticpiperacillin in the drug piperacillin/tazobactam, one of the preferred antibiotic treatments for nosocomial pneumonia caused by Pseudomonas aeruginosa.[citation needed] Tazobactam broadens the spectrum of piperacillin by making it effective against organisms that express β-lactamase and would normally degrade piperacillin.[1]

Tazobactam is a heavily modified penicillin and a sulfone.

 

References

 Yang Y, Rasmussen BA, Shlaes DM (1999). “Class A beta-lactamases—enzyme-inhibitor interactions and resistance”. Pharmacol Ther. 83: 141–151. doi:10.1016/S0163-7258(99)00027-3.
Tazobactam ball-and-stick.png
SYSTEMATIC (IUPAC) NAME
(2S,3S,5R)-3-Methyl-7-oxo-3-(1H-1,2,3-triazol-1-ylmethyl)-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid 4,4-dioxide
CLINICAL DATA
AHFS/DRUGS.COM International Drug Names
PREGNANCY
CATEGORY
  • B
LEGAL STATUS
  • (Prescription only)
ROUTES OF
ADMINISTRATION
Intravenous
IDENTIFIERS
CAS NUMBER 89786-04-9 Yes
ATC CODE J01CG02
PUBCHEM CID: 123630
DRUGBANK DB01606 Yes
CHEMSPIDER 110216 Yes
UNII SE10G96M8W Yes
KEGG D00660 Yes
CHEBI CHEBI:9421 Yes
CHEMBL CHEMBL404 Yes
CHEMICAL DATA
FORMULA C10H12N4O5S
MOLECULAR MASS 300.289 g/mol
PATENT SUBMITTED GRANTED
2-OXO-1-AZETIDINE SULFONIC ACID DERIVATIVES AS POTENT BETA-LACTAMASE INHIBITORS [EP0979229] 2000-02-16 2002-10-23
DHA-pharmaceutical agent conjugates of taxanes [US7199151] 2004-09-16 2007-04-03
Antimicrobial composition comprising a vinyyl pyrrolidinon derivative and a carbapenem antibiotic or a beta-lactamase inhibitor [EP0911030] 1999-04-28 2005-04-13
7-alkylidene-3-substituted-3-cephem-4-carboxylates as beta-lactamase inhibitors [US7488724] 2006-04-06 2009-02-10
Sustained release of antiinfectives [US7718189] 2006-04-06 2010-05-18
Conjugate of fine porous particles with polymer molecules and the utilization thereof [US2006159715] 2006-07-20
ENGINEERED BACTERIOPHAGES AS ADJUVANTS FOR ANTIMICROBIAL AGENTS AND COMPOSITIONS AND METHODS OF USE THEREOF [US2010322903] 2009-01-12 2010-12-23
Microparticles for the treatment of disease [US2010323019] 2010-08-19 2010-12-23
Packaging System [US2010326868] 2010-08-30 2010-12-30
COMBINATION ANTIBIOTIC AND ANTIBODY THERAPY FOR THE TREATMENT OF PSEUDOMONAS AERUGINOSA INFECTION [US2010272736] 2010-02-04 2010-10-28

 

MK 7655, RELEBACTAM, a β-Lactamase inhibitor

DR ANTHONY MELVIN CRASTO

MK 7655, RELEBACTAM

(1R,2S,5R)-7-Oxo-N-(4-piperidinyl)-6-(sulfooxy)-1,6-diazabicyclo[3.2.1]octane-2-carboxamide

(1R,2S,5R)-7-oxo-2-((piperidin-4-yl)carbamoyl)-1,6-diazabicyclo(3.2.1)octan-6-yl hydrogen sulfate monohydrate

Sulfuric acid, mono((1R,2S,5R)-7-oxo-2-((4-piperidinylamino)carbonyl)-1,6-diazabicyclo(3.2.1)oct-6-yl) ester, hydrate (1:1)

MF C12H22N4O7S
MW 366.39068 g/mol

CAS 1174020-13-3

β-Lactamase inhibitor

MK-7655 is a beta-lactamase inhibitor in phase III clinical studies at Merck & Co for the treatment of serious bacterial infections…….See clinicaltrials.gov, trial identifier numbers NCT01505634 and NCT01506271.

In 2014, Qualified Infectious Disease Product (QIDP) and Fast Track designations were assigned by the FDA for the treatment of complicated urinary tract infections, complicated intra-abdominal infections and hospital-acquired bacterial pneumonia/ventilator-associated bacterial pneumonia.

 

sc1

PAPER

A concise synthesis of a beta-lactamase inhibitor
Org Lett 2011, 13(20): 5480

http://pubs.acs.org/doi/abs/10.1021/ol202195n

http://pubs.acs.org/doi/suppl/10.1021/ol202195n/suppl_file/ol202195n_si_001.pdf

 

Abstract Image

 

MK-7655 (1) is a β-lactamase inhibitor in clinical trials as a combination therapy for the treatment of bacterial infection resistant to β-lactam antibiotics. Its unusual structural challenges have inspired a rapid synthesis featuring an iridium-catalyzed N–H insertion and a series of late stage transformations designed around the reactivity of the labile bicyclo[3.2.1]urea at the core of the target.

H NMR (400 MHz, DMSO-d6): δ 8.30 (br s, 2H), 8.20 (d, J = 7.8 Hz, 1H), 4.01 (s, 1H), 3.97-3.85 (m, 1H), 3.75 (d, J = 6.5 Hz, 1H), 3.28 (dd, J = 12.9, 2.5 Hz, 2H), 3.05-2.93 (m, 4H), 2.08-1.97 (m, 1H), 1.95-1.79 (m, 3H), 1.73-1.59 (m, 4H);

13C NMR (DMSO-d6, 100 MHz) δ 169.7, 166.9, 59.8, 58.3, 46.9, 44.3, 42.9, 28.5, 28.3, 20.8, 18.9;

HRMS calculated for C12H20N4O6S (M+H): 349.1182, found: 349.1183.

[α]D 25 = -23.3 (c = 1.0, CHCl3)

PATENT

WO 2009091856

http://www.google.com/patents/WO2009091856A2?cl=en

PAPER

Discovery of MK-7655, a beta-lactamase inhibitor for combination with Primaxin
Bioorg Med Chem Lett 2014, 24(3): 780

http://www.sciencedirect.com/science/article/pii/S0960894X13014856

Image for unlabelled figure

 

WO 2014200786

 

NXL104, Avibactam

Dr. Anthony Melvin Castro

NXL-104, Avibactam

trans-7-oxo-6-(sulphooxy)-1,6-diazabicyclo[3,2,1]octane-2-carboxamide sodium salt (e.g., NXL-104)

CAS 396731-20-7, 1192491-61-4

AVE-1330
AVE-1330A

PHASE 1 a broad-spectrum intravenous beta-lactamase inhibitor, was under development for the treatment of infections due to nosocomial drug resistant Gram-negative bacteria

SANOFI  INNOVATOR

Novexel holds exclusive worldwide development and commercialization rights from Sanofi.

 

NXL104; Avibactam; UNII-7352665165;

MOLECULAR FORMULA: C7H11N3O6S
MOLECULAR WEIGHT: 265.24374 g/mol

CAS 1192500-31-4, 396731-14-9

[(2S,5R)-2-carbamoyl-7-oxo-1,6-diazabicyclo[3.2.1]octan-6-yl] hydrogen sulfate

(2S,5R)-7-oxo-6-(sulfooxy)-1,6-diazabicyclo[3.2.1]octane-2-carboxamide

trans-7-oxo-6-(sulfooxy)-1,6-diazabicyclo[3.2.1]octan-2-carboxamide

1,6-Diazabicyclo(3.2.1)octane-2-carboxamide, 7-oxo-6-(sulfooxy)-, (1R,2S,5R)-rel-

Avibactam is a non-β-lactam β-lactamase inhibitor antibiotic being developed by Actavis jointly with AstraZeneca. A new drug application for avibactam incombination with ceftazidime was approved by the FDA on February 25, 2015, for treating complicated urinary tract and complicated intra-abdominal Infections caused by antibiotic resistant-pathogens, including those caused by multi-drug resistant gram-negative bacterial pathogens.[2][3][4]

Increasing resistance to cephalosporins among Gram-(-) bacterial pathogens, especially among hospital-acquired infections, results in part from the production of beta lactamase enzymes that deactivate these antibiotics. While the co-administration of a beta lactamase inhibitor can restore antibacterial activity to the cephalorsporin, previously approved beta lactamase inhibitors such astazobactam and Clavulanic acid do not inhibit important classes of beta lactamase including Klebsiella pneumoniae carbapenemases (KPCs), metallo-beta lactamases, and AmpC. Avibactam inhibits KPCs, AmpC, and some Class D beta lactamases, but is not active aganist NDM-1.[5]

U.S. Pat. No. 7,112,592 discloses novel heterocyclic compounds and their salts, processes for making the compounds and methods of using the compounds as antibacterial agents. One such compound is sodium salt of trans-7-oxo-6-(sulphooxy)-1,6-diazabicyclo[3,2,1]octane-2-carboxamide. Application WO 02/10172 describes the production of azabicyclic compounds and salts thereof with acids and bases, and in particular, trans-7-oxo-6-sulphoxy-1,6-diazabicyclo[3.2.1]octane-2-carboxamide and its pyridinium, tetrabutylammonium and sodium salts. Application WO 03/063864 and U.S. Patent Publication No. 2005/0020572 describe the use of compounds including trans-7-oxo-6-(sulphooxy)-1,6-diazabicyclo[3,2,1]octane-2-carboxamide sodium salt, as β-lactamase inhibitors that can be administered alone or in, combination with β-lactamine antibacterial agents. These references are incorporated herein by reference, in their entirety.

 

PATENT

In some embodiments, sulphaturamide or tetrabutylammonium salt of (1R,2S,5R)-7-oxo-6-(sulphooxy)-1,6-diazabicyclo[3.2.1]octane-2-carboxamide may be prepared by chiral resolution of its racemic precursor trans-7-oxo-6-(phenylmethoxy)-1,6-diazabicyclo[3.2.1]octane-2-carboxamide, the preparation of which is described in Example 33a Stage A in Application WO 02/10172. In exemplary embodiments, injection of 20 μl of a sample of 0.4 mg/mL of trans-7-oxo-6-(sulphooxy)-1,6-diazabicyclo[3.2.1]octane-2-carboxamide, eluted on a Chiralpak ADH column (5 25 cm×4.6 mm) with heptane-ethanol-diethylamine mobile phase 650/350/0.05 vol at 1 mL/min makes it possible to separate the (1R,2S,5R) and (1S,2R,5S) enantiomers with retention times of 17.4 minutes and 10.8 minutes respectively. The sulphaturamide is then obtained by conversion according to the conditions described in Example 33a Stage B then Stage C and finally in Example 33b of Application WO 02/10172.

In other embodiments, the sulphaturamide can be prepared from the mixture of the oxalate salt of (2S)-5-benzyloxyamino-piperidine-2-carboxylic acid, benzyl ester (mixture (2S,5R)/(2S,5S) ˜50/50) described in application FR2921060.

 

Figure US08835455-20140916-C00006

…..

PATENT

http://www.google.com/patents/WO2015150941A1?cl=en

 

References

  1.  “Full Prescribing Information: AVYCAZ™ (ceftazidime-avibactam) for Injection, for intravenous use”. ©2015 Actavis. All rights reserved. Retrieved 1 June 2015.
  2.  Zhanel, GG (2013). “Ceftazidime-avibactam: a novel cephalosporin/β-lactamase inhibitor combination”. Drugs 73 (2): 159-77.doi:10.1007/s40265-013-0013-7. PMID 23371303.
  3.  “Actavis Announces FDA Acceptance of the NDA Filing for Ceftazidime-Avibactam, a Qualified Infectious Disease Product”. Actavis—a global, integrated specialty pharmaceutical company—Actavis. Actavis plc. Retrieved 1 June 2015.
  4. Ehmann, DE; Jahic, H; Ross, PL; Gu, RF; Hu, J; Durand-Réville, TF; Lahiri, S; Thresher, J; Livchak, S; Gao, N; Palmer, T; Walkup, GK; Fisher, SL (2013). “Kinetics of Avibactam Inhibition against Class A, C, and D β-Lactamases”. The Journal of biological chemistry 288 (39): 27960–71.doi:10.1074/jbc.M113.485979. PMC 3784710. PMID 23913691.
  5.  “www.accessdata.fda.gov” (PDF).

 

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Biochemistry and Dysmetabolism of Aging and Serious Illness

Curator: Larry H. Bernstein, MD, FCAP

 

White Matter Lipids as a Ketogenic Fuel Supply in Aging Female Brain: Implications for Alzheimer’s Disease

Lauren P. Klosinski, Jia Yao, Fei Yin, Alfred N. Fonteh, Michael G. Harrington, Trace A. Christensen, Eugenia Trushina, Roberta Diaz Brinton
http://www.ebiomedicine.com/article/S2352-3964(15)30192-4/abstract      DOI: http://dx.doi.org/10.1016/j.ebiom.2015.11.002
Highlights
  • Mitochondrial dysfunction activates mechanisms for catabolism of myelin lipids to generate ketone bodies for ATP production.
  • Mechanisms leading to ketone body driven energy production in brain coincide with stages of reproductive aging in females.
  • Sequential activation of myelin catabolism pathway during aging provides multiple therapeutic targets and windows of efficacy.

The mechanisms underlying white matter degeneration, a hallmark of multiple neurodegenerative diseases including Alzheimer’s, remain unclear. Herein we provide a mechanistic pathway, spanning multiple transitions of aging, that links mitochondrial dysfunction early in aging with later age white matter degeneration. Catabolism of myelin lipids to generate ketone bodies can be viewed as an adaptive survival response to address brain fuel and energy demand. Women are at greatest risk of late-onset-AD, thus, our analyses in female brain address mechanisms of AD pathology and therapeutic targets to prevent, delay and treat AD in the sex most affected with potential relevance to men.

 

White matter degeneration is a pathological hallmark of neurodegenerative diseases including Alzheimer’s. Age remains the greatest risk factor for Alzheimer’s and the prevalence of age-related late onset Alzheimer’s is greatest in females. We investigated mechanisms underlying white matter degeneration in an animal model consistent with the sex at greatest Alzheimer’s risk. Results of these analyses demonstrated decline in mitochondrial respiration, increased mitochondrial hydrogen peroxide production and cytosolic-phospholipase-A2 sphingomyelinase pathway activation during female brain aging. Electron microscopic and lipidomic analyses confirmed myelin degeneration. An increase in fatty acids and mitochondrial fatty acid metabolism machinery was coincident with a rise in brain ketone bodies and decline in plasma ketone bodies. This mechanistic pathway and its chronologically phased activation, links mitochondrial dysfunction early in aging with later age development of white matter degeneration. The catabolism of myelin lipids to generate ketone bodies can be viewed as a systems level adaptive response to address brain fuel and energy demand. Elucidation of the initiating factors and the mechanistic pathway leading to white matter catabolism in the aging female brain provides potential therapeutic targets to prevent and treat demyelinating diseases such as Alzheimer’s and multiple sclerosis. Targeting stages of disease and associated mechanisms will be critical.

3. Results

  1. 3.1. Pathway of Mitochondrial Deficits, H2O2 Production and cPLA2 Activation in the Aging Female Brain
  2. 3.2. cPLA2-sphingomyelinase Pathway Activation in White Matter Astrocytes During Reproductive Senescence
  3. 3.3. Investigation of White Matter Gene Expression Profile During Reproductive Senescence
  4. 3.4. Ultra Structural Analysis of Myelin Sheath During Reproductive Senescence
  5. 3.5. Analysis of the Lipid Profile of Brain During the Transition to Reproductive Senescence
  6. 3.6. Fatty Acid Metabolism and Ketone Generation Following the Transition to Reproductive Senescence

 

4. Discussion

Age remains the greatest risk factor for developing AD (Hansson et al., 2006, Alzheimer’s, 2015). Thus, investigation of transitions in the aging brain is a reasoned strategy for elucidating mechanisms and pathways of vulnerability for developing AD. Aging, while typically perceived as a linear process, is likely composed of dynamic transition states, which can protect against or exacerbate vulnerability to AD (Brinton et al., 2015). An aging transition unique to the female is the perimenopausal to menopausal conversion (Brinton et al., 2015). The bioenergetic similarities between the menopausal transition in women and the early appearance of hypometabolism in persons at risk for AD make the aging female a rational model to investigate mechanisms underlying risk of late onset AD.

Findings from this study replicate our earlier findings that age of reproductive senescence is associated with decline in mitochondrial respiration, increased H2O2 production and shift to ketogenic metabolism in brain (Yao et al., 2010, Ding et al., 2013, Yin et al., 2015). These well established early age-related changes in mitochondrial function and shift to ketone body utilization in brain, are now linked to a mechanistic pathway that connects early decline in mitochondrial respiration and H2O2 production to activation of the cPLA2-sphingomyelinase pathway to catabolize myelin lipids resulting in WM degeneration (Fig. 12). These lipids are sequestered in lipid droplets for subsequent use as a local source of ketone body generation via astrocyte mediated beta-oxidation of fatty acids. Astrocyte derived ketone bodies can then be transported to neurons where they undergo ketolysis to generate acetyl-CoA for TCA derived ATP generation required for synaptic and cell function (Fig. 12).

Thumbnail image of Fig. 12. Opens large image

http://www.ebiomedicine.com/cms/attachment/2040395791/2053874721/gr12.sml

Fig. 12

Schematic model of mitochondrial H2O2 activation of cPLA2-sphingomyelinase pathway as an adaptive response to provide myelin derived fatty acids as a substrate for ketone body generation: The cPLA2-sphingomyelinase pathway is proposed as a mechanistic pathway that links an early event, mitochondrial dysfunction and H2O2, in the prodromal/preclinical phase of Alzheimer’s with later stage development of pathology, white matter degeneration. Our findings demonstrate that an age dependent deficit in mitochondrial respiration and a concomitant rise in oxidative stress activate an adaptive cPLA2-sphingomyelinase pathway to provide myelin derived fatty acids as a substrate for ketone body generation to fuel an energetically compromised brain.

Biochemical evidence obtained from isolated whole brain mitochondria confirms that during reproductive senescence and in response to estrogen deprivation brain mitochondria decline in respiratory capacity (Yao et al., 2009, Yao et al., 2010, Brinton, 2008a, Brinton, 2008b, Swerdlow and Khan, 2009). A well-documented consequence of mitochondrial dysfunction is increased production of reactive oxygen species (ROS), specifically H2O2 (Boveris and Chance, 1973, Beal, 2005, Yin et al., 2014, Yap et al., 2009). While most research focuses on the damage generated by free radicals, in this case H2O2 functions as a signaling molecule to activate cPLA2, the initiating enzyme in the cPLA2-sphingomyelinase pathway (Farooqui and Horrocks, 2006, Han et al., 2003, Sun et al., 2004). In AD brain, increased cPLA2 immunoreactivity is detected almost exclusively in astrocytes suggesting that activation of the cPLA2-sphingomyelinase pathway is localized to astrocytes in AD, as opposed to the neuronal or oligodendroglial localization that is observed during apoptosis (Sun et al., 2004, Malaplate-Armand et al., 2006, Di Paolo and Kim, 2011, Stephenson et al., 1996,Stephenson et al., 1999). In our analysis, cPLA2 (Sanchez-Mejia and Mucke, 2010) activation followed the age-dependent rise in H2O2 production and was sustained at an elevated level.

Direct and robust activation of astrocytic cPLA2 by physiologically relevant concentrations of H2O2 was confirmed in vitro. Astrocytic involvement in the cPLA2-sphingomyelinase pathway was also indicated by an increase in cPLA2 positive astrocyte reactivity in WM tracts of reproductively incompetent mice. These data are consistent with findings from brains of persons with AD that demonstrate the same striking localization of cPLA2immunoreactivity within astrocytes, specifically in the hippocampal formation (Farooqui and Horrocks, 2004). While neurons and astrocytes contain endogenous levels of cPLA2, neuronal cPLA2 is activated by an influx of intracellular calcium, whereas astrocytic cPLA2 is directly activated by excessive generation of H2O2 (Sun et al., 2004, Xu et al., 2003, Tournier et al., 1997). Evidence of this cell type specific activation was confirmed by the activation of cPLA2 in astrocytes by H2O2 and the lack of activation in neurons. These data support that astrocytic, not neuronal, cPLA2 is the cellular mediator of the H2O2 dependent cPLA2-sphingomyelinase pathway activation and provide associative evidence supporting a role of astrocytic mitochondrial H2O2 in age-related WM catabolism.

