Archive for the ‘Glycobiology: Biopharmaceutical Production’ Category

Glycobiology vs Proteomics: Glycobiologists Prespective in the effort to explain the origin, etiology and potential therapeutics for the Coronavirus Pandemic (COVID-19)

 Curator: Ofer Markman, PhD

 The sugars involved in a viral disease are unique in many ways when compared with the DNA/RNA or the proteins involved: they are almost totally dependent on the infected cells and thus are not affected by the viral mutation rate or by the virus at all. Nevertheless they are affected by the cells, their type and their sugar production mechanisms and in some respect to the production rate by which the virus is replicated in the infected cells. Mutations may have nevertheless major effect not on the structures of the glycans but rather on the existence of the glycosylation site, and thus the glycan at all, but not on its structures.

This may make the gycomolecule a good target for diagnostics as stability in the molecule may mean longer life shelve of diagnostics kits.

Unique sugars are already predicted/found in the virus from certain Chinese origin, in this case an o-linked glycan/s not previously detected.

  • The proximal origin of SARS-CoV-2

Kristian G. AndersenAndrew RambautW. Ian Lipkin, Edward C. Holmes & Robert F. Garry

Nature Medicine (2020)Cite this article


  • The covid-19 coronavirus epidemic has a natural origin, scientists say


  • Emerging WuHan (COVID-19) coronavirus: glycan shield and structure prediction of spike glycoprotein and its interaction with human CD26

Naveen Vankadari & Jacqueline A. Wilce


Nevertheless, if the virus can infect multiple cells once current cells are not going to be available for any reason those viruses may present other glycans.

Once one starts to treat the infected person via modulation of protein production or by other means the change in the dynamic of protein production vs. protein glycosylation may cause changes in protein glycosyation, including their structures, this is well known to biotechnologists producing glycoproteins in labs and production.

This may either be a problem in understanding the state of disease or an advantage as it may help following response to the treatment and help as a co-treatment diagnostics.

Early Studies include the following:

  • Carbohydrate-based Diagnostics: A New Approach to COVID-19 Testing?

Mar 19, 2020 | Original story from Iceni Diagnostics


Glycans may play a role in treatment as well. TAMIFLU a case in point. Tamiflu is directed to the flu enzyme Neuraminidaze that is part of the viral structures. This approach was also explored to develop treatments.

  • Pneumagen Ltd Leverages its Novel Glycan Approach to Target Coronavirus (COVID-19) Infections

March 17, 2020 PR-M03-20-NI-024


Glycans do not only effect their own involvement in treatment/diagnostics they also are effecting protein based diagnostics for this see statement by Dr. Michael Mercier of UAH

  • We’re dealing with COVID-19, but what’s a virus in the first place?

23-Mar-2020 8:45 AM EDT, by University of Alabama Huntsville



Other related articles on this topic published on this topic in this Open Access Online Scientific Journal include the following: 


The Relevance of Glycans in the Viral Pathology of COVID-19

Reporter: Ofer Markman, PhD


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The Relevance of Glycans in the Viral Pathology of COVID-19


Reporter: Ofer Markman, PhD


While we are constantly cautioning people from putting too high validity to the results seen in the bellow mentioned Study, 

The ABO blood groups are a result of various and different glycans on the surfaces of red blood cells and their definition is related to Blood Immunology and Blood Typing classification used for Blood Donations. It was the hallmark of the 1930 Nobel prize of medicine.

In the article, ABO blood group glycans modulate sialic acid recognition on erythrocytes the authors claim that the ABH(O) glycan can modulate the surface of cells and their interactions to pathogens, in this case the Malaria pathogen.

Glycans are involved in the interaction of the flu virus to the host cell and the antiviral drug Tamiflu (Oseltamivir ) is based on the inhibition of that sort of interaction/modulation.

The Relevance of Glycans in the Viral Pathology of COVID-19

Even if true, the numbers in this paper show statistical significant difference but mildly significant difference in the risk profile suggesting we are to pay too much attention to the phenomena or worry, regardless of the fact these result have no significance on behavioral instructions nor would call for testing one’s blood type in regards to a viral infection. I would neither totally ignore the finding as it may shed light on viral pathology and infection mechanisms and understanding of the later may lead us to treatment or effective vaccines. But we are still early on this path.



Original Reference

Jiao Zhao, Yan Yang, Han-Ping Huang, Dong Li, Dong-Feng Gu, Xiang-Feng Lu, Zheng Zhang, Lei Liu, Ting Liu, Yu-Kun Liu, Yun-Jiao He, Bin Sun, Mei-Lan Wei, Guang-Yu Yang,  View ORCID Profile Xinghuan Wang, Li Zhang, Xiao-Yang Zhou, Ming-Zhao Xing,  View ORCID Profile Peng George Wang

doi: https://doi.org/10.1101/2020.03.11.20031096

yet a more relevant understanding of glycans in the biology of the virus:-

this week NEJM interview shed some more light on the issue in their 2nd part of the talk when they discussed again blood group risk factors in the covid-19 context.


Audio Interview: The Challenges of Safe Reopening

List of authors.

  • Eric J. Rubin, M.D., Ph.D., 
  • Lindsey R. Baden, M.D., 
  • and Stephen Morrissey, Ph.D.

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Reporter and Curator: Dr. Sudipta Saha, Ph.D.


Obesity is a global concern that is associated with many chronic complications such as type 2 diabetes, insulin resistance (IR), cardiovascular diseases, and cancer. Growing evidence has implicated the digestive system, including its microbiota, gut-derived incretin hormones, and gut-associated lymphoid tissue in obesity and IR. During high fat diet (HFD) feeding and obesity, a significant shift occurs in the microbial populations within the gut, known as dysbiosis, which interacts with the intestinal immune system. Similar to other metabolic organs, including visceral adipose tissue (VAT) and liver, altered immune homeostasis has also been observed in the small and large intestines during obesity.


A link between the gut microbiota and the intestinal immune system is the immune-derived molecule immunoglobulin A (IgA). IgA is a B cell antibody primarily produced in dimeric form by plasma cells residing in the gut lamina propria (LP). Given the importance of IgA on intestinal–gut microbe immunoregulation, which is directly influenced by dietary changes, scientists hypothesized that IgA may be a key player in the pathogenesis of obesity and IR. Here, in this study it was demonstrate that IgA levels are reduced during obesity and the loss of IgA in mice worsens IR and increases intestinal permeability, microbiota encroachment, and downstream inflammation in metabolic tissues, including inside the VAT.


IgA deficiency alters the obese gut microbiota and its metabolic phenotype can be recapitulated into microbiota-depleted mice upon fecal matter transplantation. In addition, the researchers also demonstrated that commonly used therapies for diabetes such as metformin and bariatric surgery can alter cellular and stool IgA levels, respectively. These findings suggested a critical function for IgA in regulating metabolic disease and support the emerging role for intestinal immunity as an important modulator of systemic glucose metabolism.


Overall, the researchers demonstrated a critical role for IgA in regulating intestinal homeostasis, metabolic inflammation, and obesity-related IR. These findings identify intestinal IgA+ immune cells as mucosal mediators of whole-body glucose regulation in diet-induced metabolic disease. This research further emphasized the importance of the intestinal adaptive immune system and its interactions with the gut microbiota and innate immune system within the larger network of organs involved in the manifestation of metabolic disease.


Future investigation is required to determine the impact of IgA deficiency during obesity in humans and the role of metabolic disease in human populations with selective IgA deficiency, especially since human IgA deficiency is associated with an altered gut microbiota that cannot be fully compensated with IgM. However, the research identified IgA as a critical immunological molecule in the intestine that impacts systemic glucose homeostasis, and treatments targeting IgA-producing immune populations and SIgA may have therapeutic potential for metabolic disease.


























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Reporter and Curator: Dr. Sudipta Saha, Ph.D.


Stroke is a leading cause of death worldwide and the most common cause of long-term disability amongst adults, more particularly in patients with diabetes mellitus and arterial hypertension. Increasing evidence suggests that disordered physiological variables following acute ischaemic stroke, especially hyperglycaemia, adversely affect outcomes.


Post-stroke hyperglycaemia is common (up to 50% of patients) and may be rather prolonged, regardless of diabetes status. A substantial body of evidence has demonstrated that hyperglycaemia has a deleterious effect upon clinical and morphological stroke outcomes. Therefore, hyperglycaemia represents an attractive physiological target for acute stroke therapies.


However, whether intensive glycaemic manipulation positively influences the fate of ischaemic tissue remains unknown. One major adverse event of management of hyperglycaemia with insulin (either glucose-potassium-insulin infusions or intensive insulin therapy) is the occurrence of hypoglycaemia, which can also induce cerebral damage.


Doctors all over the world have debated whether intensive glucose management, which requires the use of IV insulin to bring blood sugar levels down to 80-130 mg/dL, or standard glucose control using insulin shots, which aims to get glucose below 180 mg/dL, lead to better outcomes after stroke.


A period of hyperglycemia is common, with elevated blood glucose in the periinfarct period consistently linked with poor outcome in patients with and without diabetes. The mechanisms that underlie this deleterious effect of dysglycemia on ischemic neuronal tissue remain to be established, although in vitro research, functional imaging, and animal work have provided clues.


While prompt correction of hyperglycemia can be achieved, trials of acute insulin administration in stroke and other critical care populations have been equivocal. Diabetes mellitus and hyperglycemia per se are associated with poor cerebrovascular health, both in terms of stroke risk and outcome thereafter.


Interventions to control blood sugar are available but evidence of cerebrovascular efficacy are lacking. In diabetes, glycemic control should be part of a global approach to vascular risk while in acute stroke, theoretical data suggest intervention to lower markedly elevated blood glucose may be of benefit, especially if thrombolysis is administered.


Both hypoglycaemia and hyperglycaemia may lead to further brain injury and clinical deterioration; that is the reason these conditions should be avoided after stroke. Yet, when correcting hyperglycaemia, great care should be taken not to switch the patient into hypoglycaemia, and subsequently aggressive insulin administration treatment should be avoided.


Early identification and prompt management of hyperglycaemia, especially in acute ischaemic stroke, is recommended. Although the appropriate level of blood glucose during acute stroke is still debated, a reasonable approach is to keep the patient in a mildly hyperglycaemic state, rather than risking hypoglycaemia, using continuous glucose monitoring.


The primary results from the Stroke Hyperglycemia Insulin Network Effort (SHINE) study, a large, multisite clinical study showed that intensive glucose management did not improve functional outcomes at 90 days after stroke compared to standard glucose therapy. In addition, intense glucose therapy increased the risk of very low blood glucose (hypoglycemia) and required a higher level of care such as increased supervision from nursing staff, compared to standard treatment.
















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Glycobiology advances

Larry H. Bernstein, MD, FCAP, Curator



The Evolution of the Glycobiology Space

The Nascent Stage of another Omics Field with Biomarker and Therapeutic Potential

Enal Razvi, Ph.D. , Gary Oosta, Ph.D



The Evolution of the Glycobiology Space


Glycobiology is an important field of study with medical applications because it is known that tumor cells alter their glycosylation pattern, which may contribute to their metastatic potential as well as potential immune evasion. [iStock/© vjanez]    http://www.genengnews.com/media/images/AnalysisAndInsight/Apr12_2016_iStock_41612310_PlasmaMembraneOfACell1211657142.jpg

There is growing interest in the field of glycobiology given the fact that epitopes with physiological and pathological relevance have glyco moieties.  We believe that another “omics” revolution is on the horizon—the study of the glyco modifications on the surface of cells and their potential as biomarkers and therapeutic targets in many disease classes. Not much industry tracking of this field has taken place. Thus, we sought to map this landscape by examining the entire ensemble of academic publications in this space and teasing apart the trends operative in this field from a qualitative and quantitative perspective. We believe that this methodology of en masse capture and publication and annotation provides an effective approach to evaluate this early-stage field.

Identifiation and Growth of Glycobiology Publications


For this article, we identified 7000 publications in the broader glycobiology space and analyzed them in detail.  It is important to frame glycobiology in the context of genomics and proteomics as a means to assess the scale of the field. Figure 1 presents the relative sizes of these fields as assessed by publications in from 1975 to 2015.

Note that the relative scale of genomics versus proteomics and glycobiology/glycomics in this graph strongly suggests that glycobiology is a nascent space, and thus a driver for us to map its landscape today and as it evolves over the coming years.

Figure 2. (A) Segmentation of the glycobiology landscape. (B) Glycobiology versus glycomics publication growth.



To examine closely the various components of the glycobiology space, we segmented the publications database, presented in Figure 2A. Note the relative sizes and growth rates (slopes) of the various segments.

Clearly, glycoconjugates currently are the majority of this space and account for the bulk of the publications.  Glycobiology and glycomics are small but expanding and therefore can be characterized as “nascent market segments.”  These two spaces are characterized in more detail in Figure 2B, which presents their publication growth rates.

Note the very recent increased attention directed at these spaces and hence our drive to initiate industry coverage of these spaces. Figure 2B presents the overall growth and timeline of expansion of these fields—especially glycobiology—but it provides no information about the qualitative nature of these fields.

Focus of Glycobiology Publications


Figure 2C. Word cloud based on titles of publications in the glycobiology and glycomics spaces.

To understand the focus of publications in this field, and indeed the nature of this field, we constructed a word cloud based on titles of the publications that comprise this space presented in Figure 2C.

There is a marked emphasis on terms such as oligosaccharides and an emphasis on cells (this is after all glycosylation on the surface of cells). Overall, a pictorial representation of the types and classes of modifications that comprise this field emerge in this word cloud, demonstrating the expansion of the glycobiology and to a lesser extent the glycomics spaces as well as the character of these nascent but expanding spaces.

Characterization of the Glycobiology Space in Journals

Figure 3A. Breakout of publications in the glycobiology/glycomics fields.   http://www.genengnews.com/Media/images/AnalysisAndInsight/April12_2016_SelectBiosciences_Figure3a_5002432117316.jpg
Having framed the overall growth of the glycobiology field, we wanted to understand its structure and the classes of researchers as well as publications that comprise this field. To do this, we segmented the publications that constitute this field into the various journals in which glycobiology research is published. Figure 3A presents the breakout of publications by journal to illustrate the “scope” of this field.

The distribution of glycobiology publications across the various journals suggests a very concentrated marketplace that is very technically focused. The majority of the publications segregate into specialized journals on this topic, a pattern very indicative of a field in the very early stages of development—a truly nascent marketplace.


Figure 3B. Origin of publications in the glycobiology/glycomics fields.
We also sought to understand the “origin” of these publications—the breakout between academic- versus industry-derived journals. Figure 3B presents this breakout and shows that these publications are overwhelmingly (92.3%) derived from the academic sector. This is again a testimonial to the early nascent nature of this marketplace without significant engagement by the commercial sector and therefore is an important field to characterize and track from the ground up.

Select Biosciences, Inc. further analyzed the growth trajectory of the glycobiology papers in Figure 3C as a means to examine closely the publications trajectory. Although there appears to be some wobble along the way, overall the trajectory is upward, and of late it is expanding significantly.

In Summary

Figure 3C. Trajectory of the glycobiology space.   http://www.genengnews.com/Media/images/AnalysisAndInsight/April12_2016_SelectBiosciences_Figure3c1236921793.jpg
Glycobiology is the study of what coats living cells—glycans, or carbohydrates, and glycoconjugates. This is an important field of study with medical applications because it is known that tumor cells alter their glycosylation pattern, which may contribute to their metastatic potential as well as potential immune evasion.