The pattern of gene expression during the shift to reproductive senescence in the female mouse hippocampus recapitulates key observations in human AD brain tissue, specifically elevation in cPLA2, sphingomyelinase and ceramidase (Schaeffer et al., 2010, He et al., 2010, Li et al., 2014). Further, up-regulation of myelin synthesis, lipid metabolism and inflammatory genes in reproductively incompetent female mice is consistent with the gene expression pattern previously reported from aged male rodent hippocampus, aged female non-human primate hippocampus and human AD hippocampus (Blalock et al., 2003, Blalock et al., 2004, Blalock et al., 2010, Blalock et al., 2011, Kadish et al., 2009, Rowe et al., 2007). In these analyses of gene expression in aged male rodent hippocampus, aged female non-human primate hippocampus and human AD hippocampus down regulation of genes related to mitochondrial function, and up-regulation in multiple genes encoding for enzymes involved in ketone body metabolism occurred (Blalock et al., 2003, Blalock et al., 2004, Blalock et al., 2010, Blalock et al., 2011, Kadish et al., 2009, Rowe et al., 2007). The comparability across data derived from aging female mouse hippocampus reported herein and those derived from male rodent brain, female nonhuman brain and human AD brain strongly suggest that cPLA2-sphingomyelinase pathway activation, myelin sheath degeneration and fatty acid metabolism leading to ketone body generation is a metabolic adaptation that is generalizable across these naturally aging models and are evident in aged human AD brain. Collectively, these data support the translational relevance of findings reported herein.

Data obtained via immunohistochemistry, electron microscopy and MBP protein analyses demonstrated an age-related loss in myelin sheath integrity. Evidence for a loss of myelin structural integrity emerged in reproductively incompetent mice following activation of the cPLA2-sphingomyelinase pathway. The unraveling myelin phenotype observed following reproductive senescence and aging reported herein is consistent with the degenerative phenotype that emerges following exposure to the chemotherapy drug bortezomib which induces mitochondrial dysfunction and increased ROS generation (Carozzi et al., 2010, Cavaletti et al., 2007,Ling et al., 2003). In parallel to the decline in myelin integrity, lipid droplet density increased. In aged mice, accumulation of lipid droplets declined in parallel to the rise in ketone bodies consistent with the utilization of myelin-derived fatty acids to generate ketone bodies. Due to the sequential relationship between WM degeneration and lipid droplet formation, we posit that lipid droplets serve as a temporary storage site for myelin-derived fatty acids prior to undergoing β-oxidation in astrocytes to generate ketone bodies.

Microstructural alterations in myelin integrity were associated with alterations in the lipid profile of brain, indicative of WM degeneration resulting in release of myelin lipids. Sphingomyelin and galactocerebroside are two main lipids that compose the myelin sheath (Baumann and Pham-Dinh, 2001). Ceramide is common to both galactocerebroside and sphingomyelin and is composed of sphingosine coupled to a fatty acid. Ceramide levels increase in aging, in states of ketosis and in neurodegeneration (Filippov et al., 2012, Blazquez et al., 1999, Costantini et al., 2005). Specifically, ceramide levels are elevated at the earliest clinically recognizable stage of AD, indicating a degree of WM degeneration early in disease progression (Di Paolo and Kim, 2011,Han et al., 2002, Costantini et al., 2005). Sphingosine is statistically significantly elevated in the brains of AD patients compared to healthy controls; a rise that was significantly correlated with acid sphingomyelinase activity, Aβ levels and tau hyperphosphorylation (He et al., 2010). In our analyses, a rise in ceramides was first observed early in the aging process in reproductively incompetent mice. The rise in ceramides was coincident with the emergence of loss of myelin integrity consistent with the release of myelin ceramides from sphingomyelin via sphingomyelinase activation. Following the rise in ceramides, sphingosine and fatty acid levels increased. The temporal sequence of the lipid profile was consistent with gene expression indicating activation of ceramidase for catabolism of ceramide into sphingosine and fatty acid during reproductive senescence. Once released from ceramide, fatty acids can be transported into the mitochondrial matrix of astrocytes via CPT-1, where β-oxidation of fatty acids leads to the generation of acetyl-CoA (Glatz et al., 2010). It is well documented that acetyl-CoA cannot cross the inner mitochondrial membrane, thus posing a barrier to direct transport of acetyl-CoA generated by β-oxidation into neurons. In response, the newly generated acetyl-CoA undergoes ketogenesis to generate ketone bodies to fuel energy demands of neurons (Morris, 2005,Guzman and Blazquez, 2004, Stacpoole, 2012). Because astrocytes serve as the primary location of β-oxidation in brain they are critical to maintaining neuronal metabolic viability during periods of reduced glucose utilization (Panov et al., 2014, Ebert et al., 2003, Guzman and Blazquez, 2004).

Once fatty acids are released from myelin ceramides, they are transported into astrocytic mitochondria by CPT1 to undergo β-oxidation. The mitochondrial trifunctional protein HADHA catalyzes the last three steps of mitochondrial β-oxidation of long chain fatty acids, while mitochondrial ABAD (aka SCHAD—short chain fatty acid dehydrogenase) metabolizes short chain fatty acids. Concurrent with the release of myelin fatty acids in aged female mice, CPT1, HADHA and ABAD protein expression as well as ketone body generation increased significantly. These findings indicate that astrocytes play a pivotal role in the response to bioenergetic crisis in brain to activate an adaptive compensatory system that activates catabolism of myelin lipids and the metabolism of those lipids into fatty acids to generate ketone bodies necessary to fuel neuronal demand for acetyl-CoA and ATP.

Collectively, these findings provide a mechanistic pathway that links mitochondrial dysfunction and H2O2generation in brain early in the aging process to later stage white matter degeneration. Astrocytes play a pivotal role in providing a mechanistic strategy to address the bioenergetic demand of neurons in the aging female brain. While this pathway is coincident with reproductive aging in the female brain, it is likely to have mechanistic translatability to the aging male brain. Further, the mechanistic link between bioenergetic decline and WM degeneration has potential relevance to other neurological diseases involving white matter in which postmenopausal women are at greater risk, such as multiple sclerosis. The mechanistic pathway reported herein spans time and is characterized by a progression of early adaptive changes in the bioenergetic system of the brain leading to WM degeneration and ketone body production. Translationally, effective therapeutics to prevent, delay and treat WM degeneration during aging and Alzheimer’s disease will need to specifically target stages within the mechanistic pathway described herein. The fundamental initiating event is a bioenergetic switch from being a glucose dependent brain to a glucose and ketone body dependent brain. It remains to be determined whether it is possible to prevent conversion to or reversal of a ketone dependent brain. Effective therapeutic strategies to intervene in this process require biomarkers of bioenergetic phenotype of the brain and stage of mechanistic progression. The mechanistic pathway reported herein may have relevance to other age-related neurodegenerative diseases characterized by white matter degeneration such as multiple sclerosis.

Blood. 2015 Oct 15;126(16):1925-9.    http://dx.doi.org:/10.1182/blood-2014-12-617498. Epub 2015 Aug 14.
Targeting the leukemia cell metabolism by the CPT1a inhibition: functional preclinical effects in leukemias.
Cancer cells are characterized by perturbations of their metabolic processes. Recent observations demonstrated that the fatty acid oxidation (FAO) pathway may represent an alternative carbon source for anabolic processes in different tumors, therefore appearing particularly promising for therapeutic purposes. Because the carnitine palmitoyl transferase 1a (CPT1a) is a protein that catalyzes the rate-limiting step of FAO, here we investigated the in vitro antileukemic activity of the novel CPT1a inhibitor ST1326 on leukemia cell lines and primary cells obtained from patients with hematologic malignancies. By real-time metabolic analysis, we documented that ST1326 inhibited FAO in leukemia cell lines associated with a dose- and time-dependent cell growth arrest, mitochondrial damage, and apoptosis induction. Data obtained on primary hematopoietic malignant cells confirmed the FAO inhibition and cytotoxic activity of ST1326, particularly on acute myeloid leukemia cells. These data suggest that leukemia treatment may be carried out by targeting metabolic processes.
Oncogene. 2015 Oct 12.   http://dx.doi.org:/10.1038/onc.2015.394. [Epub ahead of print]
Tumour-suppression function of KLF12 through regulation of anoikis.
Suppression of detachment-induced cell death, known as anoikis, is an essential step for cancer metastasis to occur. We report here that expression of KLF12, a member of the Kruppel-like family of transcription factors, is downregulated in lung cancer cell lines that have been selected to grow in the absence of cell adhesion. Knockdown of KLF12 in parental cells results in decreased apoptosis following cell detachment from matrix. KLF12 regulates anoikis by promoting the cell cycle transition through S phase and therefore cell proliferation. Reduced expression levels of KLF12 results in increased ability of lung cancer cells to form tumours in vivo and is associated with poorer survival in lung cancer patients. We therefore identify KLF12 as a novel metastasis-suppressor gene whose loss of function is associated with anoikis resistance through control of the cell cycle.
Mol Cell. 2015 Oct 14. pii: S1097-2765(15)00764-9. doi: 10.1016/j.molcel.2015.09.025. [Epub ahead of print]
PEPCK Coordinates the Regulation of Central Carbon Metabolism to Promote Cancer Cell Growth.
Phosphoenolpyruvate carboxykinase (PEPCK) is well known for its role in gluconeogenesis. However, PEPCK is also a key regulator of TCA cycle flux. The TCA cycle integrates glucose, amino acid, and lipid metabolism depending on cellular needs. In addition, biosynthetic pathways crucial to tumor growth require the TCA cycle for the processing of glucose and glutamine derived carbons. We show here an unexpected role for PEPCK in promoting cancer cell proliferation in vitro and in vivo by increasing glucose and glutamine utilization toward anabolic metabolism. Unexpectedly, PEPCK also increased the synthesis of ribose from non-carbohydrate sources, such as glutamine, a phenomenon not previously described. Finally, we show that the effects of PEPCK on glucose metabolism and cell proliferation are in part mediated via activation of mTORC1. Taken together, these data demonstrate a role for PEPCK that links metabolic flux and anabolic pathways to cancer cell proliferation.
Mol Cancer Res. 2015 Oct;13(10):1408-20.   http://dx.doi.org:/10.1158/1541-7786.MCR-15-0048. Epub 2015 Jun 16.
Disruption of Proline Synthesis in Melanoma Inhibits Protein Production Mediated by the GCN2 Pathway.
Many processes are deregulated in melanoma cells and one of those is protein production. Although much is known about protein synthesis in cancer cells, effective ways of therapeutically targeting this process remain an understudied area of research. A process that is upregulated in melanoma compared with normal melanocytes is proline biosynthesis, which has been linked to both oncogene and tumor suppressor pathways, suggesting an important convergent point for therapeutic intervention. Therefore, an RNAi screen of a kinase library was undertaken, identifying aldehyde dehydrogenase 18 family, member A1 (ALDH18A1) as a critically important gene in regulating melanoma cell growth through proline biosynthesis. Inhibition of ALDH18A1, the gene encoding pyrroline-5-carboxylate synthase (P5CS), significantly decreased cultured melanoma cell viability and tumor growth. Knockdown of P5CS using siRNA had no effect on apoptosis, autophagy, or the cell cycle but cell-doubling time increased dramatically suggesting that there was a general slowdown in cellular metabolism. Mechanistically, targeting ALDH18A1 activated the serine/threonine protein kinase GCN2 (general control nonderepressible 2) to inhibit protein synthesis, which could be reversed with proline supplementation. Thus, targeting ALDH18A1 in melanoma can be used to disrupt proline biosynthesis to limit cell metabolism thereby increasing the cellular doubling time mediated through the GCN2 pathway.  This study demonstrates that melanoma cells are sensitive to disruption of proline synthesis and provides a proof-of-concept that the proline synthesis pathway can be therapeutically targeted in melanoma tumors for tumor inhibitory efficacy. Mol Cancer Res; 13(10); 1408-20. ©2015 AACR.
SDHB-Deficient Cancers: The Role of Mutations That Impair Iron Sulfur Cluster Delivery.
BACKGROUND:  Mutations in the Fe-S cluster-containing SDHB subunit of succinate dehydrogenase cause familial cancer syndromes. Recently the tripeptide motif L(I)YR was identified in the Fe-S recipient protein SDHB, to which the cochaperone HSC20 binds.
METHODS:   In order to characterize the metabolic basis of SDH-deficient cancers we performed stable isotope-resolved metabolomics in a novel SDHB-deficient renal cell carcinoma cell line and conducted bioinformatics and biochemical screening to analyze Fe-S cluster acquisition and assembly of SDH in the presence of other cancer-causing SDHB mutations.

RESULTS:

We found that the SDHB(R46Q) mutation in UOK269 cells disrupted binding of HSC20, causing rapid degradation of SDHB. In the absence of SDHB, respiration was undetectable in UOK269 cells, succinate was elevated to 351.4±63.2 nmol/mg cellular protein, and glutamine became the main source of TCA cycle metabolites through reductive carboxylation. Furthermore, HIF1α, but not HIF2α, increased markedly and the cells showed a strong DNA CpG island methylator phenotype (CIMP). Biochemical and bioinformatic screening revealed that 37% of disease-causing missense mutations in SDHB were located in either the L(I)YR Fe-S transfer motifs or in the 11 Fe-S cluster-ligating cysteines.

CONCLUSIONS:

These findings provide a conceptual framework for understanding how particular mutations disproportionately cause the loss of SDH activity, resulting in accumulation of succinate and metabolic remodeling in SDHB cancer syndromes.

 

SR4 Uncouples Mitochondrial Oxidative Phosphorylation, Modulates AMPK-mTOR Signaling, and Inhibits Proliferation of HepG2 Hepatocarcinoma Cells

  1. L. Figarola, J. Singhal, J. D. Tompkins, G. W. Rogers, C. Warden, D. Horne, A. D. Riggs, S. Awasthi and S. S. Singhal.

J Biol Chem. 2015 Nov 3, [epub ahead of print]

 

CD47 Receptor Globally Regulates Metabolic Pathways That Control Resistance to Ionizing Radiation

  1. W. Miller, D. R. Soto-Pantoja, A. L. Schwartz, J. M. Sipes, W. G. DeGraff, L. A. Ridnour, D. A. Wink and D. D. Roberts.

J Biol Chem. 2015 Oct 9, 290 (41): 24858-74.

 

Knockdown of PKM2 Suppresses Tumor Growth and Invasion in Lung Adenocarcinoma

  1. Sun, A. Zhu, L. Zhang, J. Zhang, Z. Zhong and F. Wang.

Int J Mol Sci. 2015 Oct 15, 16 (10): 24574-87.

 

EglN2 associates with the NRF1-PGC1alpha complex and controls mitochondrial function in breast cancer

  1. Zhang, C. Wang, X. Chen, M. Takada, C. Fan, X. Zheng, H. Wen, Y. Liu, C. Wang, R. G. Pestell, K. M. Aird, W. G. Kaelin, Jr., X. S. Liu and Q. Zhang.

EMBO J. 2015 Oct 22, [epub ahead of print]

 

Mitochondrial Genetics Regulate Breast Cancer Tumorigenicity and Metastatic Potential.

Current paradigms of carcinogenic risk suggest that genetic, hormonal, and environmental factors influence an individual’s predilection for developing metastatic breast cancer. Investigations of tumor latency and metastasis in mice have illustrated differences between inbred strains, but the possibility that mitochondrial genetic inheritance may contribute to such differences in vivo has not been directly tested. In this study, we tested this hypothesis in mitochondrial-nuclear exchange mice we generated, where cohorts shared identical nuclear backgrounds but different mtDNA genomes on the background of the PyMT transgenic mouse model of spontaneous mammary carcinoma. In this setting, we found that primary tumor latency and metastasis segregated with mtDNA, suggesting that mtDNA influences disease progression to a far greater extent than previously appreciated. Our findings prompt further investigation into metabolic differences controlled by mitochondrial process as a basis for understanding tumor development and metastasis in individual subjects. Importantly, differences in mitochondrial DNA are sufficient to fundamentally alter disease course in the PyMT mouse mammary tumor model, suggesting that functional metabolic differences direct early tumor growth and metastatic efficiency. Cancer Res; 75(20); 4429-36. ©2015 AACR.

 

Cancer Lett. 2015 Oct 29. pii: S0304-3835(15)00656-4.    http://dx.doi.org:/10.1016/j.canlet.2015.10.025. [Epub ahead of print]
Carboxyamidotriazole inhibits oxidative phosphorylation in cancer cells and exerts synergistic anti-cancer effect with glycolysis inhibition.

Targeting cancer cell metabolism is a promising strategy against cancer. Here, we confirmed that the anti-cancer drug carboxyamidotriazole (CAI) inhibited mitochondrial respiration in cancer cells for the first time and found a way to enhance its anti-cancer activity by further disturbing the energy metabolism. CAI promoted glucose uptake and lactate production when incubated with cancer cells. The oxidative phosphorylation (OXPHOS) in cancer cells was inhibited by CAI, and the decrease in the activity of the respiratory chain complex I could be one explanation. The anti-cancer effect of CAI was greatly potentiated when being combined with 2-deoxyglucose (2-DG). The cancer cells treated with the combination of CAI and 2-DG were arrested in G2/M phase. The apoptosis and necrosis rates were also increased. In a mouse xenograft model, this combination was well tolerated and retarded the tumor growth. The impairment of cancer cell survival was associated with significant cellular ATP decrease, suggesting that the combination of CAI and 2-DG could be one of the strategies to cause dual inhibition of energy pathways, which might be an effective therapeutic approach for a broad spectrum of tumors.

 

Cancer Immunol Res. 2015 Nov;3(11):1236-47.    http://dx.doi.org:/10.1158/2326-6066.CIR-15-0036. Epub 2015 May 29.
Inhibition of Fatty Acid Oxidation Modulates Immunosuppressive Functions of Myeloid-Derived Suppressor Cells and Enhances Cancer Therapies.

Myeloid-derived suppressor cells (MDSC) promote tumor growth by inhibiting T-cell immunity and promoting malignant cell proliferation and migration. The therapeutic potential of blocking MDSC in tumors has been limited by their heterogeneity, plasticity, and resistance to various chemotherapy agents. Recent studies have highlighted the role of energy metabolic pathways in the differentiation and function of immune cells; however, the metabolic characteristics regulating MDSC remain unclear. We aimed to determine the energy metabolic pathway(s) used by MDSC, establish its impact on their immunosuppressive function, and test whether its inhibition blocks MDSC and enhances antitumor therapies. Using several murine tumor models, we found that tumor-infiltrating MDSC (T-MDSC) increased fatty acid uptake and activated fatty acid oxidation (FAO). This was accompanied by an increased mitochondrial mass, upregulation of key FAO enzymes, and increased oxygen consumption rate. Pharmacologic inhibition of FAO blocked immune inhibitory pathways and functions in T-MDSC and decreased their production of inhibitory cytokines. FAO inhibition alone significantly delayed tumor growth in a T-cell-dependent manner and enhanced the antitumor effect of adoptive T-cell therapy. Furthermore, FAO inhibition combined with low-dose chemotherapy completely inhibited T-MDSC immunosuppressive effects and induced a significant antitumor effect. Interestingly, a similar increase in fatty acid uptake and expression of FAO-related enzymes was found in human MDSC in peripheral blood and tumors. These results support the possibility of testing FAO inhibition as a novel approach to block MDSC and enhance various cancer therapies. Cancer Immunol Res; 3(11); 1236-47. ©2015 AACR.

 

Ionizing radiation induces myofibroblast differentiation via lactate dehydrogenase

  1. L. Judge, K. M. Owens, S. J. Pollock, C. F. Woeller, T. H. Thatcher, J. P. Williams, R. P. Phipps, P. J. Sime and R. M. Kottmann.

Am J Physiol Lung Cell Mol Physiol. 2015 Oct 15, 309 (8): L879-87.

 

Vitamin C selectively kills KRAS and BRAF mutant colorectal cancer cells by targeting GAPDH

  1. Yun, E. Mullarky, C. Lu, K. N. Bosch, A. Kavalier, K. Rivera, J. Roper, Chio, II, E. G. Giannopoulou, C. Rago, A. Muley, J. M. Asara, J. Paik, O. Elemento, Z. Chen, D. J. Pappin, L. E. Dow, N. Papadopoulos, S. S. Gross and L. C. Cantley.

Science. 2015 Nov 5, [epub ahead of print]

 

Down-regulation of FBP1 by ZEB1-mediated repression confers to growth and invasion in lung cancer cells

  1. Zhang, J. Wang, H. Xing, Q. Li, Q. Zhao and J. Li.

Mol Cell Biochem. 2015 Nov 6, [epub ahead of print]

 

J Mol Cell Cardiol. 2015 Oct 23. pii: S0022-2828(15)30073-0.     http://dx.doi.org:/10.1016/j.yjmcc.2015.10.002. [Epub ahead of print]
GRK2 compromises cardiomyocyte mitochondrial function by diminishing fatty acid-mediated oxygen consumption and increasing superoxide levels.

The G protein-coupled receptor kinase-2 (GRK2) is upregulated in the injured heart and contributes to heart failure pathogenesis. GRK2 was recently shown to associate with mitochondria but its functional impact in myocytes due to this localization is unclear. This study was undertaken to determine the effect of elevated GRK2 on mitochondrial respiration in cardiomyocytes. Sub-fractionation of purified cardiac mitochondria revealed that basally GRK2 is found in multiple compartments. Overexpression of GRK2 in mouse cardiomyocytes resulted in an increased amount of mitochondrial-based superoxide. Inhibition of GRK2 increased oxygen consumption rates and ATP production. Moreover, fatty acid oxidation was found to be significantly impaired when GRK2 was elevated and was dependent on the catalytic activity and mitochondrial localization of this kinase. Our study shows that independent of cardiac injury, GRK2 is localized in the mitochondria and its kinase activity negatively impacts the function of this organelle by increasing superoxide levels and altering substrate utilization for energy production.

 

Br J Pharmacol. 2015 Oct 27. doi: 10.1111/bph.13377. [Epub ahead of print]
All-trans retinoic acid protects against doxorubicin-induced cardiotoxicity by activating the Erk2 signalling pathway.
BACKGROUND AND PURPOSE:

Doxorubicin (Dox) is a powerful antineoplastic agent for treating a wide range of cancers. However, doxorubicin cardiotoxicity of the heart has largely limited its clinical use. It is known that all-trans retinoic acid (ATRA) plays important roles in many cardiac biological processes, however, the protective effects of ATRA on doxorubicin cardiotoxicity remain unknown. Here, we studied the effect of ATRA on doxorubicin cardiotoxicity and underlying mechanisms.

EXPERIMENTAL APPROACHES:

Cellular viability assays, western blotting and mitochondrial respiration analyses were employed to evaluate the cellular response to ATRA in H9c2 cells and primary cardiomyocytes. Quantitative PCR (Polymerase Chain Reaction) and gene knockdown were performed to investigate the underlying molecular mechanisms of ATRA’s effects on doxorubicin cardiotoxicity.

KEY RESULTS:

ATRA significantly inhibited doxorubicin-induced apoptosis in H9c2 cells and primary cardiomyocytes. ATRA was more effective against doxorubicin cardiotoxicity than resveratrol and dexrazoxane. ATRA also suppressed reactive oxygen species (ROS) generation, and restored the expression level of mRNA and proteins in phase II detoxifying enzyme system: Nrf2 (nuclear factor-E2-related factor 2), MnSOD (manganese superoxide dismutase), HO-1 (heme oxygenase1) as well as mitochondrial function (mitochondrial membrane integrity, mitochondrial DNA copy numbers, mitochondrial respiration capacity, biogenesis and dynamics). Both Erk1/2 (extracellular signal-regulated kinase1/2) inhibitor (U0126) and Erk2 siRNA, but not Erk1 siRNA, abolished the protective effect of ATRA against doxorubicin-induced toxicity in H9c2 cells. Remarkably, ATRA did not compromise the anticancer efficacy of doxorubicin in gastric carcinoma cells.

CONCLUSION AND IMPLICATION:

ATRA protected cardiomyocytes against doxorubicin-induced toxicity by activating the Erk2 pathway without compromising the anticancer efficacy of doxorubicin. Therefore, ATRA may be a promising candidate as a cardioprotective agent against doxorubicin cardiotoxicity.

 

Proteomic and Biochemical Studies of Lysine Malonylation Suggest Its Malonic Aciduria-associated Regulatory Role in Mitochondrial Function and Fatty Acid Oxidation

  1. Colak, O. Pougovkina, L. Dai, M. Tan, H. Te Brinke, H. Huang, Z. Cheng, J. Park, X. Wan, X. Liu, W. W. Yue, R. J. Wanders, J. W. Locasale, D. B. Lombard, V. C. de Boer and Y. Zhao.

Mol Cell Proteomics. 2015 Nov 1, 14 (11): 3056-71.

 

Foxg1 localizes to mitochondria and coordinates cell differentiation and bioenergetics

  1. Pancrazi, G. Di Benedetto, L. Colombaioni, G. Della Sala, G. Testa, F. Olimpico, A. Reyes, M. Zeviani, T. Pozzan and M. Costa.

Proc Natl Acad Sci U S A. 2015 Oct 27, 112(45): 13910-5.

 

Evidence of Mitochondrial Dysfunction within the Complex Genetic Etiology of Schizophrenia

  1. E. Hjelm, B. Rollins, F. Mamdani, J. C. Lauterborn, G. Kirov, G. Lynch, C. M. Gall, A. Sequeira and M. P. Vawter.

Mol Neuropsychiatry. 2015 Nov 1, 1 (4): 201-219.

 

Metabolic Reprogramming Is Required for Myofibroblast Contractility and Differentiation

  1. Bernard, N. J. Logsdon, S. Ravi, N. Xie, B. P. Persons, S. Rangarajan, J. W. Zmijewski, K. Mitra, G. Liu, V. M. Darley-Usmar and V. J. Thannickal.

J Biol Chem. 2015 Oct 16, 290 (42): 25427-38.

 

J Biol Chem. 2015 Oct 23;290(43):25834-46.    http://dx.doi.org:/10.1074/jbc.M115.658815. Epub 2015 Sep 4.
Kinome Screen Identifies PFKFB3 and Glucose Metabolism as Important Regulators of the Insulin/Insulin-like Growth Factor (IGF)-1 Signaling Pathway.

The insulin/insulin-like growth factor (IGF)-1 signaling pathway (ISP) plays a fundamental role in long term health in a range of organisms. Protein kinases including Akt and ERK are intimately involved in the ISP. To identify other kinases that may participate in this pathway or intersect with it in a regulatory manner, we performed a whole kinome (779 kinases) siRNA screen for positive or negative regulators of the ISP, using GLUT4 translocation to the cell surface as an output for pathway activity. We identified PFKFB3, a positive regulator of glycolysis that is highly expressed in cancer cells and adipocytes, as a positive ISP regulator. Pharmacological inhibition of PFKFB3 suppressed insulin-stimulated glucose uptake, GLUT4 translocation, and Akt signaling in 3T3-L1 adipocytes. In contrast, overexpression of PFKFB3 in HEK293 cells potentiated insulin-dependent phosphorylation of Akt and Akt substrates. Furthermore, pharmacological modulation of glycolysis in 3T3-L1 adipocytes affected Akt phosphorylation. These data add to an emerging body of evidence that metabolism plays a central role in regulating numerous biological processes including the ISP. Our findings have important implications for diseases such as type 2 diabetes and cancer that are characterized by marked disruption of both metabolism and growth factor signaling.

 

FASEB J. 2015 Oct 19.    http://dx.doi.org:/pii: fj.15-276360. [Epub ahead of print]
Perm1 enhances mitochondrial biogenesis, oxidative capacity, and fatigue resistance in adult skeletal muscle.

Skeletal muscle mitochondrial content and oxidative capacity are important determinants of muscle function and whole-body health. Mitochondrial content and function are enhanced by endurance exercise and impaired in states or diseases where muscle function is compromised, such as myopathies, muscular dystrophies, neuromuscular diseases, and age-related muscle atrophy. Hence, elucidating the mechanisms that control muscle mitochondrial content and oxidative function can provide new insights into states and diseases that affect muscle health. In past studies, we identified Perm1 (PPARGC1- and ESRR-induced regulator, muscle 1) as a gene induced by endurance exercise in skeletal muscle, and regulating mitochondrial oxidative function in cultured myotubes. The capacity of Perm1 to regulate muscle mitochondrial content and function in vivo is not yet known. In this study, we use adeno-associated viral (AAV) vectors to increase Perm1 expression in skeletal muscles of 4-wk-old mice. Compared to control vector, AAV1-Perm1 leads to significant increases in mitochondrial content and oxidative capacity (by 40-80%). Moreover, AAV1-Perm1-transduced muscles show increased capillary density and resistance to fatigue (by 33 and 31%, respectively), without prominent changes in fiber-type composition. These findings suggest that Perm1 selectively regulates mitochondrial biogenesis and oxidative function, and implicate Perm1 in muscle adaptations that also occur in response to endurance exercise.-Cho, Y., Hazen, B. C., Gandra, P. G., Ward, S. R., Schenk, S., Russell, A. P., Kralli, A. Perm1 enhances mitochondrial biogenesis, oxidative capacity, and fatigue resistance in adult skeletal muscle.

 

A conserved MADS-box phosphorylation motif regulates differentiation and mitochondrial function in skeletal, cardiac, and smooth muscle cells.
Exposure to metabolic disease during fetal development alters cellular differentiation and perturbs metabolic homeostasis, but the underlying molecular regulators of this phenomenon in muscle cells are not completely understood. To address this, we undertook a computational approach to identify cooperating partners of the myocyte enhancer factor-2 (MEF2) family of transcription factors, known regulators of muscle differentiation and metabolic function. We demonstrate that MEF2 and the serum response factor (SRF) collaboratively regulate the expression of numerous muscle-specific genes, including microRNA-133a (miR-133a). Using tandem mass spectrometry techniques, we identify a conserved phosphorylation motif within the MEF2 and SRF Mcm1 Agamous Deficiens SRF (MADS)-box that regulates miR-133a expression and mitochondrial function in response to a lipotoxic signal. Furthermore, reconstitution of MEF2 function by expression of a neutralizing mutation in this identified phosphorylation motif restores miR-133a expression and mitochondrial membrane potential during lipotoxicity. Mechanistically, we demonstrate that miR-133a regulates mitochondrial function through translational inhibition of a mitophagy and cell death modulating protein, called Nix. Finally, we show that rodents exposed to gestational diabetes during fetal development display muscle diacylglycerol accumulation, concurrent with insulin resistance, reduced miR-133a, and elevated Nix expression, as young adult rats. Given the diverse roles of miR-133a and Nix in regulating mitochondrial function, and proliferation in certain cancers, dysregulation of this genetic pathway may have broad implications involving insulin resistance, cardiovascular disease, and cancer biology.

 

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Biomarker Development


Biomarker Development

Curator: Larry H. Bernstein, MD, FCAP

 

 

NBDA’s Biomarker R&D Modules

http://nbdabiomarkers.org/

“collaboratively creating the NBDA Standards* required for end-to-end, evidence – based biomarker development to advance precision (personalized) medicine”

http://nbdabiomarkers.org/sites/all/themes/nbda/images/nbda_logo.jpg

http://nbdabiomarkers.org/about/what-we-do/pipeline-overview/assay-development

 

Successful biomarkers should move systematically and seamlessly through specific R&D “modules” – from early discovery to clinical validation. NBDA’s end-to-end systems approach is based on working with experts from all affected multi-sector stakeholder communities to build an in-depth understanding of the existing barriers in each of these “modules” to support decision making at each juncture.  Following extensive “due diligence” the NBDA works with all stakeholders to assemble and/or create the enabling standards (guidelines, best practices, SOPs) needed to support clinically relevant and robust biomarker development.

Mission: Collaboratively creating the NBDA Standards* required for end-to-end, evidence – based biomarker development to advance precision (personalized) medicine.
NBDA Standards include but are not limited to: “official existing standards”, guidelines, principles, standard operating procedures (SOP), and best practices.

https://vimeo.com/83266065

 

“The NBDA’s vision is not to just relegate the current biomarker development processes to history, but also to serve as a working example of what convergence of purpose, scientific knowledge and collaboration can accomplish.”

NBDA Workshop VII – “COLLABORATIVELY BUILDING A FOUNDATION FOR FDA BIOMARKER QUALIFICATION”
NBDA Workshop VII   December 14-15, 2015   Washington Court Hotel, Washington, DC

The upcoming meeting was preceded by an NBDA workshop held on December 1-2, 2014, “The Promising but Elusive Surrogate Endpoint:  What Will It Take?” where we explored in-depth with FDA leadership and experts in the field the current status and future vison for achieving success in surrogate endpoint development.  Through panels and workgroups, the attendees extended their efforts to pursue the FDA’s biomarker qualification pathway through the creation of sequential contexts of use models to support qualification of drug development tools – and ultimately surrogate endpoints.

Although the biomarker (drug development tools) qualification pathway (http://www.fda.gov/Drugs/DevelopmentApprovalProcess/DrugDevelopmentTools…) represents an opportunity to increase the value of predictive biomarkers, animal models, and clinical outcomes across the drug (and biologics) development continuum, there are myriad challenges.  In that regard, the lack of evidentiary standards to support contexts of use-specific biomarkers emerged from the prior NBDA workshop as the major barrier to achieving the promise of biomarker qualification.  It also became clear that overall, the communities do not understand the biomarker qualification process; nor do they fully appreciate that it is up to the stakeholders in the field (academia, non-profit foundations, pharmaceutical and biotechnology companies, and patient advocate organizations) to develop these evidentiary standards.

This NBDA workshop will feature a unique approach to address these problems.  Over the past two years, the NBDA has worked with experts in selected disease areas to develop specific case studies that feature a systematic approach to identifying the evidentiary standards needed for sequential contexts of use for specific biomarkers to drive biomarker qualification.   These constructs, and accompanying whitepapers are now the focus of collaborative discussions with FDA experts.

The upcoming meeting will feature in-depth panel discussions of 3-4 of these cases, including the case leader, additional technical contributors, and a number of FDA experts.  Each of the panels will analyze their respective case for strengths and weaknesses – including suggestions for making the biomarker qualification path for the specific biomarker more transparent and efficient. In addition, the discussions will highlight the problem of poor reproducibility of biomarker discovery results, and its impact on the qualification process.

 

Health Care in the Digital Age

Mobile, big data, the Internet of Things and social media are leading a revolution that is transforming opportunities in health care and research. Extraordinary advancements in mobile technology and connectivity have provided the foundation needed to dramatically change the way health care is practiced today and research is done tomorrow. While we are still in the early innings of using mobile technology in the delivery of health care, evidence supporting its potential to impact the delivery of better health care, lower costs and improve patient outcomes is apparent. Mobile technology for health care, or mHealth, can empower doctors to more effectively engage their patients and provide secure information on demand, anytime and anywhere. Patients demand safety, speed and security from their providers. What are the technologies that are allowing this transformation to take place?

 

https://youtu.be/WeXEa2cL3oA    Monday, April 27, 2015  Milken Institute

Moderator


Michael Milken, Chairman, Milken Institute

 

Speakers


Anna Barker, Fellow, FasterCures, a Center of the Milken Institute; Professor and Director, Transformative Healthcare Networks, and Co-Director, Complex Adaptive Systems Network, Arizona State University
Atul Butte, Director, Institute of Computational Health Sciences, University of California, San Francisco
John Chen, Executive Chairman and CEO, BlackBerry
Victor Dzau, President, Institute of Medicine, National Academy of Sciences; Chancellor Emeritus, Duke University
Patrick Soon-Shiong, Chairman and CEO, NantWorks, LLC

 

Mobile, big data, the Internet of Things and social media are leading a revolution that is transforming opportunities in health care and research. Extraordinary advancements in mobile technology and connectivity have provided the foundation needed to dramatically change the way health care is practiced today and research is done tomorrow. While we are still in the early innings of using mobile technology in the delivery of health care, evidence supporting its potential to impact the delivery of better health care, lower costs and improve patient outcomes is apparent. Mobile technology for health care, or mHealth, can empower doctors to more effectively engage their patients and provide secure information on demand, anytime and anywhere. Patients demand safety, speed and security from their providers. What are the technologies that are allowing this transformation to take place?

 

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Complexity of Protein-Protein Interactions

Curator: Larry H. Bernstein, MD, FCAP

Cracking the Complex

Using mass spec to study protein-protein interactions

By Jeffrey M. Perkel | November 1, 2015

http://www.the-scientist.com//?articles.view/articleNo/44317/title/Cracking-the-Complex/

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Mass spectrometry is a proteomics workhorse. By precisely measuring polypeptide masses, researchers can identify and sequence those molecules, and characterize whether and how they have been chemically modified. To twist a phrase, by their masses you shall know them.

But many proteins do not act in isolation. Critical biological processes such as DNA replication, transcription, translation, cell division, and energy generation rely on the action of massive protein assemblies, many of which comprise dozens of subunits. While these clusters are ripe for study, few traditional mass spectrometric methods can handle them.

Indeed, protein complexes are unwieldy for many types of analysis, says Philip Compton, director of instrumentation at the Proteomics Center of Excellence at Northwestern University in Evanston, Illinois. Most complexes are held together by noncovalent interactions, assemble only transiently, or are located in the cell membrane—all of which complicate sample preparation, he explains. Also, while some complexes are relatively abundant, others are rare, further thwarting detection and analysis.

For mass spectrometry specifically, however, the problem with analyzing protein complexes, which can weigh in at 500 kDa, is size. “In a mass spec, things of that size have traditionally been fairly difficult to handle,” Compton says. Even if you can deliver them into the spectrometer itself, you need a way to figure out which proteins are present, and in what stoichiometry. Plus, normal sample preparation procedures tend to denature proteins, ripping complexes apart.

Still, researchers are increasingly keen to train their mass specs on intact protein assemblies. The Scientistasked four protein-complex experts about the approaches they use in their own labs. This is what they said.

Determining subunit composition 

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GETTING TOGETHER: Lactate dehydrogenase from human skeletal muscle comprises four identical M subunits, shown here in different colors.  FVASCONCELLOS/WIKIMEDIA COMMONS

RESEARCHER: Philip Compton, Director of Instrumentation, Proteomics Center of Excellence, Northwestern University

PROJECT: High-throughput top-down proteomics

SOLUTION: If protein complexes are onions, Compton needs a way to iteratively peel off the layers to see what’s inside. Working with researchers at Thermo Fisher Scientific, Compton is developing an Orbitrap-based mass spectrometer that can do just that, or perform what is called an MS3 study.

Basically, an MS3 experiment involves weighing all the complexes in a sample fraction—there could be as many as 10 or 15 at a time—grabbing one, smashing it into inert-gas molecules to eject a subunit, weighing and sequencing the cast-off piece, and then repeating the process.

That’s the goal, but because that instrument is not yet built, Compton must temporarily content himself with what he calls a “pseudo-MS3” experiment. Basically, instead of one seamless workflow, the instrument shatters the complex, weighs the pieces that come off it, and then repeats the process, only this time capturing and fragmenting those ejected pieces for subsequent analysis (Anal Chem, 85:11163-73, 2013). “We’re kind of splitting it into these two different steps; that accomplishes essentially the same thing,” Compton says.

Compton and his team are still ironing out the kinks, but they have begun applying the approach to protein complexes involved in metabolism. One of these, lactate dehydrogenase (LDH), is a 145-kDa tetramer comprising M (muscle) and H (heart) subunits that can exist in any of five configurations (MMMM, MMMH, MMHH, MHHH, and HHHH). Using the MS3 workflow, Compton says he can differentiate these “multiproteoform assemblies,” as well as any posttranslational modifications those subunits may bear, and determine the abundance of each. Now he hopes to apply the approach to quantify LDH differences between cell and tissue types.

From Protein Complexes to Subunit Backbone Fragments: A Multi-stage Approach to Native Mass Spectrometry

Thermo Fisher Scientific, 28199 Bremen, Germany
Northwestern University, Evanston, Illinois 60208, United States
Anal. Chem., 2013, 85 (23), pp 11163–11173    DOI: http://dx.doi.org:/10.1021/ac4029328
Publication Date (Web): November 15, 2013   Copyright © 2013 American Chemical Society
Abstract Image
Native mass spectrometry (MS) is becoming an important integral part of structural proteomics and system biology research. The approach holds great promise for elucidating higher levels of protein structure: from primary to quaternary. This requires the most efficient use of tandem MS, which is the cornerstone of MS-based approaches. In this work, we advance a two-step fragmentation approach, or (pseudo)-MS3, from native protein complexes to a set of constituent fragment ions. Using an efficient desolvation approach and quadrupole selection in the extended mass-to-charge (m/z) range, we have accomplished sequential dissociation of large protein complexes, such as phosporylase B (194 kDa), pyruvate kinase (232 kDa), and GroEL (801 kDa), to highly charged monomers which were then dissociated to a set of multiply charged fragmentation products. Fragment ion signals were acquired with a high resolution, high mass accuracy Orbitrap instrument that enabled highly confident identifications of the precursor monomer subunits. The developed approach is expected to enable characterization of stoichiometry and composition of endogenous native protein complexes at an unprecedented level of detail.