At this point, glycobiology is largely basic research and thus it pales in comparison with the field of genomics. But in 10 years, we predict the study of glycobiology and glycomics will be ubiquitous and in the mainstream.

We started our analysis of this space because we’ve been focusing on many other classes of analytes, such as microRNAs, long-coding RNAs, oncogenes, tumor suppressor genes, etc., whose potential as biomarkers is becoming established. Glycobiology, on the other hand, represents an entire new space—a whole new category of modifications that could be analyzed for diagnostic potential and perhaps also for therapeutic targeting.

Today, glycobiology and glycomics are where genomics was at the start of the Human Genome Project. They respresent a nascent space and with full headroom for growth. Select Biosciences will continue to track this exciting field for research developments as well as development of biomarkers based on glyco-epitopes.

Enal Razvi, Ph.D., conducted his doctoral work on viral immunology and subsequent to receiving his Ph.D. went on to the Rockefeller University in New York to serve as Aaron Diamond Post-doctoral fellow under Professor Ralph Steinman [Nobel Prize Winner in 2011 for his discovery of dendritic cells in the early-70s with Zanvil Cohn]. Subsequently, Dr. Razvi completed his research fellowship at Harvard Medical School. For the last two decades Dr. Razvi has worked with small and large companies and consulted for more than 100 clients worldwide. He currently serves as Biotechnology Analyst and Managing Director of SelectBio U.S. He can be reached at enal@selectbio.us. Gary M. Oosta holds a Ph.D. in Biophysics from Massachusetts Institute of Technology and a B.A. in Chemistry from E. Mich. Univ. He has 25 years of industrial research experience in various technology areas including medical diagnostics, thin-layer coating, bio-effects of electromagnetic radiation, and blood coagulation. Dr. Oosta has authored 20 technical publications and is an inventor on 77 patents worldwide. In addition, he has managed research groups that were responsible for many other patented innovations. Dr. Oosta has a long-standing interest in using patents and publications as strategic technology indicators for future technology selection and new product development. To enjoy more articles like this from GEN, click here to subscribe now!

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Triglycerides: Is it a Risk Factor or a Risk Marker for Atherosclerosis and Cardiovascular Disease ? The Impact of Genetic Mutations on (ANGPTL4) Gene, encoder of (angiopoietin-like 4) Protein, inhibitor of Lipoprotein Lipase


Reporters, Curators and Authors: Aviva Lev-Ari, PhD, RN and Larry H. Bernstein, MD, FCAP


The role for triglycerides as a risk factor for cardiovascular disease is not new, going back to Donald Frederickson’s classification of hyperlipidemias, at least with respect to Type I and Type IIb. Whether there was a mechanism beyond the observations was yet an open question.  The paper that follows addresses such a question.


Large Genetic Studies Support Role For Triglycerides In Cardiovascular Disease




Two  papers published in the New England Journal of Medicine offer new genetic evidence to support the increasingly accepted though still controversial view that triglycerides play an important causal role in cardiovascular disease. If fully validated the new findings could lead to new drugs to prevent and treat cardiovascular disease, though others caution that there is still a long way to go before this could happen.

Both studies describe the impact of genetic mutations on a gene (ANGPTL4) which encodes for a protein (angiopoietin-like 4) that inhibits lipoprotein lipase, an enzyme that plays a key role in breaking down and removing triglycerides from the blood. The large studies found that people with  mutations that inactivate ANGPTL4 have lower levels of triglycerides, higher levels of HDL cholesterol, and decreased risk for cardiovascular disease.

The findings, writes Sander Kersten (Wageningen University, the Netherlands) in an accompanying editorial, “suggest that lowering plasma triglyceride levels is a viable approach to reducing the risk of coronary artery disease.”

The Genetics of Dyslipidemia — When Less Is More

Sander Kersten, Ph.D.   Mar 2, 2016;   http://dx.doi.org:/10.1056/NEJMe1601117

Two groups of investigators now describe in the Journal important genetic evidence showing a causal role of plasma triglycerides in coronary heart disease. Stitziel and colleagues2 tested 54,003 coding-sequence variants covering 13,715 human genes in more than 72,000 patients with coronary artery disease and 120,000 controls. Dewey and colleagues3 sequenced the exons of the gene encoding angiopoietin-like 4 (ANGPTL4) in samples obtained from nearly 43,000 participants in the DiscovEHR human genetics study. The two groups found a significant association between an inactivating mutation (E40K) in ANGPTL4 and both low plasma triglyceride levels and high levels of HDL cholesterol. ANGPTL4 is an inhibitor of lipoprotein lipase, the enzyme that breaks down plasma triglycerides along the capillaries in heart, muscle, and fat.4 Extensive research has shown that ANGPTL4 orchestrates the processing of triglyceride-rich lipoproteins during physiologic conditions such as fasting, exercise, and cold exposure.4 The E40K mutation in ANGPTL4 was previously shown to nearly eliminate the ability of ANGPTL4 to inhibit lipoprotein lipase, a mechanism that may result in part from the destabilization of ANGPTL4.5

The key finding in each study was that carriers of the E40K mutation and other rare mutations in ANGPTL4 had a lower risk of coronary artery disease than did noncarriers, a result that is consistent with the lower triglyceride levels and higher HDL cholesterol levels among mutation carriers. These findings confirm previous data6 and provide convincing genetic evidence that an elevated plasma triglyceride level increases the risk of coronary heart disease. In combination with extensive recent data on other genetic variants that modulate plasma triglyceride levels, the studies suggest that lowering plasma triglyceride levels is a viable approach to reducing the risk of coronary artery disease.

However, as a cautionary note, Talmud and colleagues7 previously found that the presence of the E40K variant was associated with an increased risk of coronary heart disease after adjustment for the altered plasma lipids. Consistent with this hypothesis, the overexpression of Angptl4 in mice was found to protect against atherosclerosis independent of plasma lipids.8

The studies also “implicate targeted inactivation of ANGPTL4 as a potential weapon in the war on heart disease,” though he also points to a previous study that did not support this hypothesis. Sekar Kathiresan (Broad Institute), senior author of one of the NEJM studies, told me that the previous study was small and “basically got the result wrong. Between, the two papers in this NEJM issue, we are looking at 10X more data.”

Recent large genetic studies have resulted in an important change in the field. Many researchers now believe that HDL, which was once thought to play an important protective role in atherosclerosis, is only a marker of disease. In contrast, triglycerides are now thought by many to play an important functional role.

One of the NEJM papers showed that a human monoclonal antibody to ANGPTL4 lowered triglyceride levels in animals. The study was funded by Regeneron and was performed by researchers at Regeneron and Geisinger, as part of an ongoing collaboration using deidentified genetic data from Geisinger patients. In their NEJM paper the researchers reported inflammation and other side effects in the animals treated with the antibody, but they said that no such problem has been observed in humans who have mutations that have the same functional effect as the antibody.

Coding Variation in ANGPTL4, LPL, and SVEP1and the Risk of Coronary Disease Myocardial Infarction Genetics and CARDIoGRAM Exome Consortia Investigators

March 2, 2016     http://dx.doi.org:/10.1056/NEJMoa1507652

Although genomewide association studies have identified more than 56 loci associated with the risk of coronary artery disease,1-3 the disease-associated variants are typically common (minor-allele frequency >5%) and located in noncoding sequences; this has made it difficult to pinpoint causal genes and affected pathways. This lack of a causal mechanism has in part hindered the immediate translation of the findings of genomewide association studies into new therapeutic targets. However, the discovery of rare or low-frequency coding-sequence variants that affect the risk of coronary artery disease has facilitated advances in the prevention and treatment of disease. The most recent example of such advances is the development of a new class of therapeutic agents that is based on the discovery of the gene encoding proprotein convertase subtilisin/kexin type 9 (PCSK9) as a regulator of low-density lipoprotein (LDL) cholesterol4 and the discovery that low-frequency and loss-of-function variants in this gene protect against coronary artery disease.5,6

Recently, low-frequency coding variation across the genome was systematically tabulated with the use of next-generation exome and whole-genome sequencing data from more than 12,000 persons of various ancestries (including a major contribution from the National Heart, Lung, and Blood Institute Exome Sequencing Project). Protein-altering variants (i.e., nonsynonymous, splice-site, and nonsense single-nucleotide substitutions) that were observed at least twice among these 12,000 persons were included in a genotyping array (hereafter referred to as the exome array). In addition, the exome array contains previously described variants from genomewide association studies, a sparse genomewide grid of common markers, markers that are informative with regard to ancestry (i.e., African American, Native American, and European), and some additional content. Additional information on the design of the exome array is provided at http://genome.sph.umich.edu/wiki/Exome_Chip_Design. In this study, we focused on the 220,231 autosomal variants that were present on the array and were expected to alter protein sequence (i.e., missense, nonsense, splice-site, and frameshift variants) and used these to test the contribution of low-frequency coding variation to the risk of coronary artery disease.

Low-Frequency Coding Variants Associated with Coronary Artery Disease

The discovery cohort comprised 120,575 persons (42,335 patients and 78,240 controls) (Table S1 in the Supplementary Appendix). In the discovery cohort, we found significant associations between low-frequency coding variants in theLPA and PCSK9 genes and coronary artery disease (Table 1


Low-Frequency Coding Variations Previously Associated with Coronary Artery Disease.). Both gene loci also harbor common noncoding variants associated with coronary artery disease that had previously been discovered through genomewide association studies. These variants were also present on the exome array and had significant associations with coronary artery disease in our study (Table 1). In a conditional analysis, the associations between coronary artery disease and the low-frequency coding variants in both LPA and PCSK9 were found to be independent of the associations between coronary artery disease and the more common variants (Table 1). ….

We found a significant association between SVEP1 p.D2702G and blood pressure (Table 3TABLE 3   Association between Low-Frequency Variants and Traditional Risk Factors., and Table S7 in the Supplementary Appendix). The allele associated with an increased risk of coronary artery disease was also associated with higher systolic blood pressure (0.94 mm Hg higher for each copy of the allele among allele carriers, P=3.0×10−7) and higher diastolic blood pressure (0.57 mm Hg higher for each copy of the allele among allele carriers, P=4.4×10−7). We did not find an association between SVEP1 p.D2702G and any plasma lipid trait. In contrast, ANGPTL4 p.E40K was not associated with blood pressure but instead was found to be associated with significantly lower levels of triglycerides (approximately 0.3 standard deviation units lower for each copy of the allele among allele carriers, P=1.6×10−13) (Table 3) and with higher levels of high-density lipoprotein (HDL) cholesterol (approximately 0.3 standard deviation units higher for each copy of the allele among allele carriers, P=8.2×10−11) (Table 3). In a conditional analysis, these effects appeared to be at least partially independent of each other (Table S8 in the Supplementary Appendix). We did not observe any significant association between ANGPTL4 p.E40K and LDL cholesterol level (Table 3). Both SVEP1 p.D2702G and ANGPTL4 p.E40K were nominally associated with type 2 diabetes in a direction concordant with the associated risk of coronary artery disease.

ANGPTL4 Loss-of-Function Mutations, Plasma Lipid Levels, and Coronary Artery Disease

The finding that a missense allele in ANGPTL4 reduced the risk of coronary artery disease, potentially by reducing triglyceride levels, raised the possibility that complete loss-of-function variants in ANGPTL4 may have an even more dramatic effect on triglyceride concentrations and the risk of coronary artery disease. We therefore examined sequence data for the seven protein-coding exons of ANGPTL4 in 6924 patients with early-onset myocardial infarction and 6834 controls free from coronary artery disease (details of the patients and controls are provided in Table S3 in the Supplementary Appendix). We discovered a total of 10 variants that were predicted to lead to loss of gene function (Figure 1A FIGURE 1    

Loss-of-Function Alleles in ANGPTL4 and Plasma Lipid Levels., and Table S9 in the Supplementary Appendix), carried by 28 heterozygous persons; no homozygous or compound heterozygous persons were discovered. Carriers of loss-of-function alleles had significantly lower levels of triglycerides than did noncarriers (a mean of 35% lower among carriers, P=0.003) (Figure 1B, and Table S10 in the Supplementary Appendix), with no significant difference in LDL or HDL cholesterol levels. Moreover, we found a lower risk of coronary artery disease among carriers of loss-of-function alleles (9 carriers among 6924 patients vs. 19 carriers among 6834 controls; odds ratio for disease, 0.47; P=0.04) (Table S11 in the Supplementary Appendix). A similar investigation was performed for the 48 protein-coding exons of SVEP1; however, only 3 loss-of-function allele carriers were discovered (2 carriers among 6924 patients vs. 1 carrier among 6834 controls).

Coding Variation in LPL and the Risk of Coronary Artery Disease

On the basis of the fact that a loss of ANGPTL4 function was associated with reduced risk of coronary artery disease and that ANGPTL4 inhibits lipoprotein lipase (LPL), one would expect a gain of LPL function to also be associated with a lower risk of coronary artery disease, whereas a loss of LPL function would be expected to be associated with a higher risk. In observations consistent with these expectations, we found a low-frequency missense variant in LPL on the exome array that was associated with an increased risk of coronary artery disease (p.D36N; minor-allele frequency, 1.9%; odds ratio for disease, 1.13; P=2.0×10−4) (Table S12 in the Supplementary Appendix); previous studies have shown that this allele (also known as p.D9N) is associated with LPL activity that is 20% lower in allele carriers than in noncarriers.8 We also identified a nonsense mutation in LPL on the exome array that was significantly associated with a reduced risk of coronary artery disease (p.S447*; minor-allele frequency, 9.9%; odds ratio, 0.94; P=2.5×10−7) (Table S12 in the Supplementary Appendix). Contrary to most instances in which the premature introduction of a stop codon leads to loss of gene function, this nonsense mutation, which occurs in the penultimate codon of the gene, paradoxically induces a gain of LPL function.9 …..

Through large-scale exomewide screening, we identified a low-frequency coding variant in ANGPTL4 that was associated with protection against coronary artery disease and a low-frequency coding variant in SVEP1 that was associated with an increased risk of the disease. Moreover, our results highlight LPL as a significant contributor to the risk of coronary artery disease and support the hypothesis that a gain of LPL function or loss of ANGPTL4 inhibition protects against the disease.

ANGPTL4 has previously been found to be involved in cancer pathogenesis and wound healing.10 Previous functional studies also revealed that ANGPTL4 regulates plasma triglyceride concentration by inhibiting LPL.11 The minor allele at p.E40K has previously been associated with lower levels of triglycerides and higher levels of HDL cholesterol.12 We now provide independent confirmation of these lipid effects. In vitro and in vivo experimental evidence suggests that the lysine allele at p.E40K results in destabilization of ANGPTL4 after its secretion from the cell in which it was synthesized. It may be that the p.E40K variant leads to increases in the enzymatic activity of LPL because of this destabilization.13 Previous, smaller studies produced conflicting results regarding p.E40K and the risk of coronary artery disease14,15; we now provide robust support for an association between p.E40K and a reduced risk of coronary artery disease.

An important caveat  to this research is that it is still very early. Most promising therapeutic targets do not work out. James Stein (University of Wisconsin) praised the papers but also offered a word of caution. “This is great science and important research that sheds light on the genetic regulation of TG-rich lipoproteins, serum TG levels, and CVD risk,” he said. “Since it is hard, if not impossible, to disconnect TG-rich lipoproteins from LDL, we should be humble in extrapolating these findings to clinical medicine in an era of low LDL due to statins and PCSK9 inhibitors. I hope this research identifies new targets for drug therapy and better understanding of CVD risk prediction– only time will tell.”

Previous studies with fibrates and other drugs have failed to convincingly show that lowering triglycerides is beneficial. Kathiresan said that what really seems to matter is “how you alter the plasma triglyceride-rich lipoproteins (TRLs).” Some genes that alter TRLs have other metabolic effects. As an example he cited a gene that lowers TRLs but increases the risk for type 2 diabetes. The NEJM papers, by observing the effect of specific mutations, therefore point the way to targets that may be clinically significant.

Conclusions:  The work that has been presented puts a new light on the possible role of triglycerides in the development of congenitally predetermined cardiovascular disease. It does not necessarily establish a general link to mechanism of cardiovascular disease, but it opens up new pathways to our understanding.



John Contois commented on your update
“Are triglycerides a CHD risk factor? The answer is still maybe. Triglyceride-rich lipoproteins are inextricably linked to LDL metabolism and LDL particle number (and apo B). Still these are important new data and targets for novel therapeutics.”


Risk of Dis-lipids Syndromes in Modern Society


Risk of Dis-lipids Syndrome in Modern Society

Aurelian Udristioiuᶪ, Manole Cojocaru²
¹Department of Biochemistry, Clinical Laboratory, Emergency County Hospital Targu Jiu & Titu Maiorescu University, Bucharest, Romania,
Department of Physiology, Faculty of Medicine, Titu Maiorescu University, Bucharest, Romania

Aim of this work was to emphasis the preclinical evaluation of dis-lipids syndromes types at the patients which were presented to a routine control for checking health status, in the hospital ambulatory.
Material and Method:
Were analyzed 60 patients, registered in Clinical Laboratory, assessing by running on the Hitachi 912 Analyzer, the principal biochemical parameters of lipid metabolism: Cholesterol, Triglycerides and fractions of Cholesterol, HDL and LDL. From the total of 60 patients 35 were females and 25 males.
The persons with an alarm signal of atherosclerotic process were in 28 % and persons with low HDL was in 17%. The cases with atherosclerotic index, report-LDL/HDL>3.5 for men and 2.5 for women were in 14 % , the cases with predictive value with coronary risk, report-CO/HDL>5 were presented in 5 % and the cases with dis-lipid syndrome type 2- 4, with high Cholesterol and Triglycerides, were presented in 30% percent.
Lipids controls, and its fractions, are necessary to be prevented atherosclerotic process in the incipient status of ill.








March 2, 2016 Regeneron Genetics Center Publication in New England Journal of Medicine Links ANGPTL4 Inhibition and Risk of Coronary Artery Disease Demonstrates power of large-scale Precision Medicine initiatives


e-Books by Leaders in Pharmaceutical Business Intelligence (LPBI) Group

  • Perspectives on Nitric Oxide in Disease Mechanisms, on Amazon since 6/2/12013



  • Genomics Orientations for Personalized Medicine, on Amazon since 11/23/2015


  • Milestones in Physiology: Discoveries in Medicine, Genomics and Therapeutics, on Amazon.com since 12/27/2015


  • Cardiovascular, Volume Two: Cardiovascular Original Research: Cases in Methodology Design for Content Co-Curation, on Amazon since 11/30/2015


  • Cardiovascular Diseases, Volume Three: Etiologies of Cardiovascular Diseases: Epigenetics, Genetics and Genomics, on Amazon since 11/29/2015


  • Cardiovascular Diseases, Volume Four: Regenerative and Translational Medicine: The Therapeutics Promise for Cardiovascular Diseases, on Amazon since 12/26/2015


Other related articles on this topic published in this Open Access Online Scientific Journal include the following:

Editorial & Publication of Articles in e-Books by  Leaders in Pharmaceutical Business Intelligence:  Contributions of Larry H Bernstein, MD, FCAP


Editorial & Publication of Articles in e-Books by Leaders in Pharmaceutical Business Intelligence: Contributions of Aviva Lev-Ari, PhD, RN












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newly developed oxazolidinone antibiotics

Larry H. Bernstein, MD, FCAP, Curator




New Antibacterial oxazolidinones in pipeline by Wockhardt





MF C19 H25 F2 N3 O5, MW 413.42

Acetamide, N-​[[(5S)​-​3-​[3,​5-​difluoro-​4-​[4-​hydroxy-​4-​(methoxymethyl)​-​1-​piperidinyl]​phenyl]​-​2-​oxo-​5-​oxazolidinyl]​methyl]​-

CAS 957796-51-9

Antibacterial oxazolidinones

THIS MAY BE WCK 4086?????


WO 2015173664, US8217058, WO 2012059823, 


Oxazolidinone represent a novel chemical class of synthetic antimicrobial agents.Linezolid represents the first member of this class to be used clinically. Oxazolidinones display activity against important Gram-positive human and veterinary pathogens including Methicillin-Resistant Staphylococcus aureus (MRSA), Vancomycin Resistant Enterococci (VRE) and β-lactam Resistant Streptococcus pneumoniae (PRSP). The oxazolidinones also show activity against Gram-negative aerobic bacteria, Gram-positive and Gram-negative anaerobes. (Diekema D J et al., Lancet 2001 ; 358: 1975-82).

Various oxazolidinones and their methods of preparation are disclosed in the literature. International Publication No. WO 1995/25106 discloses substituted piperidino phenyloxazolidinones and International Publication No. WO 1996/13502 discloses phenyloxazolidinones having a multisubstituted azetidinyl or pyrrolidinyl moiety. US Patent Publication No. 2004/0063954, International Publication Nos. WO 2004/007489 and WO 2004/007488 disclose piperidinyl phenyl oxazolidinones for antimicrobial use.

Pyrrolidinyl/piperidinyl phenyl oxazohdinone antibacterial agents are also described in Kim H Y et al., Bioorg. & Med. Chem. Lett., (2003), 13:2227-2230. International Publication No. WO 1996/35691 discloses spirocyclic and bicyclic diazinyl and carbazinyl oxazolidinone derivatives. Diazepeno phenyloxazolidinone derivatives are disclosed in the International Publication No. WO 1999/24428. International Publication No. WO 2002/06278 discloses substituted aminopiperidino phenyloxazolidinone derivatives.

Various other methods of preparation of oxazolidinones are reported in US Patent No. 7087784, US Patent No. 6740754, US Patent No. 4948801 , US Patent No. 3654298, US Patent No. 5837870, Canadian Patent No. 681830, J. Med. Chem., 32, 1673 (1989), Tetrahedron, 45, 1323 (1989), J. Med. Chem., 33, 2569 (1990), Tetrahedron Letters, 37, 7937-40 (1996) and Organic Process Research and Development, 11 , 739-741(2007).

Indian Patent Application No. 2534/MUM/2007 discloses a process for the preparation of substituted piperidino phenyloxazolidinones. International Publication No. WO2012/059823 further discloses the process for the preparation of phosphoric acid mono-(L-{4-[(5)-5-(acetylaminomethyl)-2-oxo-oxazolidin-3-yl]-2,6-difluorophenyl}4-methoxymethyl piperidine-4-yl)ester.

US Patent No. 8217058 discloses (5S)-N-{3-[3,5-difluoro-4-(4-hydroxy-4-methoxymethyl-piperidin-l-yl)-phenyl]-2-oxo-oxazolidin-5-ylmethyl}-acetamide as an antibacterial agent and its process for preparation.









Figure imgf000004_0001

Wockhardt Ltd,

Figure imgf000006_0001
Figure imgf000006_0002

(3) (4)



WO 2012059823


Phosphoric acid mono-(l-{4-[(S)-5-(acetylamino- methyl)-2-oxo-oxazolidin-3-yl]-2,6-difluorophenyl}-4-methoxymethyl-piperidin-4-yl) ester of Formula (A),
Figure imgf000022_0001
the process comprising the steps of:
a) Converting intermediate of Formula (1) into intermediate of Formula (3)
Figure imgf000022_0002
b) Converting intermediate of Formula (3) into intermediate of Formula (5)
Figure imgf000022_0003

c) Converting intermediate of Formula (5) into intermediate of structure (6)

Figure imgf000022_0004
(5) <6> d) Converting intermediate of Formula (6) into intermediate of Formula (10)
Figure imgf000023_0001
e) Converting intermediate of Formula (10) into intermediate of Formula (11),
Figure imgf000023_0002

f) Converting intermediate of Formula (11) into compound of Formula (A) or Pharmaceutically acceptable salts thereof

Figure imgf000023_0003



Figure imgf000006_0001
Figure imgf000006_0002
Figure imgf000006_0003


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MedChemComm articles -3rd Q 2015

Larry H. Bernstein, MD, FCAP, Curator



MedChemComm articles in July, August and September 2015.

MedChemComm medchemcomm-rsc@rsc.org


Transition metal diamine complexes with antimicrobial activity against Staphylococcus aureus and methicillin-resistant S. aureus (MRSA)
G. W. Karpin, D. M. Morris, M. T. Ngo, J. S. Merola and J. O. Falkinham III
DOI: 10.1039/C5MD00228A, Concise Article
Multivalent glycoconjugates as vaccines and potential drug candidates
Sumati Bhatia, Mathias Dimde and Rainer Haag
DOI: 10.1039/C4MD00143E, Review Article
Polypharmacology modelling using proteochemometrics (PCM): recent methodological developments, applications to target families, and future prospects
Isidro Cortés-Ciriano, Qurrat Ul Ain, Vigneshwari Subramanian, Eelke B. Lenselink, Oscar Méndez-Lucio, Adriaan P. IJzerman, Gerd Wohlfahrt, Peteris Prusis, Thérèse E. Malliavin, Gerard J. P. van Westen and Andreas Bender
DOI: 10.1039/C4MD00216D, Review Article
Towards understanding cell penetration by stapled peptides
Qian Chu, Raymond E. Moellering, Gerard J. Hilinski, Young-Woo Kim, Tom N. Grossmann, Johannes T.-H. Yeh and Gregory L. Verdine
DOI: 10.1039/C4MD00131A, Concise Article

Rational design of protein–protein interaction inhibitors
Didier Rognan
DOI: 10.1039/C4MD00328D, Review Article


Transition metal diamine complexes with antimicrobial activity againstStaphylococcus aureus and methicillin-resistant S. aureus (MRSA)

Med. Chem. Commun., 2015,6, 1471-1478     DOI: http://dx.doi.org:/10.1039/C5MD00228A


Multivalent glycoconjugates as vaccines and potential drug candidates

Med. Chem. Commun., 2014,5, 862-878   DOI: http://dx.doi.org:/10.1039/C4MD00143E

Pathogens adhere to the host cells during the first steps of infection through multivalent interactions which involve protein–glycan recognition. Multivalent interactions are also involved at different stages of immune response. Insights into these multivalent interactions generate a way to use suitable carbohydrate ligands that are attached to a basic scaffold consisting of e.g., dendrimer, polymer, nanoparticle, etc., with a suitable linker. Thus a multivalent architecture can be obtained with controllable spatial and topology parameters which can interfere with pathogen adhesion. Multivalent glycoconjugates bearing natural or unnatural carbohydrate antigen epitopes have also been used as carbohydrate based vaccines to stimulate an innate and adaptive immune response. Designing and synthesizing an efficient multivalent architecture with optimal ligand density and a suitable linker is a challenging task. This review presents a concise report on the endeavors to potentially use multi- and polyvalent glycoconjugates as vaccines as well as anti-infectious and anti-inflammatory drug candidates.


Graphical abstract: Multivalent glycoconjugates as vaccines and potential drug candidates




Polypharmacology modelling using proteochemometrics (PCM): recent methodological developments, applications to target families, and future prospects

Proteochemometric (PCM) modelling is a computational method to model the bioactivity of multiple ligands against multiple related protein targets simultaneously. Hence it has been found to be particularly useful when exploring the selectivity and promiscuity of ligands on different proteins. In this review, we will firstly provide a brief introduction to the main concepts of PCM for readers new to the field. The next part focuses on recent technical advances, including the application of support vector machines (SVMs) using different kernel functions, random forests, Gaussian processes and collaborative filtering. The subsequent section will then describe some novel practical applications of PCM in the medicinal chemistry field, including studies on GPCRs, kinases, viral proteins (e.g. from HIV) and epigenetic targets such as histone deacetylases. Finally, we will conclude by summarizing novel developments in PCM, which we expect to gain further importance in the future. These developments include adding three-dimensional protein target information, application of PCM to the prediction of binding energies, and application of the concept in the fields of pharmacogenomics and toxicogenomics. This review is an update to a related publication in 2011 and it mainly focuses on developments in the field since then.


Graphical abstract: Polypharmacology modelling using proteochemometrics (PCM): recent methodological developments, applications to target families, and future prospects



Related articles   

Experimental and computational studies of fatty acid distribution networks


Kinetics and non-exponential binding of DNA-coated colloids


Configuration of nonspherical amphiphilic particles at a fluid–fluid interface



Towards understanding cell penetration by stapled peptides

Med. Chem. Commun., 2015,6, 111-119   DOI: http://dx.doi.org:/10.1039/C4MD00131A

Hydrocarbon-stapled α-helical peptides are a new class of targeting molecules capable of penetrating cells and engaging intracellular targets formerly considered intractable. This technology has been applied to the development of cell-permeable ligands targeting key intracellular protein–protein interactions. However, the properties governing cell penetration of hydrocarbon-stapled peptides have not yet been rigorously investigated. Herein we report our studies to systematically probe cellular uptake of stapled peptides. We developed a high-throughput epifluorescence microscopy assay to quantitatively measure stapled peptide intracellular accumulation and demonstrated that this assay yielded highly reproducible results. Using this assay, we analyzed more than 200 peptides with various sequences, staple positions and types, and found that cell penetration ability is strongly related to staple type and formal charge, whereas other physicochemical parameters do not appear to have a significant effect. We next investigated the mechanism(s) involved in stapled peptide internalization and have demonstrated that stapled peptides penetrate cells through a clathrin- and caveolin-independent endocytosis pathway that involves, in part, sulfated cell surface proteoglycans, but that also seems to exploit a novel, uncharacterized pathway. Taken together, staple type and charge are the key physical properties in determining the cell penetration ability of stapled peptides, and anionic cell surface proteoglycans might serve as receptors to mediate stapled peptide internalization. These findings improve our understanding of stapled peptides as chemical probes and potential targeted therapeutics, and provide useful guidelines for the design of next-generation stapled peptides with enhanced cell permeability.

Graphical abstract: Towards understanding cell penetration by stapled peptides



Introduction Hydrocarbon stapled a-helical peptides are an exciting new class of investigational agents capable of targeting and interfering with intracellular protein–protein interactions.1,2 (For reviews on hydrocarbon stapled peptides, see ref. 3 and 4, and for reviews on synthetic a-helix stabilization in general, see ref. 5 and 6.) These peptides contain a synthetic brace, referred to as a staple, introduced across one face of an a-helix (Fig. 1), that in favorable cases can increase a-helical content and protease resistance, enhance target binding affinity, promote cell membrane penetration, and suppress clearance in vivo. 7–10

Fig. 1 All-hydrocarbon stapled peptide technology. (a) Schematic illustration of peptide stapling. Two alpha-methylated, alkenyl-bearing non-natural amino acids are incorporated at two or more positions in the peptide chain and then cross-linked by ruthenium-catalyzed ringclosing olefin metathesis. (b) Different types of alkenyl-containing non-natural amino acids with distinct stereochemistry at the a-carbon and varied lengths of alkenyl side chains. (c) Three types of stapled peptides used in this study with optimized combinations of nonnatural amino acids.