EXTEND YOUR RANGE: Compton’s team uses a souped-up version of Thermo Fisher’s Orbitrap-based Q Exactive HF mass spectrometer, which among other things features a fourfold wider mass range. Other researchers can perform similar work using Thermo’s Exactive Plus EMR Orbitrap system, an off-the-shelf, “extended mass range” instrument. But, because the EMR lacks the “high-mass isolation capabilities” of Compton’s bespoke hardware, the application range is more limited, he says. “You can still do a similar experiment to us, provided that you have one clean [purified] complex.”

Mapping protein-protein interaction interfaces
RESEARCHER: Igor Kaltashov, Professor of Chemistry, University of Massachusetts Amherst

PROJECT: Probing the interactions of candidate protein therapeutics with their molecular targets

SOLUTION: Most attempts at studying protein complexes deliver them to the mass spec intact. Kaltashov takes a different approach, using a technique called hydrogen-deuterium exchange (HDX).

It works like this: proteins (like other molecules) pass hydrogen atoms back and forth with the solvent that surrounds them. Normally, one hydrogen is simply swapped for another, and nobody is the wiser. But in deuterated (“heavy”) water, as hydrogens are swapped at the protein surface, the protein gets slightly heavier as deuterium molecules replace some of the hydrogens. This allows researchers to probe how accessible different pieces of the protein are to the solvent, based on how much deuterium they pick up from the buffer, and how quickly they do so.

As Kaltashov explains, HDX can be used to study any event that might alter the accessibility of different protein regions to the solvent that surrounds them. Those events include protein folding and aggregation, but also protein-protein interactions. “Once two proteins bind to each other, solvent would be excluded from the interface, and that would be reflected in the hydrogen-deuterium exchange kinetics,” he says. That change is evident when compared to the proteins in isolation.

In a 2009 review, Kaltashov demonstrated the process with transferrin, an iron transport protein, and its receptor. After undergoing the exchange reaction, the proteins were fragmented to peptides and analyzed piecemeal. Some peptides exhibited no hydrogen-deuterium exchange, he says. That suggests they were never exposed to solvent because they were buried inside the protein core. Other peptides exchanged hydrogens with the solvent at the same rate regardless of receptor binding, indicating they are not part of the protein-receptor interface. A third set of peptides, though, exhibited clear differences in the presence and absence of receptor, marking those as elements of the protein-protein interaction domain (Anal Chem, 81:7892-99, 2009).

“You can actually localize these sites and obtain information both on the strength of the binding [interactions] and the structural characteristics of the interface region,” Kaltashov says.

H/D exchange and mass spectrometry in the studies of protein conformation and dynamics: Is there a need for a top-down approach?

Hydrogen/deuterium exchange (HDX) combined with mass spectrometry (MS) detection has matured in recent years to become a powerful tool in structural biology and biophysics. Several limitations of this technique can and will be addressed by tapping into ever expanding arsenal of methods to manipulate ions in the gas phase offered by mass spectrometry.

Keywords: hydrogen/deuterium exchange (HDX), mass spectrometry (MS), protein ion fragmentation, collision-induced dissociation (CAD), electron-capture dissociation (ECD), electron-transfer dissociation (ETD), protein conformation, protein dynamics

Introduction: HDX MS in the context of structural proteomics

The spectacular successes of proteomics and bioinformatics in the past decade have resulted in an explosive growth of information on the composition of complex networks of proteins interacting at the cellular level and beyond. However, a simple inventory of interacting proteins is insufficient for understanding how the components of sophisticated biological machinery work together. Protein interactions with each other, small ligands and other biopolymers are governed by their higher order structure, whose determination on a genome scale is a focus of structural proteomics. Realization that “the structures of individual macromolecules are often uninformative about function if taken out of context”1 is shifting the focus of the inquiry from comprehensive characterization of individual protein structures to structural analysis of protein complexes.

X-ray crystallography remains the mainstay in this field, and high resolution structures of proteins and protein complexes often provide important clues as to how they carry out their diverse functions in vivo. However, individual proteins are not static objects, and their behavior cannot be adequately described based solely on information derived from static snapshots and without taking into consideration their dynamic character.2Conformation and dynamics of small proteins can be probed at high spatial resolution on a variety of time scales using NMR spectroscopy; however, rather unforgiving molecular weight limitations make this technique less suited for the studies of larger proteins and protein complexes.

Mass spectrometry (MS) is playing an increasingly visible role in this field, as it can provide information on protein dynamics on a variety of levels, ranging from interactions with their physiological partners by forming dynamic assemblies3 to large-scale conformational transitions within individual subunits.4 Perhaps one of the most powerful MS-based tools to characterize protein conformation and dynamics is HDX MS, a technique that combined hydrogen/deuterium exchange in solution5 with MS detection of the progress of exchange reactions.6 This technique is certainly not new,7 and in fact already made lasting impact in diverse fields ranging from structural proteomics8 to analysis of biopharmaceutical products.9 Nevertheless, HDX MS methodology is still in a phase where dramatic progress is made, fed by the continued expansion of the experimental armamentarium offered by MS. In particular, better integration of new methods of manipulating ions in the gas phase into HDX MS routine is likely to result in truly transformative changes. This sea change in HDX MS methodology will transform it to a potent tool rivaling NMR in terms of resolution, but without suffering the limitations of this technique.

What information can be deduced from HDX MS measurements? The classic “bottom-up” approach, its challenges and limitations

While the concept of HDX experiment may appear rather transparent (Figure 1), interpretation of the results is usually not. The backbone protection measured in a typical HDX MS experiment is a combination of several factors, as the exchange reaction of each labile hydrogen atom is a convolution of two processes.5The first is a protein motion that makes a particular hydrogen atom exposed to solvent and therefore available for the exchange. This could be a small-scale event, such as relatively frequent local structural fluctuations transiently exposing hydrogen atoms residing close to the protein surface, or a rare global unfolding event exposing atoms sequestered from the solvent in the protein core. The second process is a chemical reaction of exchanging the unprotected labile hydrogen atom with the solvent. The kinetics of this reaction (intrinsic exchange rate) strongly depends on solution temperature and pH (with a minimum at pH 2.5-3 for backbone amides), parameters that obviously have a great influence on the protein dynamics as well.

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Schematic representation of HDX MS experiments: bottom-up (A) and top-down (B) HDX MS.

Since the majority of HDX MS studies target protein dynamics under near-native conditions, the experiments are typically carried out at physiological pH, where the progress of the exchange is followed by monitoring the protein mass change. The direct infusion scheme offers the simplest way to carry out such measurements, either in real time7 or by using on-line rapid mixing.10 However, in many cases these straightforward approaches cannot be used, as they limit the choice of exchange buffer systems to those compatible with electrospray ionization (ESI). To avoid this, HDX can be carried out in any suitable buffer followed by rapid quenching (lowering pH to 2.5-3 and temperature to near 0°C). Dramatic deceleration of the intrinsic exchange rate for backbone amides under these conditions allows the protein solution to be de-salted prior to MS analysis. Additionally, the slow exchange conditions denature most proteins, resulting in facile removal of various binding partners, ranging from small ligands to receptors (their binding to the protein of interest inevitably complicates the HDX MS data interpretation by making accurate mass measurements in the gas phase less straightforward).

An example of such experiments is shown in Figure 2, where HDX is used to probe the higher order structure and conformational dynamics of metal transporter transferrin (Fe2Tf) alone and in the receptor-bound form. Both Tf-metal and Tf-receptor complexes dissociate under the slow exchange conditions prior to MS analysis; therefore, the protein mass evolution in each case reflects solely deuterium uptake in the course of exchange in solution. The extra protection afforded by the receptor binding to Tf persists over an extended period of time, and it may be tempting to assign it to shielding of labile hydrogen atoms at the protein-receptor interface. However, this view is overly simplistic, as the conformational effects of protein binding are frequently felt well beyond the interface region. The difference in the backbone protection levels of receptor-free and receptor-bound forms of Fe2Tf appears to grow during the initial hour of the exchange (Figure 2), reflecting significant stabilization of Fe2Tf higher order structure by the receptor binding. Indeed, while the fast phase of HDX is typically ascribed to frequent local fluctuations (transient perturbations of higher order structure) affecting relatively small protein segments, the slower phases of HDX usually reflect relatively rare, large-scale conformational transitions (transient partial or complete unfolding). This is why global HDX MS measurements similar to those presented in Figure 2 are can be used to obtain quantitative thermodynamic characteristics for protein interaction with a variety of ligands, ranging from metal ions11 and small organic molecules 12 to other proteins13 and oligonucleotides.14

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HDX MS of Fe2Tf in the presence (blue) and the absence (red) of the cognate receptor. The exchange was carried out by diluting the protein stock solution 1:10 in exchange solution (100 mM NH4HCO3 in D2O, pH adjusted to 7.4) and incubating for a certain period of time as indicated on each diagram followed by rapid quenching (lowering pH to 2.5 and temperature to near 0°C). The black trace shows unlabeled protein.

While global HDX MS measurements under near-native conditions provide valuable thermodynamic information on proteins and their interaction with binding partners, structural studies (e.g., localizing the changes in Tf that occur as a result of receptor binding) must rely on the knowledge of exchange kinetics at the local level. This is typically accomplished by carrying out proteolysis under the slow exchange conditions following the quench of HDX.6 Here we will refer to this approach as “bottom-up” HDX MS, by drawing analogy to a bottom-up approach to obtain sequence information.15 An example is shown in Figure 3, where Fe2Tf undergoes exchange in solution in the absence and in the presence of the receptor, followed by rapid quenching of HDX reactions, protein reduction and digestion with pepsin and LC/MS analysis of the deuterium content of individual proteolytic peptides.

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Localizing the influence of the receptor binding on backbone protection of Fe2Tf using bottom-up HDX MS on the physiologically relevant time scale. The panels show isotopic distributions of representative peptic fragments derived from the protein subjected to HDX in the presence (blue) and the absence (red) of the receptor and followed by rapid quenching. Dotted lines indicate deuterium content of unlabeled and fully exchanged peptides. Colored segments within the Fe2Tf/receptor complex show location of the peptic fragments.

Evolution of deuterium content of various peptic fragments in Figure 3 reveals a wide spectrum of protection, which is clearly distributed very unevenly across the protein sequence. While some peptides exhibit nearly complete protection of backbone amides (e.g., segment [396-408] sequestered in the core of the protein C-lobe), exchange in some other segments is fast (e.g., peptide [612-621] in the solvent-exposed loop of the C-lobe). The influence of the receptor binding on the backbone protection is also highly localized. While many segments appear to be unaffected by the receptor binding, there are a few regions where exchange kinetics noticeably decelerates (e.g., segment [71-81] of the N-lobe, which contains several amino acid residues that form Tf/receptor interface according to the available model of the complex based on low-resolution cryo-EM data16).

Although the increased protection of backbone amides proximal to the protein/receptor binding interface is hardly surprising, HDX MS data also reveal a less trivial trend, acceleration of exchange kinetics in some segments of the protein as a result of receptor binding (such behavior is illustrated in Figure 3 with segment [113-134], a part of the N-lobe that is distal to the receptor). Therefore, in addition to mapping binding interface regions, HDX MS also provides a means to localize the protein segments that are affected by the binding indirectly via allosteric mechanisms. However, this example also highlights one of the limitations of HDX MS, namely inadequate spatial resolution. This peptic fragment spans several distinct regions of the protein (an α-helical segment, a β-strand, and two loops). The moderate level of protection observed in this segment in the absence of the receptor binding (fast exchange of three protons followed by slow exchange of the rest) is likely to be a result of averaging out very uneven protection patterns across this peptide. Even smaller peptides may comprise two or more distinct structural elements, such as segment [71-81] spanning three distinct regions of the protein (an α-helical segment, a β-strand, and a loop connecting them).

In some favorable cases spatial resolution in HDX MS of small proteins (<15 kDa) may be enhanced up to a single residue level by analyzing deuterium content of a set of overlapping proteolytic fragments.17However, single-residue resolution has never been demonstrated in HDX MS studies of proteins falling out of the mass range routinely accessible by NMR, although overlapping peptic fragments frequently provide moderate improvement of spatial resolution.

In addition to limited spatial resolution, the “classic” HDX MS scheme frequently suffers from incomplete sequence coverage, especially when applied to larger and extensively glycosylated proteins. Proteins with multiple disulfide bonds constitute another class of targets for which adequate sequence coverage is difficult to achieve, although certain changes in experimental protocol can alleviate this problem, at least for smaller proteins.18 Typically, an 80% level of sequence coverage is considered good, although significantly lower levels may also be adequate, depending on the context of the study.

Protein processing in HDX MS experiments is carried out under the conditions that minimize the exchange rates for backbone amides. Since these slow exchange conditions are highly denaturing for most proteins, both intact protein and its proteolytic fragments lack any protection and inevitably begin to lose their labile isotopic labels, despite low (but finite) intrinsic exchange rates.19 This phenomenon, known as “back-exchange,” may be accelerated during various stages of protein processing, e.g. during the chromatographic step.20 Although back-exchange was frequently evaluated in early HDX MS studies using unstructured model peptides, the utility of this procedure is questionable, since the intrinsic exchange rates are highly sequence-dependent. In many instances, back-exchange may be estimated using algorithms based on context-specific kinetics data (e.g., http://hx2.med.upenn.edu/download.html); it may also be determined experimentally for each proteolytic fragment by processing a fully labeled protein using a series of steps that precisely reproduce those used in HDX MS measurements.9 Typical back-exchange levels reported in recent literature range from 10% to 50%, although significantly higher numbers have also been reported. Even if back-exchange can be accounted for, it nonetheless has very detrimental influence on the quality of HDX MS measurements by reducing the available dynamic range.

Finally, the classic HDX MS scheme is poorly suited for measurements that are carried out under conditions favoring correlated exchange, when HDX kinetics follows the so-called EX1 regime, leading to appearance of bimodal and convoluted multi-modal isotopic distributions of protein ions.21 Carrying out HDX MS measurements under these conditions provides a unique opportunity to visualize and characterize distinct conformational states, which can be populated either transiently10 or at equilibrium.22 The distinction among such states can be made based on the differences in their deuterium contents. However, proteolysis in solution almost always leads to a loss of correlation between the deuterium content of fragment peptides and specific conformers with distinct levels of backbone protection. Therefore, the classic HDX MS scheme does not allow protein higher order structure and dynamics to be characterized in a conformer-specific fashion.

“Top-down” HDX MS: tandem MS allows protein structure to be probed in the conformer-specific fashion but raises the specter of hydrogen scrambling

The problem of characterizing protein conformation and dynamics in a conformer-specific fashion can be addressed using methods of tandem mass spectrometry (the so-called “top-down” HDX MS). Indeed, replacement of proteolysis in solution with protein ion fragmentation in the gas phase following mass selection of precursor ions provides a means to obtain fragment ions originating from a particular conformer with a specific level of deuterium incorporation. Deuterium content of fragment ions would then provide a measure of local protection patterns, assuming there is no internal re-arrangement of labile hydrogen and deuterium atoms during ion activation (vide infra). Although the idea to use polypeptide ion dissociation in the gas phase as an alternative to proteolysis was originally proposed in early 1990s,23 its implementation for proteins only became possible24 following dramatic improvements in FTMS and hybrid TOF analyzers in the late 1990s.

An example of conformer-specific characterization of protein higher order structure using a top-down HDX MS approach is illustrated in Figure 4. The isotopic profile of a fully deuterated 18 kDa protein wt*-CRABPI is recorded following its brief exposure to the 1H-based exchange buffer. The bimodal appearance of the isotopic distribution of the molecular ion (top trace in Figure 4A) clearly indicates the presence of at least two conformers with different levels of backbone protection. Collisional activation of the entire protein ion population generates a set of fragment ions with convoluted isotopic distributions (top trace in Figure 4B). However, mass selection of precursor ions with a specific level of deuterium content allows the top-down HDX MS measurements to be carried out in a conformation-specific fashion, taking full advantage of the HDX MS ability to detect distinct conformers. For example, selective fragmentation of protein ions representing a highly protected conformation is achieved by mass-selecting a narrow population of intact protein ions with high level of retained deuterium (the blue trace in Figure 4A). Mass-selection and subsequent fragmentation of a narrow population of protein ions with significantly lower deuterium content (the red trace in Figure 4A) generates a set of fragment ions whose isotopic distributions provide information on backbone protection within non-native protein states. For example, the data presented in Figure 4 clearly indicate that the C-terminal segment of the protein represented by the y172+ ions retains significant structure even within the partially unfolded conformers: the amount of retained deuterium atoms reduces by only 30% as a result of switching from the precursor ion from highly protected (blue) to less protected (red). At the same time, selection of the precursor ion has a much more dramatic effect on the protection levels exhibited by the N-terminal segment (represented by the b425+ ion), where more than a two-fold decrease in the amount of retained deuterium atoms is observed. Extending this analysis to other protein fragments may allow detailed backbone protection maps to be created for each protein conformer, provided there is no hydrogen scrambling prior to protein ion fragmentation (vide infra).

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Characterization of local dynamics in wt*-CRABP I in a conformer-specific fashion using top-down HDX MS (fully deuterated protein was exposed to 1H2O/CH3CO2N1H4 at pH 3.1 for 10 min; the gray trace at the bottom corresponds to HDX end-point). A: mass selection of precursor ions for subsequent CAD (from top to bottom): broad-band selection of the entire ionic population (not conformer-specific); highly protected conformers; narrow population of less protected conformers; HDX end-point. B: isotopic distributions of two representative fragment ions generated by CAD of precursor ions shown in panel A. Selection of different ion populations as precursor ions for subsequent fragmentation was achieved by varying the width of a mass selection window of a quadrupole filter (Q) in a hybrid quadrupole/time-of-flight mass spectrometer (Qq-TOF MS).

The example shown above illustrates a great promise of top-down HDX MS as a technique uniquely capable of probing structure and dynamics of populations of protein conformers coexisting in solution with high selectivity. Furthermore, this approach often allows one to avoid protein handling under the slow exchange conditions prior to MS analysis, thereby eliminating back-exchange as a factor adversely influencing the quality of measurements. Nonetheless, applications of top-down HDX MS have been limited due to concerns over the possibility of hydrogen scrambling accompanying collision-activated dissociation (CAD) of protein ions. Indeed, several reports pointed out that proton mobility in the gas phase may under certain conditions influence the outcome of top-down HDX MS measurements when CAD is employed to fragment protein ions.25, 26

The occurrence (or the absence) of hydrogen scrambling in the gas phase can be reliably detected by using built-in scrambling indicators. One particularly convenient indicator is a Histag, a 6-30 residues long, histidine-rich segment appended to wild-type sequences to facilitate protein purification on metal affinity columns. Such segments are fully unstructured in solution and, therefore, should lack any backbone protection.27 Alternatively, intrinsic scrambling indicators (e.g., internal flexible loops26), as well as other approaches25 can be used to detect occurrence of scrambling. The available experimental evidence suggests that slow protein ion activation (e.g., SORI CAD) always leads to hydrogen scrambling, while fast activation allows it to be minimized or eliminated in top-down HDX MS experiments.26

Another shortcoming of top-down HDX MS schemes utilizing CAD is the limited extent of protein ion fragmentation, which may lead to sizeable gaps in sequence coverage, particularly for larger proteins,28 and insufficient level of spatial resolution (even for smaller proteins29). Our earlier attempts to solve this problem by employing multi-stage CAD (MSn) were unsuccessful due to massive hydrogen scrambling exhibited by the second generation of fragments.

Electron-induced ion fragmentation in top-down schemes: keeping hydrogen scrambling at bay while enhancing sequence coverage and spatial resolution

Some time ago we suggested that the specter of hydrogen scrambling in top-down HDX MS measurements may be alleviated by using non-ergodic fragmentation processes, where dissociation is induced by ion-electron interaction, rather than collisional activation.30 Indeed, the results of earlier work combining hydrogen exchange of polypeptide ions in the gas phase and electron capture dissociation (ECD) were consistent with the notion of intramolecular rearrangement of hydrogen atoms occurring on a slower time scale compared to ion dissociation.31 A recent study demonstrated that the extent of scrambling was indeed negligible when ECD was used as a means to obtain fragment ions in top-down HDX MS characterization of a small protein ubiquitin.32

Our own recent work suggests that hydrogen scrambling can be avoided when top-down HDX MS employs ECD in characterizing higher order structure of larger proteins (approaching 20 kDa), although experimental conditions must be carefully controlled to minimize proton mobility induced by ion-molecule collisions in the ESI interface. The point in question is illustrated in Figure 5, which shows the results of top-down HDX MS analysis of higher order structure of wt*-CRABP I. The protein retains a significant proportion of labile deuterium label following its complete deuteration and then brief exposure to the 1H-based exchange buffer, as indicated by the isotopic distribution of the surviving molecular ions (red and blue traces in Figure 5A). However, the deuterium content of fragment ions derived from the 21-residue long His-tag region of the protein (e.g., c22 in Figure 5B) is indistinguishable from that of the exchange reaction endpoint, as long as moderate ion desolvation conditions are kept in the ESI interface. This clearly signals that hydrogen scrambling does not affect the outcome of local HDX MS measurements. However, once collision-assisted desolvation of protein ions is attempted in the ESI interface, the appearance of isotopic distributions of larger fragment ions derived from the His-tag region (e.g., c22, red trace in Figure 5B) shifts, indicating apparent deuterium retention and signaling the occurrence of limited hydrogen scrambling. We also demonstrated that deuterium distribution across the protein backbone is preserved when another recently introduced fragmentation technique based on cation-electron interactions, electron transfer dissociation (ETD), is used in top-down HDX MS schemes.33

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Top-down HDX MS of wt*-CRABP I using ECD of the entire protein ion population (fully deuterated protein was exposed to1H2O/CH3CO2N1H4 at pH 3.5 for varying time periods); the black trace at the bottom of corresponds to HDX end-point). A: isotopic distributions of surviving intact protein ions. B: two representative c-ions. Minimal collision-and temperature-induced desolvation was used for acquisition of all mass spectra, except the one top (red trace).

In addition to allowing scrambling to be easily eliminated in top-down HDX MS experiments, both ECD and ETD appear to be superior to CAD in terms of sequence coverage, at least for the proteins in the 20 kDa range. Unlike CAD, protein backbone cleavage in ECD and ETD is less specific,34 leading to a higher number of fragment ions. This translates not only to improved sequence coverage, but also enhanced spatial resolution. Indeed, in some cases it becomes possible to generate patterns of deuterium distribution across the protein backbone down to the single residue level.

One example of such work is shown in Figure 6, where ETD was used as a protein ion fragmentation tool in top-down HDX MS characterization of a 16 kDa variant of CRABP I. The bar graph shows the levels of deuterium retention in a series of c-ions derived from the N-terminal segment of the protein. The bar height at position n in this diagram shows mass difference between two cn-1 fragments, one derived from the fully deuterated protein that was exposed to the protiated exchange buffer at pH 7 for 5 min and then placed under the slow exchange conditions for the duration of the data acquisition cycle, and another one representing the HDX endpoint (raw data for bars at n=14 and 35 are shown in Figure 7). Unchanged height between two adjacent bars at residues n and n+1 indicates no difference in deuterium content of cn-1 and cn fragments, signaling no backbone amide deuterium retention at residue n+1, while bar height increase by one unit indicates complete retention of deuterium at the nth amide.

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Backbone protection pattern of CRABPI mutant (without N-terminal His-tag) obtained from top-down HDX MS measurements using ETD of the entire protein ion population. HDX was initiated by exposing the fully deuterated protein to 1H2O/CH3CO2N1H4 at pH 3.5 for 5 min followed by rapid quenching.

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An example of raw HDX MS data used to generate the protection plot shown in Figure 6. Isotopic distributions of c13 and c34 fragments derived from protein subjected to 5 min HDX exchange in solution (red trace) and protein at the HDX end-point (blue trace) were used to calculate the bar heights at n=12 and 35.

The resulting backbone protection pattern in Figure 6 shows clear correlation with the known higher order structure of the protein (the amino acid sequence and the secondary structure assignment are shown at the top of the graph). Furthermore, the diagram clearly shows uneven distribution of backbone protection even within single structural elements (e.g., lower protection at the fringes vs. the middle of helix α1), as well as unequal protection of similar structural elements participating in the same structural motif (e.g., lower protection of helix α2 vs. helix α1, consistent with the available NMR data). A comparable level of spatial resolution can be achieved with ECD, as shown recently in top-down HDX MS analysis of higher order structure of myoglobin.35

The ability to characterize protein conformation and dynamics at the single residue level is certainly very exciting; however, it comes at a price. Since the protein fragmentation is carried out entirely in the gas phase, no fragment separation can be done prior to mass analysis. A large number of fragment ions with different masses and charges are usually confined to a relatively narrow m/z region, leading to inevitable overlaps of fragment ion isotopic distributions (Figure 7). This places rather stringent requirements on the resolving power of the mass analyzer, effectively narrowing the selection of mass spectrometers suitable for this work to FTMS.

Meeting in the middle: integration of top-down strategies into bottom-up HDX MS schemes

The top-down approach to HDX MS measurements clearly shows a promise to solve many problems that mar the commonly employed bottom-up methodology. The fragmentation efficiency afforded by ECD and ETD provides better spatial resolution, at least for proteins in the 20 kDa range, and this number is likely to grow as there are numerous examples of successful use of these fragmentation techniques to obtain sequence information on significantly larger proteins.36 Unlike the classic bottom-up approach, top-down HDX MS provides an elegant solution to the problem of characterizing higher order structure and dynamics in a conformer-specific fashion (see Figure 4 and discussion in the text). Finally, back-exchange can be eliminated, as outsourcing protein fragmentation to the gas phase often eliminates the need to manipulate the protein in solution under the slow exchange conditions prior to MS analysis.

The top-down/bottom-up dichotomy in HDX MS should not be viewed through the “eitheror” prism. In fact, gas phase fragmentation can enhance the quality of HDX MS data derived from experiments that are built around the bottom-up approach. The suggestion to supplement proteolysis in solution with peptide ion fragmentation in the gas phase to achieve better spatial resolution was made over 10 years ago.37 However, earlier attempts to implement this idea using CAD on a variety of platforms yielded mixed results due to apparent scrambling in some (but not all) fragment ions.37, 38 Later reports showed even more extensive scrambling in small peptide ions subjected to collisional activation,39 an obvious anathema to the proposed marriage of CAD and bottom-up HDX MS. Nonetheless, continued search for a scrambling-free solution to this problem has yielded very encouraging results, with both ECD and ETD showing minimal scrambling when applied to short peptides under carefully controlled conditions40, 41 and feasibility of supplementing proteolytic fragmentation in solution with ETD in the gas phase was recently demonstrated using a small model protein.42 Although these initial steps are relatively modest, they certainly warrant further work in this field.

The two complementary approaches to HDX MS measurements share a set of common challenges that inevitably arise as these techniques gain popularity and the scope of their applications expands. One such challenge is presented by membrane proteins, a notoriously difficult class of biological objects. HDX MS has been shown to have a great potential in this field.43 Interestingly, some initial work in this field was done nearly ten years ago using then-infant top-down HDX MS technique,44 while more recent work in this field utilizes both bottomup18 and top-down45 approaches. Another challenge faced by HDX MS is presented by highly heterogeneous proteins, such as proteins conjugated to other biopolymers and/or synthetic polymers, which constitute a significant fraction of the next generation of biopharmaceuticals. Presently, there are no biophysical techniques capable of characterizing conformation and dynamics of these systems, and there is an urgent need to fill this gap. Finally, nearly all HDX MS work reported to date was carried out in vitro under conditions that some regard as “reductionist.” Although initial HDX work with living objects was carried out over 75 years ago,46 as the years passed only one report on in vivo HDX MS studies was published.47 As mass spectrometry at large is being increasingly used in both in vivo and ex vivo studies, there is a growing pressure on HDX MS to follow the trend, although it remains to be seen how this will be done.

It probably is not an exaggeration to say that we are witnessing a renaissance of HDX MS, with the emergence of the top-down approach not only expanding our experimental arsenal by offering new capabilities, but also serving as a catalyst in enhancing the classic bottom-up methodology. The two techniques are highly complementary, and their synergism will certainly bring about new exciting discoveries and accelerate our progress in solving a variety of problems ranging from very fundamental questions in biophysics to applied problems in drug design.

see more at  http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2805115/

WATCH OUT FOR DISULFIDES: If you’re going to try bottom-up HDX experiments, be careful of disulfide bonds, Kaltashov says. Pepsin is one of the very few proteinases that can efficiently digest a protein into its composite peptides under HDX experimental conditions, but it struggles when multiple disulfide bonds are present. In 2014, Kaltashov’s lab published two solutions to that problem. The first employs a fragmentation technique called electron capture dissociation (ECD) to break the disulfide linkage in the mass spec (Anal Chem, 86:5225-31, 2014); the second skips the pepsin digestion altogether—a strategy called top-down analysis (Anal Chem, 86:7293-98, 2014).

Enhancing the Quality of H/D Exchange Measurements with Mass Spectrometry Detection in Disulfide-Rich Proteins Using Electron Capture Dissociation

Anal Chem. 2014 Jun 3; 86(11): 5225–5231.   Published online 2014 May 12. doi:  10.1021/ac500904p
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Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) has become a potent technique to probe higher-order structures, dynamics, and interactions of proteins. While the range of proteins amenable to interrogation by HDX MS continues to expand at an accelerating pace, there are still a few classes of proteins whose analysis with this technique remains challenging. Disulfide-rich proteins constitute one of such groups: since the reduction of thiol–thiol bonds must be carried out under suboptimal conditions (to minimize the back-exchange), it frequently results in incomplete dissociation of disulfide bridges prior to MS analysis, leading to a loss of signal, inadequate sequence coverage, and a dramatic increase in the difficulty of data analysis. In this work, the dissociation of disulfide-linked peptide dimers produced by peptic digestion of the 80 kDa glycoprotein transferrin in the course of HDX MS experiments is carried out using electron capture dissociation (ECD). ECD results in efficient cleavage of the thiol–thiol bonds in the gas phase on the fast LC time scale and allows the deuterium content of the monomeric constituents of the peptide dimers to be measured individually. The measurements appear to be unaffected by hydrogen scrambling, even when high collisional energies are utilized. This technique will benefit HDX MS measurements for any protein that contains one or more disulfides and the potential gain in sequence coverage and spatial resolution would increase with disulfide bond number.
———

Hydrogen/deuterium exchange (HDX) with mass spectrometry (MS) detection has evolved in the past two decades into a powerful tool that is now used to decipher intimate details of processes as diverse as protein folding, recognition and binding, and enzyme catalysis.1,2 While initially being a tool that was used exclusively in fundamental studies, HDX MS is now becoming an indispensable part of the analytical arsenal in the biopharmaceutical sector, where it is utilized increasingly in all stages of protein drug development from discovery to quality control.35 Despite this progress, several areas remain where the application of HDX MS has met with only limited success. Disulfide-rich proteins constitute one such group, where characterization of higher-order structure and dynamics is particularly difficult, because of the suboptimal conditions used for reduction of thiol–thiol bonds following a quench of the exchange reactions. Proteins containing disulfide bonds are encountered very rarely in the protein folding studies where the most popular targets are small proteins lacking cysteine residues (with a notable exception of the oxidative folding studies), as well as in many other fundamental studies focusing on proteins of prokaryotic origin. However, disulfide-rich proteins are encountered very frequently in eukaryotic proteomes6 and constitute a large segment of the biopharmaceutical products,7 where the thiol–thiol bonds are critical elements defining conformation of protein drugs, and also play an important role in stabilizing proteins by endowing them with protease resistance.

While disulfide bond reduction is a relatively trivial task that can be readily accomplished at neutral pH using a variety of reagents, the acidic, low-temperature environment where proteins are placed to quench HDX narrows down the choice to a single reducing agent, TCEP.8 However, the alkaline pH for optimal disulfide reduction by TCEP is substantially higher, compared to the acidic environment of typical “slow exchange conditions” commonly employed to minimize back exchange within proteins and their peptic fragments prior to MS analysis.9 Furthermore, disulfide reduction in HDX MS measurements is usually carried out within a relatively short period of time (a few minutes) and at low temperature (0–4 °C) to limit the extent of the back-exchange, which in many situations does not allow the complete dissociation of thiol–thiol linkages of individual peptic fragments to be achieved in solution prior to LC separation and MS analysis of their deuterium content. Incomplete reduction of disulfide bonds dramatically increases the pool of candidate peptides that should be considered when analyzing proteolytic fragments in HDX MS measurements and frequently reduces sequence coverage and/or spatial resolution. While the former problem can be solved by employing more powerful and robust search engines for peptide identification, the latter one is more difficult to circumvent and can be very detrimental for the quality of HDX MS data and may require significant changes in experimental protocols. Indeed, a complete failure to reduce a certain disulfide bond in a protein will give rise to a thiol–thiol linked peptide dimer, whose constituent monomers do not necessarily represent a contiguous segment of the protein and may have vastly different conformational and dynamic properties. The total deuterium content of the entire dimer (measured by HDX MS) would not provide any meaningful information under these conditions, thereby effectively reducing the sequence coverage in the corresponding segments of the protein.
———-

Disulfide-rich proteins have traditionally been challenging targets for HDX MS studies, because of incomplete reduction of thiol–thiol linkages, which is a consequence of the quench conditions used to minimize amide back-exchange in peptides prior to MS analysis of their deuterium content: limited time, low temperature, and low pH. Traditionally, the principal strategy to address difficult-to-reduce or high-density disulfides in the HDX MS workflow is a brute force approach utilizing high concentrations of reductant and denaturant prior to (or even in combination with) digestion. The effectiveness of this approach is protein-dependent and extended incubation times frequently employed to enhance exposure to reductant invariably result in an undesirable increase in H/D back exchange. More recently, a novel electrochemical approach to reduce disulfides in solution under quench conditions prior to LC-MS has been reported for insulin.32 While electrochemical reduction shows promise, several limitations were identified, an apparent requirement for low-salt conditions, a higher-than-optimal temperature (10 °C), and a current cell pressure limit of 50 bar. In this work, electron capture dissociation (ECD) was used to circumvent the disulfide problem, since it effectively cleaves external disulfide bonds. Dissociation of the disulfide-linked peptide dimers can be accomplished on the fast LC time scale and produces abundant signals for monomeric subunits without interchain hydrogen scrambling, even when collisional activation of ions is applied prior to ion selection and ECD fragmentation. Inclusion of ECD in the HDX MS workflow results in increased sequence coverage and spatial resolution and provides an attractive alternative to extensive chemical reduction of disulfide-rich proteins.

see more at   http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4051250/

Approach to Characterization of the Higher Order Structure of Disulfide-Containing Proteins Using Hydrogen/Deuterium Exchange and Top-Down Mass Spectrometry

Guanbo Wang† and Igor A. Kaltashov*
http://www.chem.umass.edu/people/kaltashovlab/papers/Approach.pdf

Top-down hydrogen/deuterium exchange (HDX) with mass spectrometric (MS) detection has recently matured to become a potent biophysical tool capable of providing valuable information on higher order structure and conformational dynamics of proteins at an unprecedented level of structural detail. However, the scope of the proteins amenable to the analysis by top-down HDX MS still remains limited, with the protein size and the presence of disulfide bonds being the two most important limiting factors. While the limitations imposed by the physical size of the proteins gradually become more relaxed as the sensitivity, resolution and dynamic range of modern MS instrumentation continue to improve at an ever accelerating pace, the presence of the disulfide linkages remains a much less forgiving limitation even for the proteins of relatively modest size. To circumvent this problem, we introduce an online chemical reduction step following completion and quenching of the HDX reactions and prior to the top-down MS measurements of deuterium occupancy of individual backbone amides. Application of the new methodology to the top-down HDX MS characterization of a small (99 residue long) disulfide-containing protein β2- microglobulin allowed the backbone amide protection to be probed with nearly a single-residue resolution across the entire sequence. The high-resolution backbone protection pattern deduced from the top-down HDX MS measurements carried out under native conditions is in excellent agreement with the crystal structure of the protein and high-resolution NMR data, suggesting that introduction of the chemical reduction step to the top-down routine does not trigger hydrogen scrambling either during the electrospray ionization process or in the gas phase prior to the protein ion dissociation.

Since its initial introduction in the late 1990s,1−3 top-down hydrogen/deuterium exchange (HDX) with mass spectrometric (MS) detection evolved to become a potent biophysical tool capable of providing valuable information on higher order structure and conformational dynamics of proteins at an unprecedented level of structural detail. Among the many advantages offered by top-down HDX MS compared to conventional (bottom-up) measurements are significant reduction or indeed complete elimination of the back exchange,4 high spatial resolution,5,6 and the ability to study conformational dynamics in the conformer-specific fashion.7,8 However, despite the spectacular recent advances and the broader acceptance of this technique, the scope of the proteins amenable to the analysis by top-down HDX MS remains limited, with the protein size and the presence of disulfide bonds being the two most important limiting factors. The limitations imposed by the physical size of the proteins gradually become more relaxed as the sensitivity, resolution, and dynamic range of modern MS instrumentation continue to improve at an ever accelerating pace. However, the presence of disulfides remains a much less forgiving limitation even for the proteins of relatively modest size.

In this work we demonstrated feasibility of applying top-down HDX MS measurements to characterize higher order structure and conformational dynamics of disulfide-containing proteins, which have been out of the reach of this technique so far. Use of a moderate amount of a reducing agent TCEP is compatible with the ESI process, while allowing a fraction of the protein molecules to be reduced in solution thereby enabling nearcomplete sequence coverage at high resolution. The agreement between the top-down HDX MS and NMR data sets demonstrate that the new experimental approach is capable of capturing the dynamic picture of protein conformation at high spatial resolution without compromising the quality of the data by triggering hydrogen scrambling in the gas phase. Despite its modest size, β2m is known to be able to populate a non-native state,35 which might be a key player in a variety of processes, including amyloidosis. However, the structure of this non-native state of β2m remains elusive since this conformer exists in dynamic equilibrium with the native state of the protein.36,37 Recently we demonstrated that top-down HDX MS provides an elegant way to selectively probe structure of protein states coexisting in solution at equilibrium;8 however, β2m remained out of reach of this technique until recently due to the presence of a disulfide bond. The ability to expand the scope of top-down HDX MS to disulfide-containing proteins opens up a host of exciting possibilities to explore the structure of β2m, interferon, lysozyme, and a variety of other disulfidecontaining proteins in a conformer-specific fashion, where physiologically important non-native states may play important roles in processes as diverse as folding, recognition, signaling, and amyloidosis. ■ ASSOCIATED CONTENT *S Supporting Information Representative examples of isotopic distributions of fragment ions that have (Supplementary Figure 1) and have not (Supplementary Figure 2) been used to calculate the deuterium occupancy at individual backbone amides of β2m in top-down HDX MS measurements. This material is available free of charge via the Internet at http://pubs.acs.org.

Determining surface topology of protein complexes

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SUSSING OUT THE SURFACE: Protein topology can be probed by firing low-energy electrons (white circles) at intact protein complexes within a high-resolution mass spectrometer. That reaction, called electron capture dissociation, causes the protein complex to fracture on its surface, revealing the exposed amino acid residues.     COURTESY OF PIRIYA WONGKONGKATHEP AND HUILIN LI, UCLA

RESEARCHER: Joseph Loo, Professor of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles)

PROJECT: Studying protein-ligand and protein-protein interactions

SOLUTION: Loo is less interested in complex identification than in how the protein subunits assemble. Specifically, he wants to know which amino acid residues lie on the complex’s surface and which are buried inside or interacting with ligands.

It’s a question of structural biology, he explains: “How is this thing folded in a way that these residues are on the outside?”

To work that out, Loo combines high-resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR) with electron-capture dissociation (ECD), a mass spec fragmentation method in which an ion in the mass spectrometer interacts with free electrons, causing the protein to fracture along its peptide backbone. By measuring the mass of those fragments with high precision, researchers can determine the protein’s amino acid sequence.

In Loo’s case, though, that fragmentation is not uniform along the length of the protein. Proteins usually are denatured for mass spectrometry analysis, but the protein complexes in his studies are intact—a process called native mass spectrometry. Fragmentation thus occurs preferentially on the surface of the complex, like the cracks in the shell of a hard-boiled egg. “You get limited sequence information, but that sequence information comes from regions that are specific to its 3-D structure,” he says (Anal Chem, 86:317-20, 2014).

Native Top-Down ESI-MS of 158 kDa Protein Complex by High Resolution Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) delivers high resolving power, mass measurement accuracy, and the capabilities for unambiguously sequencing by a top-down MS approach. Here, we report isotopic resolution of a 158 kDa protein complex – tetrameric aldolase with an average absolute deviation of 0.36 ppm and an average resolving power of ~520,000 at m/z 6033 for the 26+ charge state in magnitude mode. Phase correction further improves the resolving power and average absolute deviation by 1.3 fold. Furthermore, native top-down electron capture dissociation (ECD) enables the sequencing of 149 C-terminal amino acid (AA) residues out of 463 total AAs. Combining the data from top-down MS of native and denatured aldolase complexes, a total of 58% of the backbone cleavages efficiency is achieved. The observation of complementary product ion pairs confirms the correctness of the sequence and also the accuracy of the mass fitting of the isotopic distribution of the aldolase tetramer. Top-down MS of the native protein provides complementary sequence information to top-down ECD and CAD MS of the denatured protein. Moreover, native top-down ECD of aldolase tetramer reveals that ECD fragmentation is not limited only to the flexible regions of protein complexes and that regions located on the surface topology are prone to ECD cleavage.