Stapled peptides are synthesized via incorporation of two amethyl, a-alkenyl amino acids at defined positions in a synthetic peptide, followed by ring-closing olefin metathesis to close the helix-spanning hydrocarbon bridge (Fig. 1a).11,12 The two components of the staple, namely the hydrocarbon bridge and terminal methyl groups, are both important to obtain maximal effectiveness of the conformationally constrained peptide products. This technology has been successfully utilized to target several classes of proteins formerly considered intractable, including multi-component transcription factor complexes and protein–protein interactions having extended interfaces, such as the NOTCH transcription factor complex,13 the b-catenin–TCF interaction in the oncogenic Wnt signaling pathway,14 and the epigenetic modulator PRC2 complex.15 Given the difficulties of developing traditional small molecule drugs that can successfully target intracellular protein–protein interactions, hydrocarbon stapling technology is widely considered to represent a promising avenue of research for the development of chemical probes and potential targeted therapeutics.

Multiple types of hydrocarbon staples have been obtained by varying the relative placement of the cross-linking a,a-disubstituted amino acids, as well as the stereochemistry at the acarbon and the lengths of the alkenyl substituents (Fig. 1b).16,17 These staple types were optimized to provide robust a-helical stabilization and confer the potential for in vitro and in vivo activity. As a result of the combinatorial search process used to identify helix-stabilizing hydrocarbon staples, the diversity of the resulting macrocyclic bridges has revealed stapled peptides with different physicochemical properties. Recently, a new hyperstable version of stapled peptide with tandem crosslinks, referred to as a stitched peptide, was generated by introduction of S5 at the i position, B5 at the i + 4 position, and S8 at the i + 11 position (Fig. 1c) (Y.-W. Kim and G. L. Verdine, to be published).

Of the physicochemical properties demonstrated by peptide bearing hydrocarbon staples, the capacity to promote cellular membrane penetration is perhaps the most signicant and yet remains the most poorly understood. Independent of hydrocarbon-stapled peptides, several classes of cell penetrating peptides (CPPs) have been discovered, including naturally occuring transcription factor domains such as pennetratin18 and HIV-Tat19 and synthetic cationic peptides such as polyArginine peptides.20 Notably, despite extensive exploration during the past two decades, the mechanism(s) by which CPPs enter cells remain unclear.21–23 In contrast to CPPs, in which cell penetration appears to be sequence-dependent, numerous cell permeable stapled peptides have been discovered for peptide scaffolds with little sequence homology. These divergent observations regarding cell penetration is proposed to result from several features of stapled peptides that differentiate them from typical CPPs. For example, the introduction of an allhydrocarbon cross-link results in a constrained a-helical conformation, which embeds the hydrophilic amide backbone in the core of the folded structure. Furthermore, the hydrocarbon brace itself introduces a significantly hydrophobic patch to one face of the peptide. The exposure of the hydrophobic moiety as well as the masking of the hydrophilic peptide backbone may facilitate the interaction of stapled peptides with the hydrophobic interior of the cell membrane and thereby enhance the cellular uptake. As cell penetration is a critical property of stapled peptides, we sought to develop quantitative methods to correlate a battery of stapled peptide properties with the capacity for cellular uptake. A direct comparison with several well-known CPPs has revealed that stapled peptides, including some stapled versions of the CPPs, exhibit more robust cell penetration. Lastly, we have demonstrated that stapled peptides penetrate cells through a clathrin- and caveolin-independent endocytosis pathway that involves, in part, sulfated cell surface proteoglycans. These findings significantly expand our current understanding of cell penetration by stapled peptides and provide useful information for the future rational design of cell penetrating stapled peptides with novel applications.

Results and discussion

Development of a high-throughput assay to quantitatively measure cellular uptake of peptides

Understanding the internalization process of cell penetrating peptides (CPPs), especially stapled peptides, has been a subject of great interest. The majority of previous studies have been performed by either using high-resolution microscopy to show the existence of fluorophore-labeled CPPs inside cells, or by quantitatively measuring intracellular fluorescence by flow cytometry.24,25 Although these two methods can provide important information regarding cell penetration, their respective limitations prompted us to adopt an assay that combines high-resolution imaging with reliable quantitation of intracellular accumulation to better analyze and understand the cell penetration of stapled peptides. In recent years, highthroughput cell-based imaging platforms have become increasingly popular to screen for small molecule modulators of various biological processes.26,27 Taking advantage of one of these platforms, high-content epifluorescence microscopy, we developed a high-throughput quantitative assay to measure stapled peptide intracellular access.

Proof-of-principle experiments were performed to determine whether epifluorescence microscopy could be used to quantitatively compare stapled peptide intracellular access. Human U2OS osteosarcoma cells were seeded in black, clear-bottom 384-well plates and then incubated in serum-containing media supplemented with fluorescein-labeled peptides or DMSO vehicle for 12 hours. After the treatment, cells were washed thoroughly with PBS to remove excess peptide, fixed with 4% formaldehyde, and stained with Hoechst dye to visualize nuclei. Once prepared, the plates were imaged and quantified by epi- fluorescence microscopy according to a protocol developed and discussed in detail in Experimental methods. An initial z-scan was performed using the Hoechst channel to locate the cells, and the microscope parameters were subsequently adjusted to optimize the cell size and fluorescence intensity. The parameters from this acquisition were then applied to the FITC channel, and the microscope scanned and recorded images of the FITC-labeled peptides within the z-plane of the cell. This assay was performed in a high-throughput manner, resulting in a panel of Hoechst/FITC images from individual wells (Fig. S1†). The raw image data was then analyzed using MetaXpress® software (Fig. 2a). Cells were identified based on the Hoechst stain of nuclei, with the requirement that they were a contiguous fluorescent region having a specific intensity above local background as well as having a diameter between defined minimum and maximum to be designated as “positive” cells. The cytoplasm of each cell was then identied according to the spatial location of FITC signal in relation to the nuclei as well as empiric parameters (details in Experimental methods). The FITC intensities in the cytoplasm and nuclei were then quantified separately, and the sum of these two values yielded the FITC signal for the whole cell, which can be considered the relative intracellular peptide intensity. In addition, FITC negative cells were identified on the basis of a positive Hoechst stain, which was accompanied by an absence of appreciable signal in the FITC channel.

Fig. 2 Quantitative measurement of cellular peptide intensity. (a) Hoechst channel (left) showing the location and size of nuclei, FITC channel (middle) showing the fluorescence intensity of the same cells. Information about cell size and fluorescence intensity was integrated to identify the FITC positive (green mask) and negative (red mask) cell (right). For positive cells, additional parameters allowed determination of the fluorescence intensity in the nucleus (inner intense green) and the cytoplasm (outer dim green). (b) The background fluorescence the DMSO vehicle was almost identical among different experiments. (c) Four stapled peptides from different batches of synthesis generated similar intracellular fluorescence intensity in different tests. Error bars represent the S.D. of two measurements.

We found that this system generated highly reproducible and reliable results from assay-to-assay and with different stocks of the same stapled peptides. As shown in Fig. 2b, there were negligible fluorescence differences among experiments for cells treated with DMSO vehicle, which could be used as a fluorescence background for all subsequent experiments. In addition, the same stapled peptides from different batches of synthesis and stocks featured almost identical intracellular fluorescence signals in different tests (Fig. 2c), indicating that the assay developed in this study produces repeatable and reliable results that could be directly combined and compared from a large set of experiments. Furthermore, to determine how this assay performs as a screening tool, we have calculated Z0 factor of 0.54 by using the most penetrant A6 peptide as a positive control and DMSO background as negative control, which also indicates a statistically good assay quality.

Analysis of cell penetration by stapled peptides The development of this quantitative high-throughput assay enabled a broad investigation of the physicochemical properties governing the cell uptake of a diverse set of hydrocarbonstapled peptides synthesized in our laboratory. We postulated that any correlation between cellular uptake and physicochemical properties would illuminate characteristics associated with productive cellular uptake and inform the future design of stapled peptides with improved cell penetration.28 To this end, we screened and analyzed more than 200 discrete FITC-labeled peptides belonging to three different classes: wild-type (unmodified), stapled and stitched peptides. All peptides were converted to two-dimensional structures and analyzed for theoretical physicochemical properties with the publicly available Marvin View software package from ChemAxon. Properties including the molecular weight, theoretical pI, calculated 2D polar surface area (PSA), theoretical log P and formal charge at pH 7.5 were calculated for each peptide (Table S1†). In general, the unmodified, stapled and stitched peptide libraries present in this screen had relatively similar physicochemical characteristics (Fig. S2†). The mean molecular weight and calculated PSA values were nearly identical among the three peptide classes. A notable difference was observed among theoretical log P values, which were significantly higher for the stapled and stitched peptides relative to the unmodified peptides, which is not surprising as these modified peptides contain a solvent exposed hydrocarbon crosslink. Additionally, the stapled peptide class had a mean formal charge of approximately zero while the stitched and unmodified peptide classes exhibited a positive mean charge. Overall, the calculated physicochemical properties indicated that the peptide classes were quite similar in terms of their mean properties, which is useful when making comparisons among their cell penetration properties.

We next performed an intracellular access screen by treating U2OS cells with 1 mM of FITC-labeled peptide for 12 hours in duplicate. All assays contained control DMSO wells and positive control peptides, which were compared among assays to ensure plate-to-plate reproducibility (Fig. 2b and c). The primary readout of the screen was mean cellular fluorescence intensity. As the DMSO background was highly consistent between wells and experiments, a mean background value was subtracted from all data. The results of the screen were used to generate plots comparing cell penetration with peptide physicochemical parameters. Interestingly, as a class, stapled and stitched peptides exhibited significantly higher cell penetration compared with wild-type unmodified peptides, which contained several established cell penetrating peptides (CPPs; Fig. 3a). Given that all three peptide classes have similar physicochemical properties in general, the benefit in cell penetration can be largely attributed to the synthetic stabilization of the a-helical peptides with all-hydrocarbon peptide stapling technology. Furthermore, we found that peptide charge near physiologic pH exhibited a strong correlation with intracellular access and could be fitted into a Gaussian distribution with a population centroid at a formal charge of +4 (Fig. 3b). In particular, peptides exhibiting a net negative charge (7 to 1) exhibited little cellular uptake, whereas peptides of approximately neutral charge (1 to +1) displayed moderate cell penetration above background. Interestingly, peptides with a net positive charge (+1 to +7) showed significantly higher cell penetration as a group. Cellular uptake did not appear to increase linearly with charge, as the cell penetration decreases dramatically for the peptides in this study with charge greater than +7. The same trend between formal charge and cellular uptake were observed for individual stapled and stitched peptide classes as well (Fig. S3†). This observation is not consistent with previously reported models that indicate that peptides/mini-proteins with more positive charge have better penetration properties due to tighter electrostatic interactions with the negatively charged phospholipid membrane.29,30 The lower penetration for highly charged peptides in this study could result from any one of many factors including, for example, peptide aggregation in solution, the disruption of peptide packing during internalization or difficulty in dissociation from cell membrane. Additional tests with a larger number of peptides could further our understanding of this phenomenon. In addition, there was no discernible correlation between cell penetration and peptide molecular weight, log P, pI value or PSA (Fig. S4†). Taken together, these data demonstrated that the staple type and peptide charge are key physical properties correlated with peptide cell penetration ability, whereas the other parameters do not appear to be significantly associated.

In order to further investigate the cell penetration properties for stapled peptides and to systematically analyze the similarities and differences in cellular uptake between stapled peptides and other wild-type cell penetrating peptides, we compared cell penetration of several stapled peptides to that of three well known wild-type CPPs: Tat (48–60), penetratin (Antennapedia 43–58) and poly-Arg8 (Table S2†). First, we investigated the cellular uptake at varied peptide concentrations. As shown in Fig. 4a and b, both wild-type CPPs and stapled peptides showed dose-dependent increases in cell penetration. Strong intracellular fluorescence was detected in the low micromolar range, and although the levels of accumulation were different for distinct peptides, stapled peptides featured more robust dosedependent cell penetration at lower concentrations relative to wild-type CPPs, in general. It is also interesting to note that while significant increases in intracellular fluorescence were mostly evident in the 1–10 mM range for stapled peptides, distinct profiles were observed for specific peptides. For example, TNG147 showed little cell penetration at 1 mM but showed a dramatic increase at 5 mM, which might suggest that concentration-dependent peptide packing or a receptor-mediated mechanism may facilitate the cell penetration process, and these processes may be triggered at different concentrations for distinct peptides. Furthermore, it is worth noting that the stapled peptides studied here were more cell permeable than wild-type CPPs at most concentrations tested, exhibiting nearly an order of magnitude higher intracellular fluorescence at the same treatment concentrations.

Fig. 4 Effects of peptide concentration and incubation time on cellular uptake of stapled and wild-type peptides. (a) Wild-type and (b) stapled peptides showed a dose-dependent increase in cell internalization. Cellular uptake for (c) penetratin and (d) SAHM1 peptides over time at concentrations of 5 and 10 mM. (e) A pulse-chase penetration assay for SAHM1 peptide in which fresh medium containing either a new batch of peptide or DMSO vehicle were exchanged at 12 hours after initial treatment. Error bars represent the S.D. of triplicate samples.

We next performed a time-course penetration assay to better understand the kinetics of peptide internalization using a representative CPP and stapled peptide. Penetratin and SAHM1 showed distinct kinetics of uptake and stabilization throughout a 24 hour time course. 5 mM and 10 mM penetratin peptide exhibited similar intracellular cellular fluorescence after 2 hours, which then decreased until approximately 8 hours and finally stabilized at different intracellular levels until 24 hours (Fig. 4c). On the other hand, the stapled peptide SAHM1 showed time- and dose-dependent cellular uptake, which stabilized after approximately 8 hours (Fig. 4d). Compared to the wild-type penetratin, the SAHM1 profile was unique in that dose-dependent accumulation was evident at all time points and no loss of signal was observed. One explanation for the loss of signal observed with penetratin could be attributed to an equilibrium between cell penetration and subsequent intracellular proteolysis followed by export of the fluorophore. The presence of the all-hydrocarbon crosslink in its peptide sequence and lower net charge of SAHM1 relative to penetratin, could contribute to enhanced cellular uptake and reduced intracellular proteolysis, leading to continuous accumulation in cells. To further explore the equilibrium observed for stapled peptides, we performed a pulse-chase experiment using SAHM1. After 12 hours of incubation with SAHM1, cell culture medium was aspirated and the cells were extensively washed with PBS to completely remove excess peptide. Then fresh medium containing either a new batch of 1 mM peptide or DMSO vehicle was added to cells and incubated for the indicated time points (Fig. 4e). As expected, the cellular uptake increased for the first 12 hours incubation. After medium exchange, cells incubated with fresh medium containing DMSO vehicle retained the intracellular fluorescence intensity. Interestingly, the signal for cells treated with a new batch of staple peptide continued to increase up to 24 hours (Fig. 4e). This observation indicates that despite incubation over a time course previously shown to reach equilibrium, the mechanism(s) responsible for cellular uptake are not saturated, as evidenced by further uptake upon replacement with fresh stapled peptide. Taken together, these data indicate that the mechanism(s) underlying cellular uptake by both CPPs and stapled peptides exhibit time- and dose-dependency that is not saturable at early time points or low micromolar doses and, importantly, appears to be more robustly utilized by stapled peptides.15,31