“Native” mass spectrometry (MS) is an emerging technique that has been successfully used to characterize intact, noncovalently-bound protein complexes, providing stoichiometry and structural information that is complementary to data supplied by conventional structural biology techniques.13 To confidently characterize protein complexes, electrospray ionization (ESI)-MS measurements acquired with isotopic resolving power (RP) and high mass accuracy and capabilities for deriving primary structure, i.e., sequence, information would be ideal. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) is prominent for its superior resolving power and mass accuracy and its utility for tandem MS (MS/MS) with a variety of fragmentation techniques; FT-ICR MS is noted for characterizating posttranslational modifications (PTMs) and protein-ligand and protein-protein interactions.49 However, it remains challenging to isotopically resolving large biomolecules over 100 kDa due to sample heterogeneity, cation/solvent/buffer addition, space charge effects, and electric and magnetic field inhomogeneity (for FT-ICR).1013 Unit mass resolution has been achieved for a few denatured proteins, including a 112 kDa protein with 3 Da mass error using a 9.4 T FT-ICR MS,14 a 115 kDa protein by a 7 T instrument with a mass error of 5 ppm,4 and a 148 kDa protein with a mass error of 1 Da by a 9.4 T FTMS.10

Compared to denatured proteins, it is more difficult to achieve isotopic resolution for inherently lower charged (and thus, higher m/z) native protein complexes because (1) the peak height is proportional to its charge state, (2) the resolving power is inversely proportional to mass-to-charge ratio for FT-ICR MS, and (3) the broader isotope distribution of large biomolecules reduces overall signal-to-noise ratio.15 However, the introduction of a new FT-ICR analyzer cell – the ParaCell, by Nikolaev and coworkers has significantly increased the resolving power of FT-ICR MS.16, 17 By dynamically harmonizing the electric field potential at any radius of cyclotron motion in the entire cell volume, a resolving power of 39 M has been achieved for the alkaloid, resperine (m/z 609), using a 7 T system.18 In addition, a few native protein complexes, including enolase dimer (93 kDa, RP ~ 800,000 at m/z 4250), alcohol dehydrogenase tetramer (147 kDa, RP ~ 500,000 at m/z 5465), and enolase tetramer (186 kDa), have been isotopically resolved with a 12 T FT-ICR system with the new ICR cell.18 Although Mitchell and Smith reported that cyclotron phase locking due to Coulombic interactions limits the highest mass that unit mass resolution can be achieved by FT-ICR MS (Mmax ≈ 1×104B, where B is magnetic field strength),19 the ParaCell has made it significantly easier and promising to measure high resolution mass spectra for large native protein complexes.

……

Native top-down CAD and ISD were performed for the aldolase tetramer; dissociation of the tetramer to yield monomer was observed in both approaches and no sequence information was obtained. The cleavage sites from ECD (colored in red) and CAD (colored in green) of the denatured aldolase monomer (26+) are overlaid with the native ECD results for aldolase tetramer (Figure 2B). As shown in Figure 2B, in contrast to the limited number of c-ion fragments observed in the ECD of aldolase tetramer, ECD of denatured aldolase monomer induces extensive c-ion fragments in the N-terminal region and enables the assignment of first 156 N-terminal AA residues. Surprisingly, the number of z-ions observed from ECD of the denatured aldolase monomer is much less compared to the ECD of the native aldolase tetramer. Although it may be possible that the z-ions may undergo secondary fragmentation due to excess available energy, electrons, or long ion-electron reaction times during the ECD experiment, ECD experiments with reduced reaction time and bias voltages were performed and the results argue against this assumption. Overall, 58% of the total number of backbone bonds are cleaved from combining top-down MS of native aldolase complex and denatured aldolase monomer (20% for native ECD of aldolase tetramer, 37% for ECD of denatured aldolase, and 5% for CAD of denatured aldolase).

The three dimensional structure of the aldolase tetramer is shown in Figure 3. To compare the flexibility of the structure to the data from ECD of the aldolase tetramer, one of the subunits (B-chain) is presented as B-factor putty and the D-chain is shown with its native ECD backbone cleavage regions colored in red. The remaining A- and C-chains are shown in grey. Although the C-terminal region (AA 340–363) of each subunit is highly flexible based on the crystallography B-factor (see B-chain in Figure 3A), only 4 out of 75 backbone cleavage sites are from the AA 340–363 region. Instead, the native ECD fragments largely originate from surface regions of the protein structure (see D-chain in Figure 3A). The N-terminal regions are not directly involved in the interfaces between subunits, but they are located in regions that are partially buried, which is consistent with the limited c-ions observed. To better show the native ECD backbone cleavage regions, the D-chain is rotated 90 degrees clockwise (Figure 3B). It is clear that, although protein structure flexibility might play a role in the native top-down ECD fragmentation pattern, for aldolase the ECD cleavage sites are not limited to the flexible region. In addition, backbone cleavage regions from CAD (yellow) and ECD (cyan) of denatured aldolase are complementary with the native ECD results.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3908771/bin/nihms548404f3.jpg

A) Structure of tetrameric aldolase (1ZAH)29. A- and C-chains are shown as grey ribbons, the B-chain is shown in B-factor putty, and the D-chain is in cartoon with native ECD cleavage sites colored in red, CAD cleavage sites of denatured aldolase in yellow, and ECD cleavage sites of the N-terminal region from ECD of denatured aldolase in cyan. B) The D-chain is rotated 90 degrees clockwise to show the outer surface region of the subunit structure.

Also evident in such data sets are protein–small molecule interactions. As the proteins break apart, Loo explains, ligands often remain attached to the polypeptide shards that are produced. In one recent publication, for instance, his team mapped zinc binding sites in eukaryotic alcohol dehydrogenase, a 147-kDa tetrameric complex (J Am Soc Mass Spectrom, 25:2060-8, 2014).

Revealing Ligand Binding Sites and Quantifying Subunit Variants of Non-Covalent Protein Complexes in a Single Native Top-Down FTICR MS Experiment

“Native” mass spectrometry (MS) has been proven increasingly useful for structural biology studies of macromolecular assemblies. Using horse liver alcohol dehydrogenase (hADH) and yeast alcohol dehydrogenase (yADH) as examples, we demonstrate that rich information can be obtained in a single native top-down MS experiment using Fourier transform ion cyclotron mass spectrometry (FTICR MS). Beyond measuring the molecular weights of the protein complexes, isotopic mass resolution was achieved for yeast ADH tetramer (147 kDa) with an average resolving power of 412,700 at m/z 5466 in absorption mode and the mass reflects that each subunit binds to two zinc atoms. The N-terminal 89 amino acid residues were sequenced in a top-down electron capture dissociation (ECD) experiment, along with the identifications of the zinc binding site at Cys46 and a point mutation (V58T). With the combination of various activation/dissociation techniques, including ECD, in-source dissociation (ISD), collisionally activated dissociation (CAD), and infrared multiphoton dissociation (IRMPD), 40% of the yADH sequence was derived directly from the native tetramer complex. For hADH, native top-down ECD-MS shows that both E and S subunits are present in the hADH sample, with a relative ratio of 4:1. Native top-down ISD MS hADH dimer shows that each subunit (E and S chain) binds not only to two zinc atoms, but also the NAD+/NADH ligand, with a higher NAD+/NADH binding preference for the S chain relative to the E chain. In total, 32% sequence coverage was achieved for both E and S chains.

Studying how proteins interact with one another and assemble on a structural basis is key to understanding biological processes and their function. As a complementary technique to conventional technologies used in structural biology, such as nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography, and electron microscopy, “native” mass spectrometry (MS) has established its crucial role in the characterization of intact noncovalently-bound protein complexes, revealing the composition, stoichiometry, dynamics, stability, and also spatial information of subunit arrangements in protein assemblies [111]. To date, most native MS studies of protein complexes have been performed using quadrupole time-of-flight (Q-TOF) MS instruments with electrospray ionization (ESI). Because of the efficient transmission of high mass and highm/z ions using TOF analyzers, large proteins with molecular weights up to 18 MDa have been studied [12,13]. The coupling of ion mobility spectrometry (IMS) with mass spectrometry provides a new dimension to the analysis of biomolecules [14]. With IMS, ions are separated based on size and shape, and the IMS-derived collision cross-section information can be used to understand the topological properties of gas phase protein complexes. Surface induced dissociation (SID) has been recently added for the purposes of disassembling protein complexes into sub-complexes that appear to better reflect the structure of the solution phase complexes [1517]. The capability of Orbitrap MS has been extended significantly for the analysis of macromolecules, with greatly improved mass (and m/z) range and resolving power to measure the binding of ADP and ATP to the 800 kDa GroEL complex [18].

Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) is known for its superior resolving power and mass accuracy and its capabilities for tandem MS (MS/MS) with a variety of fragmentation techniques. Particularly, after the introduction of electron capture dissociation (ECD) [19], FTICR MS quickly established its utility for protein top-down protein sequencing, post-translational modification characterization, and protein gas phase studies [2034]. Polypeptide backbone bonds are cleaved by ECD, but non-covalent interactions are preserved, which therefore makes the native top-down MS study of the non-covalent interaction sites of protein-ligands complexes more feasible. Our group and others have successfully applied top-down ECD-MS to pinpoint the interaction sites of several protein-ligand system [3538], and this can be enhanced by “supercharging” [35]. An early attempt of applying ECD-MS to the study of large protein complexes was made by Heeren and Heck, but little topology and sequence information was derived [39]. However, the Gross group starting in 2010 made the first breakthrough for the study of large protein complexes using native top-down ECD with FTICR MS. Besides obtaining molecular weight, sequence, and metal-binding site information in a single MS experiment, they correlated the origins of ECD product ions to the flexible regions of proteins as determined by the “B-factor” from the X-ray crystal structures of protein complexes [40, 41]. Therefore, native top-down ECD has been proposed as a tool to probe the flexible regions of protein complexes. Our group recently also demonstrated the capability of obtaining sequence information and isotopic mass resolution of a noncovalently-bound protein complex of 158 kDa using native top-down FTICR MS, and most importantly, we found that the origin of ECD fragments is not limited only to the flexible region of the protein complex (e.g., tetrameric aldolase), but also largely from the surface of the complex [42].

The application of FTICR MS for native top-down interrogation of large non-covalent bound protein complexes is still in its infancy. Here, for the purpose of further exploring the capability of FTICR MS in the analysis of large protein complexes, various fragmentation techniques including in-source dissociation (ISD), collisionally activated dissociation (CAD), ECD, and infrared multiphoton dissociation (IRMPD) were applied in the native top-down MS studies of a 80 kDa dimeric protein complex and a 147 kDa tetrameric protein complex. The results demonstrate that with the superior resolving power, mass accuracy, and versatile fragmentation techniques of FTICR MS, rich information, including isotopic mass resolution, amino acid sequence, point mutations, metal/ligand binding sites, and identification and quantification of subunit variants can be accomplished in a single native top-down FTICR MS experiment.

see more at   http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4444062/

Still, Loo admits, the technique “is not really ready for prime time.” His team is collecting ECD data on a bank of proteins of known structure to ensure the data they collect really do reflect protein topology. In the meantime, they are working to extend the size of the complexes they can analyze. The technique’s current limit is 800 kDa.

GO NATIONAL: FTICR mass spectrometers offer top-of-the-line accuracy and resolution, with price tags to match. Few researchers have direct access to them, Loo says, but they can always try the national laboratories. Both the National High Magnetic Field Laboratory at Florida State University and the Environmental Molecular Sciences Laboratory at the Pacific Northwest National Laboratory have user facilities open to worthy projects.

Determining the architecture of protein complexes

RESEARCHER: Vicki Wysocki, Ohio Eminent Scholar and Professor of Chemistry and Biochemistry, Ohio State University

PROJECT: Instrumentation development for whole-complex analysis

SOLUTION: An analytical chemist by training, Wysocki focuses on instrumentation development for protein-complex analysis. Among the discoveries in her lab is a method called surface-induced dissociation (SID).

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HIT THE WALL, JACK: When it comes to molecular collision in a mass spectrometer, size matters. Collide a complex with small gas molecules, and proteins in the complex will simply unravel (top). By smacking them into a “wall”—a process called surface-induced dissociation—the complex dissociates to reveal its underlying architecture.  COURTESY OF VICKI WYSOCKI

Like many other fragmentation approaches, SID works by forcing an ion in the mass spectrometer to collide with another object. Usually that object is a small gas molecule, with the energy of collision sufficient to crack the peptide backbone. But for large protein complexes, bigger is better, and the collision partner in SID is as big as it can get: the method slams protein ions of interest into a nonreactive surface inside the instrument—essentially, a wall—causing complexes to fracture into subcomplexes that reveal the assembly’s inner architecture.

Wysocki combined this approach with ion-mobility separation—a kind of gas-phase electrophoresis that resolves molecules by their size and shape—to dissect an enzyme involved in antibiotic production. The enzyme, they found, has two copies each of three subunits, alpha, beta, and gamma, arranged as a pair of triads sitting on top of one another, with the alpha and beta subunits of one triad linked more tightly to each other than either is to gamma (Anal Chem, 83:2862-65, 2011).

Such information can be valuable to protein engineers, Wysocki says, especially as this particular complex otherwise falls into a structural biology knowledge gap: “It doesn’t crystallize, and it’s too small for the cryoEM and a little bit large for NMR,” she says. “And so, mass spec turned out to be a great tool.”

Revealing the Quaternary Structure of a Heterogeneous Noncovalent Protein Complex through Surface-Induced Dissociation

Anne E. Blackwell, Eric D. Dodds,† Vahe Bandarian, and Vicki H. Wysocki*
https://research.cbc.osu.edu/wysocki.11/wp-content/uploads/2012/09/Blackwell-2011-Revealing-the-Quater.pdf

As scientists begin to appreciate the extent to which quaternary structure facilitates protein function, determination of the subunit arrangement within noncovalent protein complexes is increasingly important. While native mass spectrometry shows promise for the study of noncovalent complexes, few developments have been made toward the determination of subunit architecture, and no mass spectrometry activation method yields complete topology information. Here, we illustrate the surface-induced dissociation of a heterohexamer, toyocamycin nitrile hydratase, directly into its constituent trimers. We propose that the single-step nature of this activation in combination with high energy deposition allows for dissociation prior to significant unfolding or other large-scale rearrangement. This method can potentially allow for dissociation of a protein complex into subcomplexes, facilitating the mapping of subunit contacts and thus determination of quaternary structure of protein complexes.

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http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/ancham/2011/ancham.2011.83.issue-8/ac200452b/production/pdfimages_v02/normal.img-000.jpg

The majority of proteins exist and perform their functions as multimers of varing stoichiometries and architecture.1 However, very few methods are available that can provide insights into subunit interactions. Native mass spectrometry (MS) is increasingly being used to study noncovalent protein complexes, as many structural features found in solution may be maintained in the gas phase.2,3 While subunit stoichiometries are readily obtainable by mass measurement alone, the determination of subunit arrangement within protein complexes remains a significant challenge. This is particularly true for heterogeneous complexes with multiple types of subunits. Considerable progress has been made using solution-phase disruption to divide the original protein complex into smaller subcomplexes, which may be readily measured by MS.4,5 The composition of the stable subcomplexes provides insight on the topology of the protein complex. However, MS activation methods used to date have fallen short of providing subunit topology. Here, we present the first evidence for subunit arrangement obtained directly from gas-phase experiments on a heterogeneous complex via surfaceinduced dissociation (SID). We have demonstrated previously the ability of SID to yield unique dissociation pathways for protein complexes, resulting in complementary information to collision-induced dissociation (CID).68 While the SID process is not yet well understood for macromolecules, there is a large body of work concerning SID of small molecules; influential factors such as collision energy, surface composition, and translational-to-vibrational energy conversion have been well-studied.911 The higher effective mass of a surface relative to that of neutral gas atoms used in CID (typically argon) results in significantly higher energy deposited through a single surface collision.9 As SID is a single-collision activation process, rather than activation via thousands of less energetic collisions as in CID, dissociation pathways other than those of the lowest energies become accessible

……

This is the only study to date demonstrating an ion activation method capable of yielding extensive dissociation, as well as the release of intact subcomplexes, thus providing relevant substructure information on a noncovalent, hetero-oligomeric protein complex. The capacity to produce intact, charge-symmetric subcomplexes suggests that dissociation occurs faster than subunit unfolding and that a significant degree of secondary and tertiary structure is maintained up to the point of dissociation and for some period of time afterward. Identification of trimeric substructure in TNH provides insight into a protein with little previous structural characterization and indicates a promising advancement of MS as a tool for structural biology.

Such information can be valuable to protein engineers, Wysocki says, especially as this particular complex otherwise falls into a structural biology knowledge gap: “It doesn’t crystallize, and it’s too small for the cryoEM and a little bit large for NMR,” she says. “And so, mass spec turned out to be a great tool.”

CHOOSE MASS: Mass spec may not be the only method for quickly working out protein structure, but it surely is the fastest, Wysocki says. She recalls one instance when a colleague sent over a complex that his group couldn’t crack. “In one afternoon, my student gave them a prediction of the structure: this one’s a heptamer, with a large subunit sitting atop a hexameric ring.” Even if the experiment doesn’t work, she adds, that fast turnaround time can be a boon, as collaborators can get rapid feedback for tweaking their experimental conditions. “Mass is a great thing.”

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Irreconciliable Dissonance in Physical Space and Cellular Metabolic Conception


Irreconciliable Dissonance in Physical Space and Cellular Metabolic Conception

Curator: Larry H. Bernstein, MD, FCAP

Pasteur Effect – Warburg Effect – What its history can teach us today. 

José Eduardo de Salles Roselino

The Warburg effect, in reality the “Pasteur-effect” was the first example of metabolic regulation described. A decrease in the carbon flux originated at the sugar molecule towards the end of the catabolic pathway, with ethanol and carbon dioxide observed when yeast cells were transferred from an anaerobic environmental condition to an aerobic one. In Pasteur´s studies, sugar metabolism was measured mainly by the decrease of sugar concentration in the yeast growth media observed after a measured period of time. The decrease of the sugar concentration in the media occurs at great speed in yeast grown in anaerobiosis (oxygen deficient) and its speed was greatly reduced by the transfer of the yeast culture to an aerobic condition. This finding was very important for the wine industry of France in Pasteur’s time, since most of the undesirable outcomes in the industrial use of yeast were perceived when yeasts cells took a very long time to create, a rather selective anaerobic condition. This selective culture media was characterized by the higher carbon dioxide levels produced by fast growing yeast cells and by a higher alcohol content in the yeast culture media.

However, in biochemical terms, this finding was required to understand Lavoisier’s results indicating that chemical and biological oxidation of sugars produced the same calorimetric (heat generation) results. This observation requires a control mechanism (metabolic regulation) to avoid burning living cells by fast heat released by the sugar biological oxidative processes (metabolism). In addition, Lavoisier´s results were the first indications that both processes happened inside similar thermodynamics limits. In much resumed form, these observations indicate the major reasons that led Warburg to test failure in control mechanisms in cancer cells in comparison with the ones observed in normal cells.

[It might be added that the availability of O2 and CO2 and climatic conditions over 750 million years that included volcanic activity, tectonic movements of the earth crust, and glaciation, and more recently the use of carbon fuels and the extensive deforestation of our land masses have had a large role in determining the biological speciation over time, in sea and on land. O2 is generated by plants utilizing energy from the sun and conversion of CO2. Remove the plants and we tip the balance. A large source of CO2 is from beneath the earth’s surface.]

Biology inside classical thermodynamics places some challenges to scientists. For instance, all classical thermodynamics must be measured in reversible thermodynamic conditions. In an isolated system, increase in P (pressure) leads to increase in V (volume), all this occurring in a condition in which infinitesimal changes in one affects in the same way the other, a continuum response. Not even a quantic amount of energy will stand beyond those parameters.

In a reversible system, a decrease in V, under same condition, will led to an increase in P. In biochemistry, reversible usually indicates a reaction that easily goes either from A to B or B to A. For instance, when it was required to search for an anti-ischemic effect of Chlorpromazine in an extra hepatic obstructed liver, it was necessary to use an adequate system of increased biliary system pressure in a reversible manner to exclude a direct effect of this drug over the biological system pressure inducer (bile secretion) in Braz. J. Med. Biol. Res 1989; 22: 889-893. Frequently, these details are jumped over by those who read biology in ATGC letters.

Very important observations can be made in this regard, when neutral mutations are taken into consideration since, after several mutations (not affecting previous activity and function), a last mutant may provide a new transcript RNA for a protein and elicit a new function. For an example, consider a Prion C from lamb getting similar to bovine Prion C while preserving  its normal role in the lamb when its ability to change Human Prion C is considered (Stanley Prusiner).

This observation is good enough, to confirm one of the most important contributions of Erwin Schrodinger in his What is Life:

“This little book arose from a course of public lectures, delivered by a theoretical physicist to an audience of about four hundred which did not substantially dwindle, though warned at the outset that the subject matter was a difficult one and that the lectures could not be termed popular, even though the physicist’s most dreaded weapon, mathematical deduction, would hardly be utilized. The reason for this was not that the subject was simple enough to be explained without mathematics, but rather that it was much too involved to be fully accessible to mathematics.”

After Hans Krebs, description of the cyclic nature of the citrate metabolism and after its followers described its requirement for aerobic catabolism two major lines of research started the search for the understanding of the mechanism of energy transfer that explains how ADP is converted into ATP. One followed the organic chemistry line of reasoning and therefore, searched for a mechanism that could explain how the breakdown of carbon-carbon link could have its energy transferred to ATP synthesis. One of the major leaders of this research line was Britton Chance. He took into account that relatively earlier in the series of Krebs cycle reactions, two carbon atoms of acetyl were released as carbon dioxide ( In fact, not the real acetyl carbons but those on the opposite side of citrate molecule). In stoichiometric terms, it was not important whether the released carbons were or were not exactly those originated from glucose carbons. His research aimed at to find out an intermediate proteinaceous intermediary that could act as an energy reservoir. The intermediary could store in a phosphorylated amino acid the energy of carbon-carbon bond breakdown. This activated amino acid could transfer its phosphate group to ADP producing ATP. A key intermediate involved in the transfer was identified by Kaplan and Lipmann at John Hopkins as acetyl coenzyme A, for which Fritz Lipmann received a Nobel Prize.

Alternatively, under possible influence of the excellent results of Hodgkin and Huxley a second line of research appears. The work of Hodgkin & Huxley indicated that the storage of electrical potential energy in transmembrane ionic asymmetries and presented the explanation for the change from resting to action potential in excitable cells. This second line of research, under the leadership of Peter Mitchell postulated a mechanism for the transfer of oxide/reductive power of organic molecules oxidation through electron transfer as the key for the energetic transfer mechanism required for ATP synthesis.
This diverted the attention from high energy (~P) phosphate bond to the transfer of electrons. During most of the time the harsh period of the two confronting points of view, Paul Boyer and followers attempted to act as a conciliatory third party, without getting good results, according to personal accounts (in L. A. or Latin America) heard from those few of our scientists who were able to follow the major scientific events held in USA, and who could present to us later. Paul  Boyer could present how the energy was transduced by a molecular machine that changes in conformation in a series of 3 steps while rotating in one direction in order to produce ATP and in opposite direction in order to produce ADP plus Pi from ATP (reversibility).

However, earlier, a victorious Peter Mitchell obtained the result in the conceptual dispute, over the Britton Chance point of view, after he used E. Coli mutants to show H+ gradients in the cell membrane and its use as energy source, for which he received a Nobel Prize. Somehow, this outcome represents such a blow to Chance’s previous work that somehow it seems to have cast a shadow over very important findings obtained during his earlier career that should not be affected by one or another form of energy transfer mechanism.  For instance, Britton Chance got the simple and rapid polarographic assay method of oxidative phosphorylation and the idea of control of energy metabolism that brings us back to Pasteur.

This metabolic alternative result seems to have been neglected in the recent years of obesity epidemics, which led to a search for a single molecular mechanism required for the understanding of the accumulation of chemical (adipose tissue) reserve in our body. It does not mean that here the role of central nervous system is neglected. In short, in respiring mitochondria the rate of electron transport linked to the rate of ATP production is determined primarily by the relative concentrations of ADP, ATP and phosphate in the external media (cytosol) and not by the concentration of respiratory substrate as pyruvate. Therefore, when the yield of ATP is high as it is in aerobiosis and the cellular use of ATP is not changed, the oxidation of pyruvate and therefore of glycolysis is quickly (without change in gene expression), throttled down to the resting state. The dependence of respiratory rate on ADP concentration is also seen in intact cells. A muscle at rest and using no ATP has a very low respiratory rate.   [When skeletal muscle is stressed by high exertion, lactic acid produced is released into the circulation and is metabolized aerobically by the heart at the end of the activity].

This respiratory control of metabolism will lead to preservation of body carbon reserves and in case of high caloric intake in a diet, also shows increase in fat reserves essential for our biological ancestors survival (Today for our obesity epidemics). No matter how important this observation is, it is only one focal point of metabolic control. We cannot reduce the problem of obesity to the existence of metabolic control. There are numerous other factors but on the other hand, we cannot neglect or remove this vital process in order to correct obesity. However, we cannot explain obesity ignoring this metabolic control. This topic is so neglected in modern times that we cannot follow major research lines of the past that were interrupted by the emerging molecular biology techniques and the vain belief that a dogmatic vision of biology could replace all previous knowledge by a new one based upon ATGC readings. For instance, in order to display bad consequences derived from the ignorance of these old scientific facts, we can take into account, for instance, how ion movements across membranes affects membrane protein conformation and therefore contradicts the wrong central dogma of molecular biology. This change in protein conformation (with unchanged amino acid sequence) and/or the lack of change in protein conformation is linked to the factors that affect vital processes as the heart beats. This modern ignorance could also explain some major pitfalls seen in new drugs clinical trials and in a small scale on bad medical practices.

The work of Britton Chance and of Peter Mitchell have deep and sound scientific roots that were made with excellent scientific techniques, supported by excellent scientific reasoning and that were produced in a large series of very important intermediary scientific results. Their sole difference was to aim at very different scientific explanations as their goals (They have different Teleology in their minds made by their previous experiences). When, with the use of mutants obtained in microorganisms P Mitchell´s goal was found to survive and B Chance to succumb to the experimental evidence, all those excellent findings of B Chance and followers were directed to the dustbin of scientific history as an example of lack of scientific consideration.  [On the one hand, the Mitchell model used a unicellular organism; on the other, Chance’s work was with eukaryotic cells, quite relevant to the discussion.]

We can resume the challenge faced by these two great scientists in the following form: The first conceptual unification in bioenergetics, achieved in the 1940s, is inextricably bound up with the name of Fritz Lipmann. Its central feature was the recognition that adenosine triphosphate, ATP, serves as a universal energy  “currency” much as money serves as economic currency. In a nutshell, the purpose of metabolism is to support the synthesis of ATP. In microorganisms, this is perfect! In humans or mammals, or vertebrates, by the same reason that we cannot consider that gene expression is equivalent to protein function (an acceptable error in the case of microorganisms) this oversimplifies the metabolic requirement with a huge error. However, in case our concern is ATP chemistry only, the metabolism produces ATP and the hydrolysis of ATP pays for the performance of almost, all kinds of works. It is possible to presume that to find out how the flow of metabolism (carbon flow) led to ATP production must be considered a major focal point of research of the two contenders. Consequently, what could be a minor fall of one of the contenders, in case we take into account all that was found during their entire life of research, the real failure in B Chance’s final goal was amplified far beyond what may be considered by reason!

Another aspect that must be taken into account: Both contenders have in the scientific past a very sound root. Metabolism may produce two forms of energy currency (I personally don´t like this expression*) and I use it here because it was used by both groups in order to express their findings. Together with simplistic thermodynamics, this expression conveys wrong ideas): The second kind of energy currency is the current of ions passing from one side of a membrane to the other. The P. Mitchell scientific root undoubtedly have the work of Hodgkin & Huxley, Huxley &  Huxley, Huxley & Simmons

*ATP is produced under the guidance of cell needs and not by its yield. When glucose yields only 2 ATPs per molecule it is oxidized at very high speed (anaerobiosis) as is required to match cellular needs. On the other hand, when it may yield (thermodynamic terms) 38 ATP the same molecule is oxidized at low speed. It would be similar to an investor choice its least money yield form for its investment (1940s to 1972) as a solid support. B. Chance had the enzymologists involved in clarifying how ATP could be produced directly from NADH + H+ oxidative reductive metabolic reactions or from the hydrolysis of an enolpyruvate intermediary. Both competitors had their work supported by different but, sound scientific roots and have produced very important scientific results while trying to present their hypothetical point of view.

Before the winning results of P. Mitchell were displayed, one line of defense used by B. Chance followers was to create a conflict between what would be expected by a restrictive role of proteins through its specificity ionic interactions and the general ability of ionic asymmetries that could be associated with mitochondrial ATP production. Chemical catalyzed protein activities do not have perfect specificity but an outstanding degree of selective interaction was presented by the lock and key model of enzyme interaction. A large group of outstanding “mitochondriologists” were able to show ATP synthesis associated with Na+, K+, Ca2+… asymmetries on mitochondrial membranes and any time they did this, P. Mitchell have to display the existence of antiporters that exchange X for hydrogen as the final common source of chemiosmotic energy used by mitochondria for ATP synthesis.

This conceptual battle has generated an enormous knowledge that was laid to rest, somehow discontinued in the form of scientific research, when the final E. Coli mutant studies presented the convincing final evidence in favor of P. Mitchell point of view.

Not surprisingly, a “wise anonymous” later, pointed out: “No matter what you are doing, you will always be better off in case you have a mutant”

(Principles of Medical Genetics T D Gelehrter & F.S. Collins chapter 7, 1990).

However, let’s take the example of a mechanical wristwatch. It clearly indicates when the watch is working in an acceptable way, that its normal functioning condition is not the result of one of its isolated components – or something that can be shown by a reductionist molecular view.  Usually it will be considered that it is working in an acceptable way, in case it is found that its accuracy falls inside a normal functional range, for instance, one or two standard deviations bellow or above the mean value for normal function, what depends upon the rigor wisely adopted. While, only when it has a faulty component (a genetic inborn error) we can indicate a single isolated piece as the cause of its failure (a reductionist molecular view).

We need to teach in medicine, first the major reasons why the watch works fine (not saying it is “automatic”). The functions may cross the reversible to irreversible regulatory limit change, faster than what we can imagine. Latter, when these ideas about normal are held very clear in the mind set of medical doctors (not medical technicians) we may address the inborn errors and what we may have learn from it. A modern medical technician may cause admiration when he uses an “innocent” virus to correct for a faulty gene (a rather impressive technological advance). However, in case the virus, later shows signals that indicate that it was not so innocent, a real medical doctor will be called upon to put things in correct place again.

Among the missing parts of normal evolution in biochemistry a lot about ion fluxes can be found. Even those oscillatory changes in Ca2+ that were shown to affect gene expression (C. De Duve) were laid to rest since, they clearly indicate a source of biological information that despite the fact that it does not change nucleotides order in the DNA, it shows an opposing flux of biological information against the dogma (DNA to RNA to proteins). Another, line has shown a hierarchy, on the use of mitochondrial membrane potential: First the potential is used for Ca2+ uptake and only afterwards, the potential is used for ADP conversion into ATP (A. L. Lehninger). In fact, the real idea of A. L. Lehninger was by far, more complex since according to him, mitochondria works like a buffer for intracellular calcium releasing it to outside in case of a deep decrease in cytosol levels or capturing it from cytosol when facing transient increase in Ca2+ load. As some of Krebs cycle dehydrogenases were activated by Ca2+, this finding was used to propose a new control factor in addition to the one of ADP (B. Chance). All this was discontinued with the wrong use of calculus (today we could indicate bioinformatics in a similar role) in biochemistry that has established less importance to a mitochondrial role after comparative kinetics that today are seen as faulty.

It is important to combat dogmatic reasoning and restore sound scientific foundations in basic medical courses that must urgently reverse the faulty trend that tries to impose a view that goes from the detail towards generalization instead of the correct form that goes from the general finding well understood towards its molecular details. The view that led to curious subjects as bioinformatics in medical courses as training in sequence finding activities can only be explained by its commercial value. The usual form of scientific thinking respects the limits of our ability to grasp new knowledge and relies on reproducibility of scientific results as a form to surpass lack of mathematical equation that defines relationship of variables and the determination of its functional domains. It also uses old scientific roots, as its sound support never replaces existing knowledge by dogmatic and/or wishful thinking. When the sequence of DNA was found as a technical advance to find amino acid sequence in proteins it was just a technical advance. This technical advance by no means could be considered a scientific result presented as an indication that DNA sequences alone have replaced the need to study protein chemistry, its responses to microenvironmental changes in order to understand its multiple conformations, changes in activities and function. As E. Schrodinger correctly describes the chemical structure responsible for the coded form stored of genetic information must have minimal interaction with its microenvironment in order to endure hundreds and hundreds years as seen in Hapsburg’s lips. Only magical reasoning assumes that it is possible to find out in non-reactive chemical structures the properties of the reactive ones.

For instance, knowledge of the reactions of the Krebs cycle clearly indicate a role for solvent that no longer could be considered to be an inert bath for catalytic activity of the enzymes when the transfer of energy include a role for hydrogen transport. The great increase in understanding this change on chemical reaction arrived from conformational energy.

Again, even a rather simplistic view of this atomic property (Conformational energy) is enough to confirm once more, one of the most important contribution of E. Schrodinger in his What is Life:

“This little book arose from a course of public lectures, delivered by a theoretical physicist to an audience of about four hundred which did not substantially dwindle, though warned at the outset that the subject matter was a difficult one and that the lectures could not be termed popular, even though the physicist’s most dreaded weapon, mathematical deduction, would hardly be utilized. The reason for this was not that the subject was simple enough to be explained without mathematics, but rather that it was much too involved to be fully accessible to mathematics.”

In a very simplistic view, while energy manifests itself by the ability to perform work conformational energy as a property derived from our atomic structure can be neutral, positive or negative (no effect, increased or decreased reactivity upon any chemistry reactivity measured as work)

Also:

“I mean the fact that we, whose total being is entirely based on a marvelous interplay of this very kind, yet if all possess the power of acquiring considerable knowledge about it. I think it possible that this knowledge may advance to little just a short of a complete understanding -of the first marvel. The second may well be beyond human understanding.”

In fact, scientific knowledge allows us to understand how biological evolution may have occurred or have not occurred and yet does not present a proof about how it would have being occurred. It will be always be an indication of possible against highly unlike and never a scientific proven fact about the real form of its occurrence.

As was the case of B. Chance in its bioenergetics findings, we may get very important findings that indicates wrong directions in the future as was his case, or directed toward our past.

The Skeleton of Physical Time – Quantum Energies in Relative Space of S-labs

By Radoslav S. Bozov  Independent Researcher

WSEAS, Biology and BioSystems of Biomedicine

Space does not equate to distance, displacement of an object by classically defined forces – electromagnetic, gravity or inertia. In perceiving quantum open systems, a quanta, a package of energy, displaces properties of wave interference and statistical outcomes of sums of paths of particles detected by a design of S-labs.

The notion of S-labs, space labs, deals with inherent problems of operational module, R(i+1), where an imagination number ‘struggles’ to work under roots of a negative sign, a reflection of an observable set of sums reaching out of the limits of the human being organ, an eye or other foundational signal processing system.

While heavenly bodies, planets, star systems, and other exotic forms of light reflecting and/or emitting objects, observable via naked eye have been deduced to operate under numerical systems that calculate a periodic displacement of one relative to another, atomic clocks of nanospace open our eyes to ever expanding energy spaces, where matrices of interactive variables point to the problem of infinity of variations in scalar spaces, however, defining properties of minute universes as a mirror image of an astronomical system. The first and furthermost problem is essentially the same as those mathematical methodologies deduced by Isaac Newton and Albert Einstein for processing a surface. I will introduce you to a surface interference method by describing undetermined objective space in terms of determined subjective time.

Therefore, the moment will be an outcome of statistical sums of a numerical system extending from near zero to near one. Three strings hold down a dual system entangled via interference of two waves, where a single wave is a product of three particles (today named accordingly to either weak or strong interactions) momentum.

The above described system emerges from duality into trinity the objective space value of physical realities. The triangle of physical observables – charge, gravity and electromagnetism, is an outcome of interference of particles, strings and waves, where particles are not particles, or are strings strings, or  are waves waves of an infinite character in an open system which we attempt to define to predict outcomes of tomorrow’s parameters, either dependent or independent as well as both subjective to time simulations.

We now know that aging of a biological organism cannot be defined within singularity. Thereafter, clocks are subjective to apparatuses measuring oscillation of defined parameters which enable us to calculate both amplitude and a period, which we know to be dependent on phase transitions.

The problem of phase was solved by the applicability of carbon relative systems. A piece of diamond does not get wet, yet it holds water’s light entangled property. Water is the dark force of light. To formulate such statement, we have been searching truth by examining cooling objects where the Maxwell demon is translated into information, a data complex system.

Modern perspectives in computing quantum based matrices, 0+1 =1 and/or 0+0=1, and/or 1+1 =0, will be reduced by applying a conceptual frame of Aladdin’s flying anti-gravity carpet, unwrapping both past and future by sending a photon to both, placing present always near zero. Thus, each parallel quantum computation of a natural system approaching the limit of a vibration of a string defining 0 does not equal 0, and 1 does not equal 1. In any case, if our method 1+1 = 1, yet, 1 is not 1 at time i+1. This will set the fundamentals of an operational module, called labris operator or in simplicity S-labs. Note, that 1 as a result is an event predictable to future, while interacting parameters of addition 1+1 may be both, 1 as an observable past, and 1 as an imaginary system, or 1+1 displaced interactive parameters of past observable events. This is the foundation of Future Quantum Relative Systems Interference (QRSI), taking analytical technologies of future as a result of data matrices compressing principle relative to carbon as a reference matter rational to water based properties.

Goedel’s concept of loops exist therefore only upon discrete relative space uniting to parallel absolute continuity of time ‘lags’. ( Goedel, Escher and Bach: An Eternal Golden Braid. A Metaphorical Fugue on Minds and Machines in the Spirit of Lewis Carroll. D Hofstadter.  Chapter XX: Strange Loops, Or Tangled Hierarchies. A grand windup of many of the ideas about hierarchical systems and self-reference. It is concerned with the snarls which arise when systems turn back on themselves-for example, science probing science, government investigating governmental wrongdoing, art violating the rules of art, and finally, humans thinking about their own brains and minds. Does Gödel’s Theorem have anything to say about this last “snarl”? Are free will and the sensation of consciousness connected to Gödel’s Theorem? The Chapter ends by tying Gödel, Escher, and Bach together once again.)  The fight struggle in-between time creates dark spaces within which strings manage to obey light properties – entangled bozons of information carrying future outcomes of a systems processing consciousness. Therefore, Albert Einstein was correct in his quantum time realities by rejecting a resolving cube of sugar within a cup of tea (Henri Bergson 19th century philosopher. Bergson’s concept of multiplicity attempts to unify in a consistent way two contradictory features: heterogeneity and continuity. Many philosophers today think that this concept of multiplicity, despite its difficulty, is revolutionary.) However, the unity of time and space could not be achieved by deducing time to charge, gravity and electromagnetic properties of energy and mass.

Charge is further deduced to interference of particles/strings/waves, contrary to the Hawking idea of irreducibility of chemical energy carrying ‘units’, and gravity is accounted for by intrinsic properties of   anti-gravity carbon systems processing light, an electromagnetic force, that I have deduced towards ever expanding discrete energy space-energies rational to compressing mass/time. The role of loops seems to operate to control formalities where boundaries of space fluctuate as a result of what we called above – dark time-spaces.

Indeed, the concept of horizon is a constant due to ever expanding observables. Thus, it fails to acquire a rational approach towards space-time issues.

Richard Feynman has touched on issues of touching of space, sums of paths of particle traveling through time. In a way he has resolved an important paradigm, storing information and possibly studying it by opening a black box. Schroedinger’s cat is alive again, but incapable of climbing a tree when chased by a dog. Every time a cat climbs a garden tree, a fruit falls on hedgehogs carried away parallel to living wormholes whose purpose of generating information lies upon carbon units resolving light.

In order to deal with such a paradigm, we will introduce i+1 under square root in relativity, therefore taking negative one ( -1 = sqrt (i+1), an operational module R dealing with Wheelers foam squeezed by light, releasing water – dark spaces. Thousand words down!

What is a number? Is that a name or some kind of language or both? Is the issue of number theory possibly accountable to the value of the concept of entropic timing? Light penetrating a pyramid holding bean seeds on a piece of paper and a piece of slice of bread, a triple set, where a church mouse has taken a drop of tear, but a blood drop. What an amazing physics! The magic of biology lies above egoism, above pride, and below Saints.