Given that stapled peptides exhibit better cell penetration properties in general than parent unmodified peptides, we wondered whether the peptide stapling strategy could be applied generally to improve cellular uptake of parent unmodified peptides. To test this hypothesis, we designed a panel of stapled peptides based on Tat (48–60), penetratin and poly-Arg8 (Fig. 5a). These stapled peptides and their parent unmodified peptides were incubated in U2OS cells for 12 hours with a concentration range from 10 nM to 20 mM, mirroring the dosedependent uptake studies shown in Fig. 4. As expected, all peptides showed dose-dependent cell penetration (Fig. 5b–d). Interestingly, stapled peptides derived from penetratin and poly-Arg8 showed improved cell permeability at concentrations starting from 1 mM for stapled penetratin and 5 mM for stapled poly-Arg8. It is noteworthy that the staple position also affected the cellular uptake as the two stapled penetratin peptides with different crosslink positions exhibited varied cell penetration, though both were superior to wild-type penetratin. In contrast, reduced cellular uptake was observed for both stapled peptide variants derived from the Tat sequence (Fig. 5b). This could result from several possible effects, including disruption of peptide secondary structure, masking of residues essential for surface recognition or altering peptide packing interactions involved in cell penetration. Further focused study of these variants is warranted to elucidate the source of altered cellular uptake, however these data clearly demonstrate that peptide stapling may be a general method to further improve the cell permeability of CPPs, which could serve as more efficient transduction domains for molecular cargoes. In addition, while increasing the helical content of stabilized peptides has been stated to be a guiding principle in the successful design of biologically active stapled peptides, it has not been shown to be generally correlated with cell penetration. To specifically address whether increasing the helical content of a peptide is correlated with augmented cell penetration, we have measured the relative helicity of hydrocarbon stapled variants of Tat, penetratin and poly-Arg8 (Fig. S5†). Notably, we did not observe a general correlation between increased helical character and cell penetration of these peptides. Peptide stapling increased the helical content of both Tat and poly-Arg8 peptide sequences, which were largely unstructured when unmodified. In contrast, the unmodified penetratin peptide had signicant helical content (>50%), and the hydrocarbon stapled variants of this sequence largely retained their helicity, albeit lower overall helicity. Intriguingly, these species demonstrated the differing effect of hydrocarbon stapling and increased helical content on cell penetration since introduction of the hydrocarbon staple increased the cellular uptake of both penetratin and poly-Arg8 sequences, while it decreased uptake for Tat peptides. Therefore, we cannot conclusively state, a priori, that the incorporation of a hydrocarbon staple or increased a-helicity will lead to more productive cellular penetration, although in general stapling can increase the uptake of specific sequences (Fig. 5) and as a class stapled and stitched peptides are more cell penetrant (Fig. 3a). A more comprehensive follow-up study with CD analyses on a larger peptide library is needed to better address this question.

Fig. 5 Effects of all-hydrocarbon staples on cell penetration by wild-type cell penetrating peptides. (a) List of wild-type cell penetrating peptides and their stapled derivatives investigated in this study. (b–d) Dose-dependent cell penetration assays showed that stapling strategy greatly improves the cellular uptake of penetratin and poly-Arg8 peptides. Experiments were performed in triplicate, and error bars represent S.D. of three measurements.

Mechanistic studies of cell penetration by stapled peptides The aforementioned studies indicate that stapled peptides exhibit better cellular uptake properties than wild-type peptides in general, and that internalization correlated primarily with hydrocarbon staple type and formal peptide charge. However, the mechanism(s) utilized by peptides to translocate across the cell membrane are still unclear. Therefore, we sought to investigate the uptake mechanism(s) for stapled peptides. The uptake mechanism(s) of wild-type CPPs have been extensively studied. Some evidence indicates that they enter cells via energy-dependent endocytosis, which is an active transport process, however data suggesting passive diffusion for CPPs have also been reported; hence, the mechanism(s) of cell uptake by CPPs remains ambiguous.32–34 We first sought to determine whether cell penetration by stapled peptides and wild-type CPPs occurs via ATP-dependent endocytosis.2 Cells were pre-treated with NaN3 and 2-deoxyglucose (2-DG) to reduce cellular ATP levels, and then incubated with FITC-labeled peptides (wildtype and stapled) for 4 hours and compared to normal cells for intracellular fluorescence. Cellular ATP levels were confirmed to be decreased by approximately 90% after NaN3 and 2-DG treatment (Fig. S6†), but Tat and poly-Arg8 exhibited almost identical cellular uptake in ATP-depleted and normal cells, supporting the model that they utilize passive diffusion to translocate across the cell membrane. However, penetratin and all stapled peptides showed 20–50% lower accumulation in ATP-depleted cells, indicating an active trans-membrane process requiring cellular ATP (Fig. 6a). These data indicate that there may be more than one uptake mechanism for CPPs and stapled peptides, but that for the most robust cell penetrating peptides (penetratin and stapled peptides studied here), the internalization mechanism(s) involves ATP-dependent endocytosis.

Fig. 6 Mechanistic study of cell penetration by stapled peptides and wild-type cell penetrating peptides. (a) Cellular uptake in normal and ATP-depleted cells indicated that stapled peptides penetrate cells via an ATP-dependent endocytosis. (b) Impaired uptake was observed in NaClO3 treated cells, which inhibit proteoglycan biosynthesis. (c) Cell penetration of wild-type and stapled peptides in wild-type CHO and proteoglycan-deficient CHO cells. Experiments were performed in triplicate, and error bars represent S.D. of three measurements.*P < 0.05, **P < 0.01, ***P < 0.001.

Next, we sought to identify the specific pathway(s) utilized for cellular uptake, since energy-dependent endocytosis can be accomplished by several different pathways including caveolinand clathrin-mediated endocytosis. We repeated the cell penetration experiments under a variety of conditions that each blocked a different endocytosis pathway (Table S3†).35–37 We found that uptake was partially blocked in cells treated with sodium chlorate (Fig. 6b), which aborts the decoration of cells with sulfated proteoglycans, but was unaffected by inhibitors of other endocytic pathways (Fig. S7†). It thus appears that interaction with sulfated proteoglycans is responsible for some, but not all, endocytic uptake of stapled peptides and wild-type CPPs. It is reasonable to connect this result with the previous discovery that peptide charge is a key factor determining cell penetration. Proteoglycans are negatively charged under physiologic conditions due to the occurrence of sulfate groups, and these might form electrostatic pairs with positively charged peptides to facilitate anchoring on the cell membrane.38–40 To further confirm that sulfated proteoglycans are important to mediate cellular uptake for peptides, we performed a secondary assay using wild-type CHO cells (CHO-K1) and proteoglycan deficient CHO cells (pgsA-745) which harbor a defect in xylosyltransferase, thereby preventing glycosaminoglycan biosynthesis. All peptides showed similar penetration properties in wild-type CHO cells, but uptake was decreased by approximately 50% in proteoglycan-deficient CHO cells, consistent with the experiment using a small molecule inhibitor (Fig. 6c). Taken together, our data suggest that CPPs and stapled peptides penetrate cells through a clathrin- and caveolin-independent endocytosis pathway that is in part mediated by interaction with anionic cell surface proteoglycans. This result is very similar to the previous reports on the mechanism of cellular uptake for supercharged GFP (scGFP), which likewise does not utilize clathrin- or caveolin-mediated endocytosis.41 Notably, scGFP internalization requires actin polymerization, which may not be required for peptide penetration (Fig. S7c†) types, and distinct physicochemical properties. As a result, we found that stapled peptides penetrate cells more efficiently than unmodified peptides, including well-characterized cell penetrating peptides. For the panel of peptides used in this study, only staple type and formal charge were significantly correlated with cell penetration potential, whereas the other physical parameters did not appear to have a signicant effect. We further studied the relationships between cellular uptake and

In conclusion, we sought to investigate the cell penetration properties of stapled peptides, which is one of the most significant yet poorly understood aspects of peptide stapling technology and cellular transduction technologies in general. In order to address this problem, we developed a high-throughput assay to quantitatively measure stapled peptide intracellular accumulation. Using this assay, we analyzed more than 200 discrete peptides with various sequences, staple positions and peptide concentration or incubation time, revealing that stapled peptides accumulate in cells in a dose-dependent fashion and reach steady intracellular levels over a course of a few hours. These studies revealed similar time- and dosedependent behavior for CPPs and stapled peptides, but stapled peptides, including stapled versions of CPPs, were shown to be 10- to 20-fold more penetrant, measured by intracellular fluorescence level at a given dose, than the most potent CPP. We also propose that the specific intracellular accumulation and stabilization kinetics of stapled peptides or unmodified CPPs may be a consequence of equilibria between peptide penetration, cellular proteolysis and/or retrograde transport of the species. Finally, we investigated the mechanism(s) involved in the internalization of stapled peptide and unmodified CPPs and demonstrated that cell penetration occurs through a clathrinand caveolin-independent, energy-dependent endocytosis pathway that utilizes, in part, sulfated cell surface proteoglycans. This dataset provides significant insight into the physicochemical properties correlated with productive cellular penetration as well as a more detailed understanding of the mechanism(s) utilized by stapled peptides to access intracellular compartments, which together should aid in the design of and characterization of novel stapled peptides in the future.


Rational design of protein–protein interaction inhibitors

Med. Chem. Commun., 2015,6, 51-60    DOI: http://dx.doi.org:/10.1039/C4MD00328D
Protein–protein interactions are at the heart of most physiopathological processes. As such, they have attracted considerable attention for designing drugs of the future. Although initially considered as high-value but difficult to identify, low molecular weight compounds able to selectively and potently modulate protein–protein interactions have recently reached clinical trials. Along with high-throughput screening of compound libraries, combining structural and computational approaches has boosted this formerly minor area of research into a currently tremendously active field. This review highlights the very recent developments in the rational design of protein–protein interaction inhibitors.
Graphical abstract: Rational design of protein–protein interaction inhibitors

Didier Rognan heads the Laboratory of Structural Chemogenomics at the Faculty of Pharmacy of Strasbourg (France). He studied Pharmacy at the University of Rennes (France) and did a Ph.D. in Medicinal Chemistry in Strasbourg (France) under the supervision of Prof. C.G. Wermuth. Aer a postdoctoral fellowship at the University of Tubingen (Ger- ¨ many), he moved as an Assistant Professor to the Swiss Federal Institute of Technology (ETH) until October 2000. He was then appointed Research Director at the CNRS to build a new group in Strasbourg. He is mainly interested in all aspects (method development and applications) of structurebased drug design, notably on G protein-coupled receptor ligands and protein–protein interaction inhibitors.

Introduction Drug discovery is a long, costly, multi-step endeavour which aims at reducing all possible risks to deliver a novel therapeutic solution to previously unmet clinical needs. To reduce chemical risks, empirical rules are used to filter the chemical space and retain drug-like low molecular weight compounds. Reduction of the biological risk is addressed by considering privileged target families (e.g., G protein-coupled receptors and kinases) whose activation/inhibition by drug-like compounds is likely to correct or reverse pathological states. Until recently, mostly single macromolecules (proteins and nucleic acids) have been considered as potential drug targets. Out of 68 000 proteins currently annotated in UniProt for the human proteome,1 only about 300 targets2 have been addressed by current drugs, and the large majority of single targets is still awaiting first-in class drugs.

Besides single targets, large scale genomics and proteomics3 have identified complex networks of targets and pathways regulating physiopathological processes in a coordinated manner. The current human protein–protein interactome has been estimated between 130 000 (ref. 4) and 650 000 (ref. 5) complexes, out of which only a tiny amount is known, and only a very few6–8 have been the object of a drug discovery initiative. Protein–protein interactions (PPIs) therefore describe a totally new biological space that attracts more and more attention, with 26PPI inhibitors9,10 already under clinical development, notably in the oncology field.11 Despite PPIs may adopt quite different sizes, shapes and electrostatics,12 identifying highaffinity PPI inhibitors is a considerable challenge for many reasons: (i) in contrast to conventional targets, a medicinal chemist cannot start inhibitor design from the structure of endogenous ligands, (i) PPIs often involve flat surfaces delocalized over multiple epitopes, usually lack well-defined buried cavities13 typical of conventional targets, and are significantly larger (ca. 1000–3000 A˚2 ) than enzyme/receptor pockets (300– 1000 A˚2 ), (iii) high-throughput screening of traditional compound libraries often returns no viable hits14 for the main reason that PPI inhibitor chemical space is quite different from that described by traditional drug-like compounds.10 Nonetheless, thanks to bioinformatics and proteomics-guided prioritization of therapeutically relevant protein–protein complexes, more and more PPI inhibitors are currently reported. Several excellent reviews6,7,9,11,15–18 have already been published on experimental methods (high throughput screening, biochemical and cellular assays, and fragment-based approaches) suitable to discover PPI inhibitors. The present report will only cover computer-aided approaches, with a major emphasis on structure-based methods and recent discoveries (2012–2014).

Databases Preliminary access to experimentally validated data is key to launch a drug discovery program on PPI modulators. A multitude of databases storing genomics, proteomics and structural data are currently available to help the medicinal chemist. We will here briefly review these archives, focusing mostly on easily interpretable structural data.

PPI databases Many experimental methods with different throughputs (from low to high) have been developed to characterize binary interactomes in various species, among which the most prominent has been the yeast two-hybrid (Y2H) assays, and mass-spectrometry (MS) coupled with co-immunoprecipitation or coaffinity purification.19 These experimental data are stored in many primary databases (Table 1) that are difficult to mine due to their large heterogeneity. Metadatabases have been derived thereof to facilitate their analysis, among which the most popular are APID and PRIMOS (Table 1). These metadatabases cover a wide range of organisms and notably offer the possibility to mine experimental PPI data according to disease relevance or inter-organism crosstalk, and provide graphic tools to visualize complex networks of interacting proteins and identifying important protein nodes (hubs). It is however very difficult, from this large amount of data, to clearly prioritize PPIs for a drug discovery program. Attempts to classify the PPIs by structural druggability25 (although ligandability26 is probably a better term) are worth mentioning but should be taken with care due to the still insufficient number of existing PPI three-dimensional (3D) structures.

Table 1 Protein–protein interaction databases

Database                 Interactions                         Website                                                             References

BIND                             32 211           http://bond.unleashedinformatics.com                       20

DIP                                 78 191           http://dip.doe-mbi.ucla.edu/dip/Main.cgi                  21

HPRD                           41 327            http://www.hprd.org/                                                           22

IntAct                      448 986             http://www.ebi.ac.uk/intact/                                            17

MIPS                              9 835             http://mips.helmholtz-muenchen.de/proj/ppi/      23

APID                         196 700             http://bioinfow.dep.usal.es/apid/index.htm             24

PRIMOS                  384 127             http://primos.fh-hagenberg.at/                                        19



Table 2 Database of low molecular-weight PPI inhibitors
Database                 Ligands                             Website                                                                  References

2P2I                                 71                   http://2p2idb.cnrs-mrs.fr/                                                12

iPPI-DB                      1650               http://www.ippidb.cdithem.fr/                                        10

TIMBAL                      6896              http://mordred.bioc.cam.ac.uk/ timbal                        29

Ligand databases Initially limited to a limited subset of inhibitors able to disrupt few PPIs (e.g. p53/MDM2, Bcl-Xl/Bak, and IL-2/IL-2Ra),7,27 the repertoire of PPI inhibitors rises constantly thanks to exciting developments in biophysical fragment screening.15,28 Three publicly available databases storing information on PPIs and their inhibitors (Table 2) may be used to better describe the structural properties of druggable PPIs and the chemical space associated with their disruptors.

The 2P2Idb database12 is a hand-curated repository of protein–protein complexes of known X-ray structures (X-ray diffraction and nuclear magnetic resonance spectroscopy) for which at least one low molecular weight orthosteric inhibitor has been co-crystallized with one of the two protein partners. It currently describes 71 inhibitors for 14 PPIs, clustered in two groups (Fig. 1) with respect to the nature of the interface (protein–peptide and protein–protein). Companion tools (2P2I inspector,30 2P2I score,30 and 2P2I hunter31) are provided to analyse PPIs at a structural level, predict their structural druggability and design PPI focussed libraries, respectively.

Fig. 1 Prototypical examples of class I (left panel) and class II PPIs (right panel), exemplified by the Bcl-Xl/Bak (PDB id 1BXL) and integrase/LEDGF (PDB id 2B4J) complexes, respectively. Class I PPIs involve the interaction of a globular protein with a peptide or a single secondary structure (a-helix and b-strand) of a second protein partner. Class II PPIs are characterized by the interaction of two globular proteins.

The iPPI-DB10 is another manually curated database from world patents and the medicinal chemistry literature, focussing on low molecular weight orthosteric inhibitors, disease-related protein–protein interfaces and a clear biochemical readout (e.g. fluorescence polarisation and enzyme-linked immunosorbent assay). The database archives 1650 PPI inhibitors targeting 13 families of homologous PPI targets mainly involved in cancer, immune disorders and infectious diseases.

Finally, the TIMBAL database29 reports ca. 7000 inhibitors for 50 known PPIs. In contrast to the two other databases, TIMBAL is maintained through a predefined list of PPIs and automated searches in ChEMBL32 and the Protein Data Bank.33 In contrast to the other databases, TIMBAL also registers short peptides with an upper molecular weight limit of 1200 Da. It should be pointed that most of the 15 000 uncurated biological data present in TIMBAL arise from a single target family (integrins) and should be considered with care.

Analysing the content of these databases enables a first comparison of PPI inhibitors versus drugs, as well as PPIs amenable to disruption versus standard heterodimers. PPI surfaces disrupted by inhibitors tend to be smaller, more hydrophobic and accessible than standard heterodimers.12 As a consequence, low molecular weight PPI inhibitors tend to be larger, more hydrophobic and more aromatic-rich than standard drugs. Interestingly, many of them (ca. 60%) still comply with Lipinski’s rule-of-five, 10 revealing some hopes in the developability of such compounds.

However, it should be stated that the set of empirical rules designed to discriminate druggable from non-druggable PPIs, as well as to distinguish PPI inhibitors from conventional druglike compounds still rely on a very limited set of highly homologous data (PPIs, inhibitors), and should therefore be regarded with caution. Increasing coverage of the PPI repertoire by future experimental screens will undoubtedly lead to a better denition of PPI biological and chemical spaces. We therefore expect in the future the above-mentioned rules to be rened and be more descriptive of the true world of PPI inhibitors, notably with respect to rational design of PPI focussed libraries.


Rational design of PPI modulators

Sequence-based approaches Whatever the nature of the PPI (type I or type II, see the definition above), PPI interfaces are often characterized by the presence of hotspots,34 in other words anchor residues that contribute the most to the binding free energy of the protein– protein complex. The interaction of a single modified amino acid with a single anchor residue might be sufficient to disrupt a PPI as elegantly demonstrated by Lin et al. in a recent study.35 Capitalizing on the presence of a reactive cysteine (C246) at the interface of the complex between caspase-7 (CASP7) and the Xlinked inhibitor of apoptosis protein (XIAP), they designed the N-iodoacetyl-lysine amino acid derivative 1 (Fig. 2) that covalently traps C246 and further disrupts the XIAP–CASP7 complex, therefore triggering CASP7-dependent apoptosis and killing MCF-7 breast cancer cells (EC50 ¼ 0.64 mM) previously resistant to chemotherapy.

The easiest way to inhibit a PPI is to start with the amino acid sequence of one interacting epitope, notably if the latter is part of regular secondary structures (a-helix, b-strand, and b-turn). For example, a-helical peptides mimicking the sequence of protein transmembrane domains may disrupt PPIs quite efficiently.36,37

Fig. 2 Peptidomimetics as PPI disruptors


Due to poor pharmacokinetic profiles, linear peptides are good in vitro tools but usually not efficient clinical candidates. Chemical modifications are required to stabilize their secondary structures in physiological media and prevent early degradation. Among the most exciting developments in this area38,39 is the design of stapled peptides.40,41 Stapled peptides are synthetic analogues of a-helical protein epitopes involved in a PPI, and in which a covalent hydrocarbon linkage connects adjacent turns of the helix. Stapling is known to significantly increase the in vivo half-life of the natural peptide (increasing proteolytic stability), decrease the entropic cost of binding, and even enable cellular uptake.42 Many stapled peptides with potent in vivo activities have already been reported.39 One of these stapled peptides (ATSP-7041, compound 2, Fig. 2) just entered clinical development as a dual nM MDM2/MDMX inhibitor for p53-dependent cancer therapy.43

Heterocyclic scaffolds mimicking secondary structures can also be obtained by solution-phase synthesis to afford peptidomimetic libraries amenable to PPI inhibition. Whitby et al. notably reported the design of 8000 member 4-acetamido-3- alkoxy-benzamide focused library featuring weak p53/MDM2 inhibitors and potent HIV-1/gp41 inhibition (compound 3, Fig. 2).44 When the peptide epitope is not structured, developing macrocyclic analogues is more difficult but still feasible as recently demonstrated by Glas et al.38 who successfully improved 14-3-3 binding of a 11-mer peptide from a bacterial ExoS virulence factor by cross-linking binding amino acids with polymethylene linkers, up to an in vitro 40 nM disruptor of the ExoS/14-3-3 interaction (compound 4, Fig. 2). Interestingly, the cross-linker was not only chosen to rigidify the natural ExoS peptide structure but also to directly provide additional hydrophobic interactions to the 14-3-3 binding site.38 Only in exceptional cases the natural unmodified peptide is directly usable as a PPI inhibitor. One recent example is the 28 amino acid cell-penetrating peptide (p28) from a bacterial azurin redox protein, that binds to the DNA-binding domain of the p53 tumor suppressor and inhibits p53 degradation by interfering with the Cop1-mediated ubiquitination,45 thereby enhancing p53 levels in cancer cells and exhibiting antitumoral efficacy in patients with advanced solid tumors.46

Pharmacophore-based approaches As defined by the IUPAC,47 a pharmacophore is “an ensemble of steric and electronic features that are necessary to ensure the optimal supramolecular interactions with a specific biological target and to trigger (or block) its biological response.” Although pharmacophores are mainly used to align and compare ligands sharing the same target,48 the same concept can be easily transferred to PPIs in which one partner is the “receptor” and the second one the “ligand”. Pharmacophore features (hydrophobic, aromatic, H-bond donor and H-bond acceptor, positively and negatively ionisable) can therefore be manually or automatically mapped to atoms of the ligand in direct interactions with the receptor. The resulting pharmacophore can then be used to identify a compound library for hits fulfilling the defined query. Several tools (e.g. LigandScout,49 Discovery Studio,50 and Pocket Query51) can be directly used to map PPI pharmacophores onto protein–protein X-ray structures (Fig. 3).

Fig. 3 Example of a PPI pharmacophore mapped onto interacting atoms of human LEDGF (yellow ribbons) bound to HIV-1 integrase (red ribbons, PDB ID 2B4J). The PPI pharmacophore is composed of 2 Hbond donors (magenta balls), two H-bond acceptors (green balls), one hydrophobic feature (cyan ball) and 6 exclusion volumes (gray balls).

Using a manual PPI pharmacophore defined from the X-ray structure of the Annexin A2/S100A10 complex, a pro-angiogenic complex, Reddy et al.52 derived a simple pharmacophore (2 hydrophobes, 2 H-bond donors, and 2 H-bond acceptors) using the Unity program,53 and screened a library of 700 000 compounds to select 586 hits which were further docked to the Annexin A2 binding site to retain only 190 candidates with both a good docking and pharmacophore fitness score (Table 3). Out of 190 tested compounds, 7 hits blocked the interaction between S100A10 and the Annexin A2 N-terminus in a competitive fluorescent binding assay, with the most potent PPI inhibitor (compound 5, Fig. 4) exhibiting an IC50 of 24 mM.52 Geppert et al.54 reported the rational discovery of a low molecular weight inhibitor of the complex between interferon-a (IFN-a) and its receptor (IFNAR2). Fortunately, the PPI interface was small enough (ca. 800 A˚2 ) to be targeted by a small heterocyclic compound. After identifying major hotspots at the IFN-a surface, a fuzzy receptor-based pharmacophore was determined using the VirtualLigand approach,55 which assigns pharmacophoric features to Gaussian densities. Screening a collection of 556 000 commercially available compounds retained six virtual hits, out of which two were weak IFN-a inhibitors, but one (compound 6, Fig. 4) was confirmed by NMR and surface plasmon resonance (SPR) to bind to IFN-a with a dissociation constant (Kd) of 4 mM and to inhibit IFN-a responses in various cell assays. The novel inhibitor may be useful to reduce IFN-a titers in autoimmune disorders.


Table 3 Protein–protein pharmacophore searches to identify PPI inhibitors

Target                                     Library size                          Tested                        Hits                      Ref.

Annexin A2/S100A        10 700 000                              190                               7                          52

INFAR2/IFN-a                       556 000                                   6                               3                          54

p53/MDM2                                  21 287                                  15                               6                          56

Nrf2/Keap1                                   21 199                                  17                                1                          57

PKC3/RACK2                          330 000                                 19                                1                          58


Due to the inherent complexity of PPI pharmacophores (many features covering a large surface), combining several pharmacophores into a consensus model may help to retrieve essential features and simplify pharmacophore queries. Xue et al. applied this approach to the identification of p53–MDM2 inhibitors.56 The p53–MDM2 complex has become a prototypical PPI for its biological background (this interaction plays an important role in regulating the transcriptional activity of tumour cells) and many high affinity low molecular-weight inhibitors of this PPI identified by various screening approaches.59 Starting from a set of 15 MDM2-peptide X-ray structures, a common feature structure-based pharmacophore (2 H-bond donors, one H-bond acceptor, 2 aromatic rings, and one hydrophobe) was first identified. In addition, a receptorligand pharmacophore (five hydrophobes, one aromatic, and one H-bond donor) was generated from a separate set of 10 MDM2-non peptide complexes. Merging both pharmacophores and retaining the most common features led to an ensemble pharmacophore definition (two aromatic rings, two hydrophobes, and one H-bond donor) taking into account both peptide and non-peptide binding. This pharmacophore was used to screen a collection of 21 287 commercially available compounds, and led to a hit list of 15 compounds out of which 6 were confirmed as p53–MDM2 inhibitors using an in vitro uorescence polarization assay.56 The most potent inhibitor (compound 7, Fig. 4) is a 180 nM MDM2 inhibitor. Despite a good selectivity in a MTT tumour cell proliferation assay (p53+/+ vs. p53/ cells), compound 7 was a weak inhibitor (IC50 ¼ 85 mM) of tumour cell growth, because of poor pharmacokinetic properties.

Fig. 4 PPI inhibitors identified by pharmacophore-based virtual screening.

Along the same lines, two X-ray structures were used to derive inhibitors of the PPI between Keap1 and Nrf2, a complex involved in the response to oxidative stress.57 The two PPI pharmacophores were merged into a single query consisting of one H-bond donor, two H-bond acceptors and three negative ionisable centers. To afford some fuzziness in the search, up to two features were allowed to be missed by virtual hits. Since the Keap1-binding epitope of Nrf2 is composed of several acidic residues, only compounds bearing a negative charge were searched among a full commercial library of 251 774 compounds. The remaining 21 199 hit list was matched to the pharmacophore, and led after confirmation with docking and MM-PBSA scoring, to a list of 17 potential hits which were tested for Keap1–Nrf2 inhibition using an in vitro fluorescence polarization assay. A single compound (compound 9, Fig. 3) was confirmed in vitro as a moderately potent Keap1–Nrf2 inhibitor with an EC50 of 9.8 mM.57 Interestingly, the inhibitor activated the Nrf2 transcriptional activity .

When both protein partners involved in the PPI have not been co-crystallized, it is still possible to rationally discover PPI inhibitors, starting from the sole X-ray structure of one of the two proteins. This approach was followed by Rechfeld et al. in the discovery of PKC3–RACK2 inhibitors.58 Starting from the Xray structure of the PKC3 octameric epitope binding to RACK2 (a receptor for activated protein kinase C), a simple peptide-based pharmacophore model (3 H-bond donor/acceptor, one hydrophobe) was defined and used to screen a collection of 330 000 compounds. Out of 19 virtual hits, a thienoquinoline was found to disrupt the PPI in vitro and served as a query for a secondary screen for chemically similar analogues, which led to compound 8 (Fig. 4) as a micromolar potent PKC3-RACK2 inhibitor (IC50 ¼ 5.9 mM) which also inhibited PKC3 downstream signalling, HeLa cancer cell migration and invasion.58

Finally, pharmacophore searches may be used to prioritize privileged scaffolds for synthesizing PPI-focused libraries. For example, Fry et al. reported a rational approach to PPI library design targeting a-helical binding epitopes.60 Starting from the known X-ray structure of an a-helical p53 epitope binding to MDM2, a three point pharmacophore, featuring the three important hydrophobic side chains (Phe19, Trp23, and Leu26) of the p53 peptide, was designed and used to find heterocyclic scaffolds among the CSD database61 of small molecule X-ray structures. Several small-sized libraries (ca. 100 members) were synthesized from each hit and tested for general inhibition of PPIs involving an a-helical epitope (e.g. MDM2, BCL2, BCL-XL, and MCL1). Although no potent hit could be discovered, the average hit rate was far superior (4%) to what should be expected from a random screen. Moreover, many starting hits exhibited good ligand efficiencies,60 and are therefore interesting starting points for hit leading optimization.

Despite its apparent simplicity, PPI-based pharmacophore search is a fast, cost-effective and simple in silico approach to discover the very first inhibitors of a particular PPI. Of course, all successful examples mentioned above imply that the PPI is of manageable size and does not involve a too large and complex binding epitope. Beside the existence of a X-ray or NMR structure of the protein–protein (peptide) complex, it is therefore equally important to properly select PPIs amenable to pharmacophore-based searches, notably with respect to the complexity of the query (5–6 features) and its hydrophobic/ hydrophilic balance.

Docking-based approaches At the first sight, protein–ligand docking should be considered as the most intuitive and logical computational tool to predict likely ligands of any target of known 3D structures.62 Unfortunately, severe drawbacks associated with the scoring of protein– ligand interactions render that tool usually suitable for positioning a ligand into a binding site, but rarely to predict binding free energies or to precisely rank ligands by decreasing affinity.63 Moreover, the ability of docking algorithms to anchor ligands to flat PPI surfaces has long remained elusive. In a benchmark study, Kruger ¨ et al. used two popular docking tools (AutoDock and Glide) to reproduce the known X-ray structure of PPI inhibitors to their target.64 Surprisingly, the performance of these standard docking programs with respect to the positioning of the ligand (rmsd to the X-ray structure) was only moderately affected by switching from conventional targets to PPIs. Although PPI inhibitors with more than 10 rotatable bonds were found more difficult to properly dock, a good pose was generated in ca. 54% of the 80 PPI inhibitors considered. Docking to PPIs providing at least one charge residue was favoured over those purely hydrophobic.64 There are therefore no particular reasons to discard docking-based approaches from rational PPI inhibitor discovery scenarios. Many of the following success stories support this assumption.

We will not here review the many recent reports describing docking as a mean to predict the binding mode of a PPI inhibitor discovered by an experimental screening method.59,65–68 The next section will only focus on inhibitors discovered by a docking-based virtual screening campaign (Table 4).


Table 4 Protein–protein inhibitors discovered by docking-based screening

Target                   Library size                                     Tested                             Hits                   Ref.