We will set up the twelve parameters seen through 3+1 in classic realities:

–              discrete absolute energies/forces – no contradiction for now between Newtonian and Albert Einstein mechanics

–              mass absolute continuity – conservational law of physics in accordance to weak and strong forces

–              quantum relative spaces – issuing a paradox of Albert Einstein’s space-time resolved by the uncertainty principle

–              parallel continuity of multiple time/universes – resolving uncertainty of united space and energy through evolving statistical concepts of scalar relative space expansion and vector quantum energies by compressing relative continuity of matter in it, ever compressing flat surfaces – finding the inverse link between deterministic mechanics of displacement and imaginary space, where spheres fit within surface of triangles as time unwraps past by pulling strings from future.

To us, common human beings, with an extra curiosity overloaded by real dreams, value happens to play in the intricate foundation of life – the garden of love, its carbon management in mind, collecting pieces of squeezed cooling time.

The infinite interference of each operational module to another composing ever emerging time constrains unified by the Solar system, objective to humanity, perhaps answers that a drop of blood and a drop of tear is united by a droplet of a substance separating negative entropy to time courses of a physical realities as defined by an open algorithm where chasing power subdue to space becomes an issue of time.

Jose Eduardo de Salles Roselino

Some small errors: For intance an increase i P leads to a decrease in V ( not an increase in V)..

 

Radoslav S. Bozov  Independent Researcher

If we were to use a preventative measures of medical science, instruments of medical science must predict future outcomes based on observable parameters of history….. There are several key issues arising: 1. Despite pinning a difference on genomic scale , say pieces of information, we do not know how to have changed that – that is shift methylome occupying genome surfaces , in a precise manner.. 2. Living systems operational quo DO NOT work as by vector gravity physics of ‘building blocks. That is projecting a delusional concept of a masonry trick, who has not worked by corner stones and ever shifting momenta … Assuming genomic assembling worked, that is dealing with inferences through data mining and annotation, we are not in a position to read future in real time, and we will never be, because of the rtPCR technology self restriction into data -time processing .. We know of existing post translational modalities… 3. We don’t know what we don’t know, and that foundational to future medicine – that is dealing with biological clocks, behavior, and various daily life inputs ranging from radiation to water systems, food quality, drugs…

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Phase I/II Hepato-specific Glucokinase Activator

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Advinus Therapeutics announced that it has successfully completed a 14-day POC study in 60 Type II diabetic patients on its lead molecule, GKM-001, a glucokinase activator. The results of the trial show effective glucose lowering across all doses tested without any incidence of hypoglycemia or any other clinically relevant adverse events.

GKM-001 is differentiated from most other GK molecules that are in development, or have been discontinued, due to its novel liver selective mechanism of action.

GKM-001 belongs to a novel class of molecules for treatment of type II diabetes. It is an activator of Glucokinase (GK), a glucose-sensing enzyme found mainly in the liver and pancreas. Being liver selective, GKM-001 mostly activates GK in the liver and not in pancreas, which is its key differentiation from most competitor molecules that activate GK in pancreas as well.

GKM 001 in pipeline for Diabetes by Advinus

by DR ANTHONY MELVIN CRASTO Ph.D

ad 1
GKM 001

Advinus Therapeutics Private L,

A glucokinase activator for treatment of type II diabetes, currently in PI. Advinus is actively exploring partnership options to expedite further development and WW marketing of GKM-001.

Company Advinus Therapeutics Ltd.
Description Activator of glucokinase (GCK; GK)
Molecular Target Glucokinase (GCK) (GK)
Mechanism of Action Glucokinase activator
Therapeutic Modality Small molecule
Latest Stage of Development Phase I/II
Standard Indication Diabetes
Indication Details Treat Type II diabetes

PATENT

https://www.google.co.in/patents/WO2009047798A2?cl=en

Example Cl : (-)-{5-ChIoro-2-[2-(4-cyclopropanesulfonylphenyI)-2-(2,4- difluorophenoxy)acetylamino]thiazol-4-yl}-acetic acid, ethyl ester
1H NMR(400 MHz, CDCl3): δ 1.06-1.08 (m, 2H), 1.30 (t, J=7.2 Hz, 3H), 1.33-1.38 (m, 2H), 2.42-2.50 (m, IH), 3.73 (d, J=2 Hz, 2H), 4.22 (q, J=7.2 Hz ,2H), 5.75 (s, IH), 6.76- 6.77 (m, IH), 6.83-6.86 (m, IH), 6.90-6.98 (m, IH), 7.73 (d, J=8.4 Hz, 2H), 7.96 (d, J=8.4 Hz, 2H), 9.96 (bs, IH). MS (EI) m/z: 571.1 and 573.1 (M+ 1; for 35Cl and 37Cl respectively).

Examples C2 and C3 were prepared in analogues manner of example (Cl) from the appropriate chiral intermediate:

Figure imgf000044_0002

Example Dl : (+)-{5-Chloro-2-[2-(4-cyclopropanesulfonylphenyl)-2-(2,4- difluorophenoxy)acetylamino]thiazol-4-yl}acetic acid, ethyl ester

Advinus’ GK-activator Achieves Early POC for Diabetes

November 29 2011

Partnership Dialog Actively Underway

Advinus Therapeutics, a research-based pharmaceutical company founded by globally experienced industry executives and promoted by the TATA Group, announced that it has successfully completed a 14-day POC study in 60 Type II diabetic patients on its lead molecule, GKM-001, a glucokinase activator. The results of the trial show effective glucose lowering across all doses tested without any incidence of hypoglycemia or any other clinically relevant adverse events.

The clinical trials on GKM-001 validate the company’s pre-clinical hypothesis that a liver selective Glucokinase activator would not cause hypoglycemia (very low blood sugar), while showing robust efficacy.

“GKM-001 is differentiated from most other GK molecules that are in development, or have been discontinued, due to its novel liver selective mechanism of action. GKM-001 has a prolonged pharmacological effect and a half-life that should support a once a day dosing as both mono and combination therapy.” said Dr. Rashmi Barbhaiya, MD & CEO, Advinus Therapeutics. He added that Advinus is actively exploring partnership options to expedite further development and global marketing of GKM-001.

GKM-001 belongs to a novel class of molecules for treatment of type II diabetes. It is an activator of Glucokinase (GK), a glucose-sensing enzyme found mainly in the liver and pancreas. Being liver selective, GKM-001 mostly activates GK in the liver and not in pancreas, which is its key differentiation from most competitor molecules that activate GK in pancreas as well. The resulting increase in insulin secretion creates a potential for hypoglycemia-a risk GKM-001 is designed to avoid. Advinus has the composition of matter patent on GKM-001 for all major markets globally. Both the Single Ascending Dose data, in healthy and type II diabetics, and the Multiple Ascending Dose Study in Type II diabetics has shown that the molecule shows effective glucose lowering in a dose dependent manner and has excellent safety and tolerability profile over a 40-fold dose range. The pharmacokinetic properties of the molecule support once a day dosing. GKM-001 has the potential to be “First-in-Class” drug to address this large, growing and yet poorly addressed market.

Advinus also has identified a clinical candidate as a back-up to GKM-001, which is structurally different. In its portfolio, the company has a growing pipeline for COPD, sickle cell disease, inflammatory bowel disease, type 2 diabetes, acute and chronic pain and rheumatoid arthritis in various stages of late discovery and pre-clinical development.

Advinus Therapeutics team discovers novel molecule for treatment of diabetes

  • The first glucokinase modulator discovered and developed in India 
  • A new concept for the management of diabetes for patients, globally 
  • 100 per cent ‘made in India’ molecule for the treatment of diabetes 
  • IND approved by DGCI, Phase I clinical trial shows excellent safety and tolerance profiles with efficacy

Bangalore: Advinus Therapeutics (Advinus), the research-based pharmaceutical company founded by leading global pharmaceutical executives and promoted by the Tata group, today, announced the discovery of a novel molecule for the treatment of type II diabetes — GKM-001.The molecule is an activator of glucokinase; an enzyme that regulates glucose balance and insulin secretion in the body.

GKM-001 is a completely indigenously developed molecule and the initial clinical trials have shown excellent results for both safety and efficacy.

“Considering past failures of other companies on this target, our discovery programme primarily focused on identifying a molecule that would be efficacious without causing hypoglycaemia; a side effect associated with most compounds developed for this target.

“Recently completed Phase I data indicate that Advinus’ GKM–001 is a liver selective molecule that has overcome the biggest clinical challenge of hypoglycaemia. GKM-001 is differentiated from most other GK molecules in development due to this novel mechanism of action,” said Dr Rashmi Barbhaiya, MD and CEO, Advinus Therapeutics.

He further added, “We are very proud that GKM-001 is 100 per cent Indian. Advinus’s discovery team in Pune discovered the molecule and entire preclinical development was carried out at our centre in Bangalore. The Investigational New Drug (IND) application was filed with the DGCI for approval to initiate clinical trials in India within 34 months of initiation of the discovery programme. Subsequent to the approval of the IND, we have completed the Phase I Single Ascending Dose study in India within two months.”

GKM-001 is a novel molecule for the treatment of type II diabetes. It is the first glucokinase modulator discovered and developed in India and has potential to be both first or best in class. The success in discovering GKM-001 is attributed to the science-driven efforts in Advinus laboratories and ‘breaking the conventional mold’ for selection of a drug candidate. Advinus has ‘composition of matter’ patent on the molecule for all major markets globally. Glucokinase as a class of target is considered to be novel as currently there is no product in the market or in late clinical trials. The strategy for early clinical development revolved around assessing safety (particularly hypoglycaemia) and early assessment of therapeutic activity (glucose lowering and other biomarkers) in type II diabetics. The Phase I data, in both healthy and type II diabetics, shows excellent safety and tolerability over a 40-fold dose range and desirable pharmacokinetic properties consistent with ‘once a day’ dosing. The next wave of clinical studies planned continues on this strategy of early testing in type II diabetics.

Right behind the lead candidate GKM-001, Advinus has a rich pipeline of back up compounds on the same target. These include several structurally different compounds with diverse potency, unique pharmacology and tissue selectivity. Having discovered the molecule with early indication of wide safety margins, desired efficacy and pharmacokinetic profiles, the company now seeks to out-licence GKM-001 and its discovery portfolio.

Kasim A. Mookhtiar, , Debnath Bhuniya, Siddhartha De, Anita Chugh, Jayasagar
Gundu, Venkata Palle, Dhananjay Umrani, Nimish Vachharajani, Vikram
Ramanathan and Rashmi H. Barbhaiya
Advinus Therapeutics Ltd, Hinjewadi, Pune – 411057, and Peenya Industrial Area,
Bangalore – 560058, India
REFERENCES

patent

wo 2008104994

wo 2008 149382

wo 2009047798
WO2008104994A2* 25 Feb 2008 4 Sep 2008 Advinus Therapeutics Private L 2,2,2-tri-substituted acetamide derivatives as glucokinase activators, their process and pharmaceutical application

///////GKM 001, pipeline, Diabetes, Advinus, type II diabetes, glucokinase modulator, Rashmi Barbhaiya

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Liposomes, Lipidomics and Metabolism

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Building a Better Liposome

Computational models suggest new design for nanoparticles used in targeted drug delivery.

http://www.technologynetworks.com/Metabolomics/news.aspx?ID=184147

Using computational modeling, researchers at Carnegie Mellon University, the Colorado School of Mines and the University of California, Davis have come up with a design for a better liposome. Their findings, while theoretical, could provide the basis for efficiently constructing new vehicles for nanodrug delivery.

Liposomes are small containers with shells made of lipids, the same material that makes up the cell membrane. In recent years, liposomes have been used for targeted drug delivery. In this process, the membrane of a drug-containing liposome is engineered to contain proteins that will recognize and interact with complementary proteins on the membrane of a diseased or dysfunctional cell. After the drug-containing liposomes are administered, they travel through the body, ideally connecting with targeted cells where they release the drug.

liposome_853x480-min.jpg

This packaging technique is often used with highly toxic nanodrugs, like chemotherapy drugs, in an attempt to prevent the free drug from damaging non-cancerous cells. However, studies of this model of delivery have shown that in many cases less than 10 percent of the drugs transported by liposomes end up in tumor cells. Often, the liposome breaks open before it reaches a tumor cell and the drug is absorbed into the body’s organs, including the liver and spleen, resulting in toxic side effects.

“Even with current forms of targeted drug delivery, treatments like chemotherapy are still very brutal. We wanted to see how we could make targeted drug delivery better,” said Markus Deserno, professor of physics at Carnegie Mellon and a member of the university’s Center for Membrane Biology and Biophysics.

Deserno and colleagues propose that targeted drug delivery can be improved by making more stable liposomes. Using three different types of computer modeling, they have shown that liposomes can be made sturdier by incorporating a nanoparticle core made of a material like gold or iron and connecting that core to the liposome’s membrane using polymer tethers. The core and tethers act as a hub-and-spoke-like scaffold and shock-absorber system that help the liposome to weather the stresses and strains it encounters as it travels through the body to its target.

Francesca Stanzione and Amadeu K. Sum of the Colorado School of Mines conducted a fine-grained simulation that looked at how the polymer tethers anchor the liposome’s membrane at an atomistic level. Roland Faller of UC Davis did a meso-scale simulation that looked how a number of tethers held on to a small patch of membrane. Each of these simulations allowed researchers to look at smaller components of the liposome, nanoparticle core and tethers, but not the entire structure.

To see the entire structure, Carnegie Mellon’s Deserno and Mingyang Hu developed a coarse-grained model that represents groupings of components rather than individual atoms. For example, one lipid in the cell membrane might have 100 atoms. In a fine-grain simulation, each atom would be represented. In Deserno’s coarse grain simulation, those atoms might be represented by only three pieces instead of 100.

“Its unfeasible to look at the complete construct at an atomistic level. There are too many atoms to consider, and the timescale is too long. Even with the most advanced supercomputer, we wouldn’t have the power to run an atom-level simulation,” Deserno said. “But the physics that matters isn’t locally specific. It’s more like soft matter physics, which can be described at a much coarser resolution.”

Deserno’s simulation allowed the researchers to see how the entire reinforced liposome construct responded to stress and strain. They proposed that if a liposome was given the right-sized hub and tethers, its membrane would be much more resilient, bending to absorb impact and pressure.

Additionally, they were able to simulate how to best assemble the liposome, hub and tether system. They found that if the hub and tether are attached and placed in a solution of lipids, and solvent conditions are suitably chosen, a correctly sized liposome would self-assemble around the hub and tethers.

The researchers hope that chemists and drug developers will one day be able to use their simulations to determine what size core and polymer tethers they would need to effectively secure a liposome designed to deliver a specific drug or other nanoparticle. Using such simulations could narrow down the design parameters, speed up the development process and reduce costs.

 

Lipotype GmbH and NIHS Collaborate

http://www.technologynetworks.com/Metabolomics/news.aspx?ID=184363

NIHS to use the Lipotype Shotgun Lipidomics Technology for lipid analysis.

Lipotype GmbH and the Nestlé Institute of Health Sciences (NIHS) have collaborated to employ the innovative Lipotype Shotgun Lipidomics Technology to analyze lipids in blood for nutritional research. Recently, Lipotype and NIHS have jointly published results of the robustness of the Lipotype Technology. Lipotype envisions a future use of its technology in clinical diagnostics screens for establishing reliable lipid diagnostic biomarkers.

Innovative Lipotype Technology for lipid analysis
The purpose of this collaboration is to enable NIHS to use the Lipotype Shotgun Lipidomics Technology for lipid analysis. The mass spectrometry-based Lipotype technology covers a broad spectrum of lipid molecules and delivers quantitative results in high-throughput. The Nestlé Institute of Health Sciences uses this technology platform for nutritional research. NIHS is a specialized biomedical research institute and is part of Nestlé’s global Research & Development network.

Joint research project reveals robustness of Lipotype Technology
During the collaboration, Lipotype and NIHS conducted a joint research project and demonstrated that the Lipotype technology was robust enough to deliver data with high precision and negligible technical variation between different sites. In addition, important features are the high coverage and throughput, which were confirmed when applying the Lipotype technology.

Lipotype envisions these as important features, required for future use in clinical diagnostics screens, in order to establish and validate reliable lipid diagnostic biomarkers. The results have been published in October 2015, in the European Journal of Lipid Science and Technology (Surma et al. “An Automated Shotgun Lipidomics Platform for High Throughput, Comprehensive, and Quantitative Analysis of Blood Plasma Intact Lipids.”).

Lipids play an important role for health and disease
Lipotype is a spin-off company of the Max-Planck-Institute of Molecular Cell Biology and Genetics in Dresden, Germany. Prof. Kai Simons, CEO of Lipotype explains: “We developed a novel Shotgun-Lipidomics technology to analyze lipids in blood and other biological samples. Our analysis is quick and covers hundreds of lipid molecules at the same time. Our technology can be used to identify disease related lipid signatures.”

 

New Treatment for Obesity Developed

http://www.technologynetworks.com/Metabolomics/news.aspx?ID=183998

Researchers at the University of Liverpool, working with a global healthcare company, have helped develop a new treatment for obesity.
The treatment, which is a once-daily injectable derivative of a metabolic hormone called GLP-1 conventionally used in the treatment of type 2 diabetes, has proved successful in helping non-diabetic obese patients lose weight.

Professor John Wilding, who leads Obesity and Endocrinology research in the Institute of Ageing and Chronic Disease, investigates the pathophysiology and treatment of both obesity and type 2 diabetes and is applying his expertise in this area to work with, and often act as a consultant for, a number of large pharmaceutical companies looking to develop new treatments for obesity and diabetes.

Exciting development

Professor Wilding, said: “The biology of GLP-1 has been a focus of my research for 20 years; in particular when I was working at Hammersmith Hospital in London, I was part of the team that demonstrated that it was involved in appetite regulation; work on GLP-1 has continued during my time in Liverpool. Being involved in the development of a treatment, from the basic research right through to clinical trials in patients is very exciting”.

“It is likely that the treatment will be used initially in very specific situations, such as helping patients who are severely obese. It differs from current treatments used for diabetes, as it has stronger appetite regulating effects but no greater effect on glucose control.”

In 2014 more than 1.9 billion adults worldwide were classed as obese by the World Health Organisation; in the UK numbers have more than tripled since 1980. This Obesity can lead to other serious health-related illnesses including type 2 diabetes, hypertension and obstructive sleep apnoea as well as increasing the risk for many common cancers.

The drug has been approved in the European Union, but has not yet launched in the UK.

Professor Wilding added: “Consultancy like this can help relationship and reputation building and informs my research keeping it at the forefront of developments. It also brings many other benefits such as publications and income generation, which can help support other research, for example by such as funding for pilot projects that can lead to grant applications and investigator-initiated trials funded by the company”.

 

Evidence of How Incurable Cancer Develops

http://www.technologynetworks.com/Metabolomics/news.aspx?ID=184346

Researchers in the West Midlands have made a breakthrough in explaining how an incurable type of blood cancer develops from an often symptomless prior blood disorder.

The findings could lead to more effective treatments and ways to identify those most at risk of developing the cancer.

All patients diagnosed with myeloma, a cancer of the blood-producing bone marrow, first develop a relatively benign condition called ‘monoclonal gammopathy of undetermined significance’ or ‘MGUS’.

MGUS is fairly common in the older population and only progresses to cancer in approximately one in 100 cases. However, currently there is no way of accurately predicting which patients with MGUS are likely to go on to get myeloma.

Myeloma is diagnosed in around 4,000 people each year in the UK. It specifically affects antibody-producing white blood cells found in the bone marrow, called plasma cells. The researcher team from the University of Birmingham, New Cross and Heartlands Hospitals compared the cellular chemistry of bone marrow and blood samples taken from patients with myeloma, patients with MGUS and healthy volunteers.

Surprisingly, the researchers found that the metabolic activity of the bone marrow of patients with MGUS was significantly different to plasma from healthy volunteers, but there were very few differences at all between the MGUS and myeloma samples. The research was funded by the blood cancer charity Bloodwise, which changed its name from Leukaemia & Lymphoma in September.

The findings suggest that the biggest metabolic changes occur with the development of the symptomless condition MGUS and not with the later progression to myeloma.

Dr Daniel Tennant, who led the research at the University of Birmingham, said, “Our findings show that very few changes are required for a MGUS patient to progress to myeloma as we now know virtually all patients with myeloma evolve from MGUS. A drug that interferes with these specific initial metabolic changes could make a very effective treatment for myeloma, so this is a very exciting discovery.”

The research team found over 200 products of metabolism differed between the healthy volunteers and patients with MGUS or myeloma, compared to just 26 differences between MGUS patients and myeloma patients. The researchers believe that these small changes could drive the key shifts in the bone marrow required to support myeloma growth.

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