TLR4/MD-           2 50 000                                           14                                      3                      69

uPA–uPAR      5 000 000                                            50                                      3                     70

IL-6/gp130                          9                                                2                                     2                     71

Keap1–Nrf2            153 611                                              65                                     9                     72

CRYAB/VEGF       139 735                                             40                                     4                    73

NRP-1/VEGF-        429 623                                        1317                                   56                   74

PPxY/Nedd4       4 800 000                                          20                                       1                  75

p53/MDM2                  87 430                                        295                                       1                 76


Despite an apparent unsuitable large and concave cavity, the MD-2-binding site at the surface of the toll-like receptor 4 (TLR4) was selected for pharmacophore-constrained FlexX77 docking of a library of 49 600 compounds pre-filtered for 3D shape similarity to an existing TLR4 antagonist.69 40 virtual hits were selected for in vitro TLR4 binding and functional antagonism, and 3 of them could be confirmed experimentally. The most potent antagonist (compound 10, Fig. 5) blocked TLR4 in a gene receptor assay with an IC50 of 16.6 mM and inhibited proinflammatory cytokine release (e.g. TNF-a) from human peripheral blood mononuclear cells upon LPS activation. Due to unfavourable aqueous solubility, the compound could not be tested in vivo but represent a good starting hit for developing small molecule TLR4 antagonists for the treatment of neuropathic pain and sepsis.

Fig. 5 PPI inhibitors identified by docking-based virtual screening

To account for the conformational flexibility of proteins, Khanna et al. reported a cascade docking-based virtual screening for discovering inhibitors of the interaction between the urokinase-type plasminogen activator (uPA) and the urokinase receptor (uPAR).70 Two X-ray structures of the uPAR were first used for docking a collection of 5 million commercially available compounds using AutoDock4.78 10 000 top-ranked virtual hits were further docked, still with AutoDock, to 50 molecular dynamics snapshots of the uPAR structure, leading to 500 top-ranked compounds which, in a third step, were docked using a different program (Glide) on the 50 receptor conformers. After clustering the top 250 compounds by chemical similarity, the highest scoring compounds from each of the top 50 clusters were finally selected, purchased and evaluated in vitro in a fluorescence polarization assay. Among the three validated hits, the most potent inhibitor (compound 11, Fig. 5) binds to uPRA with a submicromolar affinity (Kd ¼ 310 nM) and inhibits the uPA–uPAR interaction with an IC50 of 10 mM.70 The hit blocked invasion of breast cancer cells but not their migration or adhesion. A close analogue of compound 11 was recently shown to be efficient in an in vivo breast cancer metastasis assay.79

Docking is not limited to the study of single protein–ligand interactions. In an elegant study, Li et al. reports a computational method enabling the simultaneous docking of multiple fragments to a single binding site, by analogy to experimental fragment screening.71 When applied to the PPI between IL-6 and gp130, simultaneous docking of two fragment pools (6 and 3 fragments, respectively) targeting two different hotspots at the PPI, two theoretical ligands could be reconstructed after tethering the best fragments at each hotspot. Searching for known drugs80 which are chemically similar to the two virtual hits suggested than two estrogen receptor modulators (raloxifene and bazedoxifene) may bind to the gp130/IL-6 PPI. Effective binding of both drugs to gp130 was confirmed experimentally, as well as inhibition of IL-6 induced STAT3 phosphorylation in various cancer cell lines defective in estrogen receptor expression. Bazedoxifene (compound 12, Fig. 5) was the most efficient (IC50 ¼ 25 mM) in inhibiting the ER-independent IL6-induced breast cancer cell proliferation, thereby offering some repositioning potential in the treatment of IL-6/gp130/STAT3 dependent tumours.71

The Nrf2–Keap1 complex, previously investigated using a pharmacophore-based approach (see the previous section), was also used for docking 300 000 commercially available compounds with the program Glide. Among the chemically diverse 65 top-ranking hits, 9 compounds were confirmed to be PPI inhibitors, the most potent disruptor (compound 13, Fig. 5) exhibiting a Kd of 2.9 mM in a fluorescence anisotropy-based assay.

A major hurdle in PPI inhibitor development is the frequently objected high molecular weight and unfavourable pharmacokinetic properties. Chen et al. strikingly contradicted this dogma by reporting a very low molecular weight inhibitor of the aB-crystallin (CRYAB)/VEGF-A interaction.73 CRYAB is a protein overexpressed in triple-negative breast cancer cells that acts as a chaperone to several proteins including the proangiogenic vascular endothelial growth factor (VEGF). Disrupting the interaction between CRYAB and VEGF-A is therefore a potential approach to cancer cell proliferation and invasion. The VEGF-binding site on the surface of the CRYAB X-ray structure was therefore targeted by docking 140 000 compounds from the NCI database using the Dock6.5 program (UCSF). Despite a very modest molecular weight (161.16 Da), one compound (compound 14, Fig. 5) was identified as an in vitro disruptor of the CRYAB/VEGF-A interface with an IC50 of ca. 20 mM. Intraperitoneal injection of compound 14 (200 mg kg1 ) remarkably suppresses tumour growth in vivo in human breast cancer xenograph models. VEGF-A is an important angiogenic factor that interacts with many other partners, notably the family of neuropilin receptors (NRP-1, NRP-2) whose inhibition leads to cancer cell apoptosis. The PPI between the C-terminal end of VEGF-A165 and the tandem b1 and b2 domains of NRP-1 was targeted for docking 430 000 molecules with a consensus docking approach relying on two docking programs (SurflexDock81 and ICM82). A consensus list of 1317 top-scoring compounds was retained for their in vitro anti-proliferative activity and binding to NRP-1 using a chemiluminescent assay.74 56 molecules (hit rate of 4.2%) antagonized the NRP-1/ VEGF-A interaction by at least 30% at the concentration of 10 mM. The best hit (compound 15, Fig. 5) is the first non-peptide NRP-1/VEGF-A antagonist (IC50 ¼ 34 mM) and displays remarkable anti-proliferative effects (IC50 ¼ 0.2 mM) on breast cancer cells. Administered at the dose of 50 mg kg1 in NOG xenographed mice, compound 15 strongly inhibits tumour growth inhibition by inducing cell apoptosis, without any effect on pro-angiogenic kinases.

Although most of the above reported therapeutical indications remain in the oncology field, PPI inhibitors have clear potential in other areas, notably infectious diseases as recently demonstrated by Han et al.75 who reported the structure-based discovery of antiviral compounds inhibiting viral–host interactions. The PPI target is the complex between the conserved Ldomain PPxY sequence of several viral matrix proteins (e.g. Ebola, Marburg, Lassa fever, and VSV) and the ubiquitin ligase Nedd4 protein. Docking ca. 5 million compounds (ZINC database)83 on the Nedd4 X-ray structure with the AutoDock4 program, yielded to the evaluation of 20 compounds, out of which one molecule was confirmed as a true inhibitor of the PPI in a cellular assay. Acquiring close analogs of the initial hit led to two more potent inhibitors (compounds 16 and 17, Fig. 5) as submicromolar inhibitors of the PPxY–Nedd4 interaction in vitro. 75 Both compounds exhibit antibudding activity against Ebola, Lassa fever, Marburg and VSV viruses, thereby decreasing viral titers, without apparent cytotoxicity on HEK293T cells.

Natural compounds are also a major source of potentially interesting PPI inhibitors. By docking a library of commercially available compounds to the p53 binding site, Vogel et al. recently reported lithocholic acid (compound 18, Fig. 5), a secondary bile acid, as a weak binder (Kd of 15 mM) to MDM4 and MDM2 proteins with a slight preference for MDM4.76 The natural compound was further shown to inhibit p53–MDM4 interactions and promote apoptosis in a p53-dependent manner by inducting caspase3/7.


We should acknowledge that peptides usually remain a good starting point to derive PPI inhibitors. Given the increasing number of high resolution X-ray structures of biologically relevant protein–protein complexes, the number of potentially increasing PPIs is likely to significantly rise in the next years. Provided that molecular rules exist to prioritize the most interesting anchoring residues at the interface, continuous protein epitopes can be easily converted into linear peptides for quick experimental validation. Recent progress in peptide stabilisation by chemical stapling next opens an immense eld for deriving either pharmacological tools or drug candidates. Numerous successes in identifying non-peptide PPI inhibitors also exist. The present review has only considered inhibitors mostly discovered by a rational structure-based virtual screening approach. Despite the few cases described herein (15 in total), examples are pretty much indicative of results than can be reasonably achieved. Comparing the properties of PPIs (Fig. 6A and B) and their inhibitors (Fig. 6C) with previously reported larger PPI data,64 some trends could be verified. Considering success as the availability of low micromolar nonpeptide inhibitors, successfully targeted PPIs present a higher proportion of charged residues with respect to conventional targets (sc-PDB data).84 Unsurprisingly, PPI inhibitors bind to smaller cavities (200–350 A˚3 ) than that presented by conventional targets (450–800 A˚3 range). Consequently, PPI inhibitors present a high proportion of aromatic rings, amide moieties and charged groups (Fig. 4 and 5) that hamper their druggability potential, as estimated here by the QED metric85 (Fig. 6C). We notice a significant proportion of negatively charged compounds, suggesting that a strong electrostatic interaction with the target is often mandatory to reach detectable affinity to PPI-participating cavities.

Fig. 6 Properties of PPIs and their inhibitors: (A) cavity properties expressed in percentage according to the cavity detection VolSite program86 (Hydro, hydrophobic; Aro, aromatic; H-bond, H-bond accepting/donating properties; Neg: negatively charged; Pos, positively charged, Du: fully accessible); (B) cavity volumes targeted by PPI inhibitors (this review) and conventional ligands (sc-PDB data84). The box delimits the 25th and 75th percentiles, and the whiskers delimit the 5th and 95th percentiles. The median and mean values are indicated by a horizontal line and an empty square in the box; (C) quantitative estimate of druggability (QED)85 of the inhibitors. QED values for true drug-like compounds should be over 0.5 (red broken line).

However, the current survey also indicates that there is no absolute dogma with respect to PPI inhibitor identification. Very low molecular weight compounds (compounds 1, 6 and 14) have been successfully identified as PPI disruptors.

Beside interfacial inhibitors, there exist promising alternative ways of inhibiting PPIs. For example, PPI stabilizers87,88 (e.g. paclitaxel, rapmycine, and forskolin) bind to rim exposed pockets at or very close to the interface, and also lead to the functional inactivation of the protein–protein complex. Such stabilizers are frequent in the nature, and this area still has not been fully exploited until now. Likewise, the allosteric inhibition of PPIs, at pockets remote from the interface, clearly deserves some consideration. Such pockets have been shown to be frequent at the close vicinity of two protein chains in close interaction,89 and represent, at least for some of them, more ligandable pockets than those presented by PPIs.

Although dominated by a continent of flat and featureless interfaces, the PPI world is also populated by very different islands in terms of shape and electrostatics that should not been discarded. Many factors are likely to increase our knowledge of PPIs and their inhibitors among which: (i) the increasing number of biologically relevant and crystallized protein–protein complexes, (ii) the development of label-free experimental screening techniques, and (iii) the significant contribution of molecular simulations to detect transient interfaces. Medicinal chemistry will be a key factor to transform moderately potent PPI inhibitor hits into clinical candidates with desired pharmacokinetic properties.


1 http://www.uniprot.org/uniprot/
?query¼organism% 3A9606+AND+keyword:%22Complete+proteome+[KW- 0181]%22, (accessed 17/07/2014).

2 J. P. Overington, B. Al-Lazikani and A. L. Hopkins, Nat. Rev. Drug Discovery, 2006, 5, 993–996.

3 P. Legrain and J. C. Rain, J. Proteomics, 2014, 107, 93–97.

4 K. Venkatesan, J. F. Rual, A. Vazquez, U. Stelzl, I. Lemmens, T. Hirozane-Kishikawa, T. Hao, M. Zenkner, X. Xin, K. I. Goh, M. A. Yildirim, N. Simonis, K. Heinzmann, ….A. L. Barabasi and M. Vidal, Nat. Methods, 2009, 6, 83–90.

5 M. P. Stumpf, T. Thorne, E. de Silva, R. Stewart, H. J. An, M. Lappe and C. Wiuf, Proc. Natl. Acad. Sci. U. S. A., 2008, 105, 6959–6964




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Proteins, Imaging and Therapeutics

Larry H Bernstein, MD, FCAP, Curator



Dissecting the Structure of Membrane Proteins

Kathy Liszewski

  • EM for Structural Analysis

Electron microscopy (EM) not only provides a straightforward approach to scrutinize the ultrastructure of cells and tissues, but it is also gaining momentum as a means to derive structural information on membrane proteins.

According to Bridget Carragher, Ph.D., co-director, Simons Electron Microscopy Center, New York Structural Biology Center, “EM is a widely applied technique to study the structure of proteins and membranes, but it is still less common than X-ray diffraction of prepared crystals. However, crystallization of membrane proteins has been particularly challenging. Since EM does not require obtaining crystals, it is becoming an increasingly used tool for performing structural analysis of membrane proteins and their complexes.”

As an example, Dr. Carragher described the use of single particle EM to directly visualize the conformational spectra of two homologous ATP-binding cassette (ABC) exporters. Single particle EM determines structure from multiple images of individual particles and uses methods like multivariate statistical analysis to separate heterogeneous particles into homogeneous classes.

“ABC transporters constitute a large family of membrane proteins that use the energy of ATP hydrolysis to translocate (either export or import) substances such as nutrients, lipids, and ions across the lipid bilayers,” said Dr. Carragher. “They are medically important because they also transport drugs and contribute to antibiotic or antifungal resistance.

“In a collaborative study, we utilized an unbiased approach employing newly developed amphiphiles in complex with lipids to create a membrane-mimicking environment for stabilizing membrane proteins. Visualization of the complexes using single particle EM analysis revealed striking conformational differences between the two transporters with respect to the effect of binding nucleotides and substrates. Overall, these studies provided a comprehensive view of the conformational flexibility of these two ABC exporters.”

As improvements continue to be made in the technology, resolution is nearing the 3 to 5 angstrom range, at least for some proteins and protein complexes.

“EM is becoming competitive with X-ray diffraction for solving some protein structures. It is not likely to replace other techniques, but rather will be complementary to them,” she added.

  • Bacterial Membrane Dynamics

reengineered nanopore


Structural model of a re-engineered nanopore
[Lukas Tamm, Ph.D., University of Virginia]

 The outer membranes of gram negative bacteria, such as Pseudomonas and E. coli, consist of multiple proteins and densely packed lipopolysaccharides (LPS or endotoxin). This structure provides a formidable barrier to antibiotics, most of which are targeted to intracellular processes.

  • “Understanding outer membrane structure and how molecules are recognized and transported across the bacterial membrane are critical to creating more effective antibiotics,” noted Lukas K. Tamm, Ph.D., professor molecular physiology and biological physics, University of Virginia.
  • The Tamm laboratory studies the dynamics of membrane proteins especially via solution NMR spectroscopy. His laboratory provided the first structure of the outer membrane ion channel of E. coli, OmpA. The group also studies OmpG, an outer membrane protein whose single polypeptide chain forms a membrane nanopore.
  • “Engineered protein nanopores have attracted interest to detect rare metal ions and neurotransmitters in solution, to sequence DNA and RNA, and to measure folding and unfolding kinetics of single proteins,” he explained. “We developed a new approach to loop immobilization that revealed cross-talk patterns between different loops of the OmpG nanopore. This will be useful to engineer new functions into OmpG and for analyzing other membrane nanopores.”
  • Dr. Tamm also studies the outer membrane protein H (OprH) from Pseudomonas aeruginosa, a multidrug resistant pathogen that is the most common cause of pneumonia and mortality in cystic fibrosis patients. It is the major cause of hospital-acquired infections.
  • “The impermeability of this pathogen’s outer membrane contributes substantially to its notorious antibiotic resistance. We utilized in vivo and in vitro assays that demonstrated the importance of the interaction of OprH with LPS in the outer membrane. Additionally, beyond determining the structure of OprH, our studies revealed that solution NMR can be a powerful tool for investigating interaction of integral membrane proteins with specific lipids. This cannot be easily done by crystallography.”
  • Dr. Tamm explained that there are many challenges remaining before antibiotic resistance can be overcome.
  • “The substrate is unknown for many of the outer membrane proteins. To develop better targeted antibiotics, it will be important to define specific substrates. Also, determining the structure of outer membrane proteins will likely also provide new insights for understanding how protein-lipid interactions contribute to antibiotic resistance. We aren’t there yet, but we are close to getting better answers.”
  • Membrane proteins, such as receptors, ion channels, and transporters, comprise nearly 30% of all proteins in eukaryotic cells. They also constitute more than 50% of all drug targets.

Yet, membrane proteins continue to present considerable challenges to the field of structural biology. Their surface is relatively hydrophobic, usually requiring potentially harmful detergent solubilization. Conformational flexibility and instability also may create roadblocks for the expression and purification required for structural analysis.

The recent Argonne National Laboratory Conference on Membrane Protein Structures highlighted advances in the field such as use of smaller and more intense beams for X-ray micro-crystallography, novel protein engineering of fusion proteins for structure determination, nanodiscs that mimic native cell environments, visualization strategies employing single particle electron microscopy, and bacterial nanopore studies that may help surmount antibiotic resistance.

  • X-Ray Micro-Crystallography

membrane proteins structure


Schematic view of the planned upgrade of the GM/CA beamline 23-ID-D at the Advanced Photon Source (APS) at Argonne National Laboratory. Top panel: cartoon of the X-ray optics to focus the beam. Bottom panel: elevation view of the endstation focusing optics, sample goniometry, and detector. The beam line upgrade will reduce the minimum beam size from 5 µm to 1 µm in the near future. The proposed APS-MBA upgrade will allow the beam to be focused to <500 nm with a 100-fold increase in intensity. The small, high intensity X-ray beam will enable structure determination for some of the most challenging problems in structural biology.


  • Many physiological processes are controlled and regulated by conformational changes in GPCRs and other integral membrane proteins. “We are studying at the atomic level how allosteric changes in such proteins regulate cell signaling,” explained Daniel M. Rosenbaum, Ph.D., assistant professor, biophysics, biochemistry, University of Texas Southwestern Medical Center.X-ray crystallography has been a workhorse technology for structural biologists for many years. Scientists generate a minute crystal by carefully optimizing conditions, shoot a high-powered X-ray beam at it, measure the angle and intensity of the diffracted beams, and derive a complete or partial structure by analyzing the results with sophisticated analytical programs.
  • “Membrane proteins are notoriously difficult to crystallize, and often yield very small, weakly diffracting, radiation-sensitive crystals that are intractable to large-beam crystallography. However, high-resolution structures can be obtained by using a micro-beam,” noted Robert F. Fischetti, Ph.D., associate division director and group leader, X-ray Science Division, Argonne National Laboratory.
  • Dr. Fischetti said the Advanced Photon Source (APS), a DOE user facility at Argonne, leads the field in deriving membrane protein structures.
  • “G-Protein Coupled Receptors (GPCRs) are one very important class of membrane proteins. There are more than 800 GPCRs, and over 40% of all drugs target them. Of the 30 known protein structures, 21 were solved at the APS.”
  • According to Dr. Fischetti, a number of key improvements and innovative approaches are needed.
  • “Stability of the beam intensity and the relative alignment of the beam and crystal are paramount in micro-crystallography. One problem is that X-ray beams cause both primary and secondary (diffusional) structural damage to the crystal. To overcome that issue smaller, hotter beams and more rapid detectors are being used in the race against radiation damage.”
  • Dr. Fischetti said the field is also seeing the emergence of breakthrough techniques, including novel sample delivery systems such as the acoustic drop and microfluidic technologies. Further, throughput is advancing.
  • “We’re approaching the ability to perform data collection on many thousands of microcrystals complexed to a variety of compounds. This is enabling pharmaceutical applications.”
  • One of the most exciting changes at APS and throughout the scientific community is the development of a new storage ring magnet lattice design, the multibend achromat (MBA). The technology promises a revolutionary increase in brightness that could reach two to three orders of magnitude beyond the current capability.
  • According to Dr. Fischetti, “This fourth generation storage ring will be nearly diffraction-limited and provide key improvements such as focusing X-rays down to the nanometer level with much higher intensity than is currently available. We expect the proposed MBA to be available in the 2020s. With this and other advances, it is clear that we are entering a new frontier in X-ray science.”
  • Disease-Related Receptors

In particular, Dr. Rosenbaum and his laboratory use protein engineering, X-ray crystallography, and NMR spectroscopy to study the structure and dynamics of molecules involved in hormone signaling and lipid homeostasis.

“GPCRs and other membrane proteins are not easily amenable to structural studies,” he said. “This limitation can often be overcome by protein engineering methods such as creating fusion proteins or thermostable mutants and using lipid-mediated crystallization methods.”

For example, Dr. Rosenbaum and colleagues studied the human β2 adrenergic receptor (β2AR) that binds epinephrine and is involved in the fight or flight response. Using the inactive structure of β2AR as guide, the team designed a β2AR agonist that could be covalently attached to a specific site on the receptor. “With this approach, we were able to crystallize a covalent agonist-bound β2AR fusion protein in lipid bilayers and determine its structure at 3.5 angstroms resolution.”

Another example of using fusion proteins to overcome membrane protein crystallization limitations is that of the human orexin receptor, OX2R. The orexin system modulates behaviors in mammals such as sleep, arousal, and feeding. Dysfunctions can lead to narcolepsy and cataplexy. The FDA recently approved the first-in-class drug, suvorexant, which became available in early 2015.

Dr. Rosenbaum and colleagues used lipid-mediated crystallization and protein engineering with a novel fusion chimera to solve the structure of the OX2R, bound to suvorexant at 2.5 angstom resolution.

“Elucidation of the molecular architecture of the human OX2R enhances our knowledge of how it recognizes ligands. Such studies provide powerful tools for designing improved therapeutics that can activate or inactivate orexin signaling.”

These studies have an overarching significance as well. “Looking at the bigger picture, these methods may lead to the design of new classes of small molecules that modulate key signaling pathways by controlling protein conformational changes within cellular membranes,” Dr. Rosenbaum concluded.

  • Nanodisc Technology

Although membrane proteins can be purified following cell lysis and detergent solubilization or after expression in heterologous systems, their true structure and function can be significantly compromised or lost entirely in the process. Ideally one would like membrane proteins to remain in a solubilized state for easier purification, functional assays, and structure determination. However, the native membrane environment is often necessary for full functionality. Detergents pose many technical obstacles including being hazardous for protein stability and interfering in many assay techniques.

Enter Nanodisc technology, a new approach for providing accessibility to the world of membrane proteins.

“We’ve always had a dream of engineering a process that would not only incorporate any membrane protein into a soluble bilayer structure, but also one that would employ a self-assembly process that would be applicable to all individual membrane proteins regardless of their structure and topology,” explained Stephen G. Sligar, Ph.D., director of the School of Molecular and Cellular Biology, University of Illinois, Urbana Champaign.

“Recently, that dream became realized by the creation of Nanodisc technology. Nanodiscs are self-assembling nanoscale phospholipid bilayers that are stabilized using engineered membrane scaffold proteins. The Nanodiscs allow membrane proteins to remain soluble and thus closely mimics native environment.”

There are many uses for the new technology according to Dr. Sligar. “Technological applications can take advantage of Nanodisc properties such as its small size, reduced light scattering, faster diffusion, enhanced stability, access to both sides of the bilayer and for surface attachment (e.g., surface plasmon resonance studies).”

Dr. Sligar and colleagues even demonstrated how to utilize the new technology for high throughput screening (HTS) assays.

“We wanted to identify antagonists that would interfere with the binding of membrane proteins to Alzheimer’s-associated amyloid β oligomers (AβOs), which are the neurotoxic ligands that instigate Alzheimer’s dementia. In collaboration with Professor William Klein and co-workers at Northwestern University, we created a solubilized membrane protein library (SMPL). This consisted of a complete set of membrane proteomes derived from biological tissue containing a heterogeneous mixture of individual proteins.

“Screening an extensive library of drug-like compounds and natural products identified yielded several ‘hits’, thus providing proof of concept for using SMPLs in HTS applications. An initial publication appeared recently in PLOS ONE.”

The results need to be confirmed in animal studies, Dr. Sligar noted. Overall, he is enthusiastic about the Nanodisc platform for uses that range from determination of structure/function to HTS applications.

“The unique properties of Nanodiscs make them ideal candidates to address important functional and structural questions involving membrane proteins in a more native environment.”


Twists and Turns in Protein Expression

In Early Drug Discovery it’s Often Unclear Which Recombinant Proteins Will Be Affected by Changing the Host Cell


  • When drug developers use different cell lines for manufacturing and preclinical research, they risk generating inconsistent results, proteins with various structures and functions. Then, confounded by variability, drug developers may lavish attention on irrelevant candidates and overlook promising candidates.

To avoid misleading themselves, drug developers must find ways to avoid or account for protein variants, which include post-translational modifications, particularly alternative glycosylations. Such variants occur all too frequently among different host cell lines, an extensive body of literature documents.

“Variability is most evident when comparisons are made between mammalian and nonmammalian cells,” says James Brady, Ph.D., vice president of technical applications and customer support at MaxCyte. “But depending on the protein that is being produced, even different mammalian cell lines, such as HEK and CHO, will exhibit substantial differences in post-translational modifications.” Differences can lead to altered protein stability, activity, or in vivo half-life.

It is often unclear during the early drug discovery process which recombinant proteins will be affected by changing the host cell. However, misleading early-stage data are associated with significant costs and extended timelines. It therefore makes sense to adopt a single host cell for all stages of the development pipeline. That is the rationale behind MaxCyte’s flow electroporation transfection platform.

  • Large-Scale Electroporation

Chemical transfection based on lipids or polymers are the most common alternatives to electroporation for large-scale transient transfection. However, reagent costs, lot-to-lot reagent variability, scale-up difficulties, and low transfection efficiency with certain cell types often are significant challenges of chemical transfection, particularly in biomanufacturing-relevant cells such as CHO.

Viral transfection vectors are another possibility. “While viral vectors may be more effective than chemical methods for introducing genes into certain difficult-to-transfect cell types, producing viral vectors often requires the development of packaging or producer cell lines,” Dr. Brady explains. “There are also biosafety concerns associated with some viral vectors.”

Unlike stable transfection, transient gene expression does not involve integration of the transgene into the host chromosome. Therefore, influences of the integration site on protein expression levels or other protein attributes are not evident. Rather the host cell’s genetic background, media/feed formulation, and culture conditions are the most significant factors influencing product quality, regardless of whether the protein is produced by stable or transient expression.

While high-end titers for stably transfected cells are now advancing into the low double-digit grams per liter, average titers are still in the lower single digits. Thus, the titers of 2–3 g/L that have recently been reported for transient expression via flow electroporation in nonengineered CHO cells are beginning to rival those of stable cell lines.

“So far, upper limits to titer by stable or transient expression have not been reached,” Dr. Brady tells GEN. “It is likely that innovations in vector design, advances in cell-line engineering, and improvements to cell-culture processes will lead to continued advances in both stable and transient titers.”

  • Monitoring Expression
  • Analytical methods are crucial for quantifying not only protein expression but also quality. A group at Fujifilm Diosynth Biotechnologies led by Greg Adams, Ph.D., the company’s director of analytical development, is promoting analytical techniques applicable throughout a molecule’s life cycle.

A scientist at Fujifilm Diosynth Biotechnologies operating an ambr250 mini-bioreactor system from Sartorius Stedim Biotech business unit TAP Biosystems.


  • Depending on the expression system, the Fujifilm Diosynth team focuses mostly on aggregation, glycosylation, and heterogeneity. The team employs a mix of rapid and conventional analyses, for example, mass spectrometry, ultra-performance liquid chromatography (UPLC), glycan analysis with rapid 2-aminobenzamide (2-AB) labeling and normal-phase UPLC, and capillary electrophoresis (CE) techniques such as imaged CE (iCE) and the CE-sodium dodecyl sulfate (CE-SDS) method. “Our objective,” declares Dr. Adams, “is same-day quality attribute analysis for understanding what’s happening in a bioreactor while designing the upstream process.”
  • Note that all the aforementioned techniques are standard analysis methods. The novelty is the context in which Fujifilm Diosynth uses them. Another distinction is the company’s high-throughput approach. The company uses liquid-handling workstations with pre-loaded tips for culture purification over protein A. The 30–60-minute preparation provides purified, active, concentrated antibody that may be analyzed in a number of ways. “We are able to analyze multiple ambr™ minireactor or 2 L bioreactor samples in hours versus days,” asserts Dr. Adams.
  • When it is applied to cell-line development, the rapid analysis philosophy holds that the same methods should be used from early development through GMP manufacturing. In practice, this is easier with antibodies because molecules of this class lend themselves to affinity purification and rapid method optimization through design of experiment (DOE), potentially beginning with transfectant pool material.
  • “Hopefully, we can have a method that we don’t have to change for the lifetime of the program,” Dr. Adams says. “It certainly helps to be able to trace data back through clinical phases and not have to worry about chromatographic profile and column changes. This has been very successful in several programs using the newer techniques, where the development phase is assisted by the speed by which you can run each method.”
  • The next challenge is to transfer this methodology to products expressed in microbial fermentation, which Dr. Adams refers to as the “next generation” of this approach to analytics.
  • Improving Solubility

Escherichia coli became the workhorse of recombinant protein expression because of its simple genetics, ease of culturing, scalability, rapid expression, and prodigious productivity. Negatives include a lack of eukaryotic post-translational machinery, codon usage bias, and difficulty with high-molecular-weight proteins.

Pros and cons must be weighed in terms of the target protein’s intended use. Quality and purity requirements for research-only proteins vary significantly, and may be worlds apart from therapeutic proteins. “The end application dictates to a large degree the choice of expression host, purity requirements, how you design the construct, and which tags to use,” says Keshav Vasanthavada, marketing specialist at GenScript.

A disadvantage in E. coli on par with low expression is insoluble expression, which results in aggregates (inclusion bodies). Researchers can deal with this phenomenon at the process level or molecular level. But before they embark on an improvement project, they should, Vasanthavada advises, check the literature to see if other researchers have produced the target protein in adequate yield and at acceptable quality. If so, it would be worthwhile to look at the other researchers’ methods and see if they can be reproduced.

Process-level strategies, which do not require target reengineering, include changing expression conditions, in vitro protein refolding, switching E. coli strains, adjusting media and buffers, or incorporating chaperone co-expression. Molecular-level approaches involve eliminating undesirable elements through truncations or mutations.

“The easiest approach is adoption of a fusion partner-based strategy,” Vasanthavada tells GEN. “It involves the use of a solubilizing partner upstream of the target protein to enhance target protein solubility.”

While this approach is generally beneficial, it has its drawbacks. For example, while a fusion partner will solubilize the target protein, there is no guarantee that the target protein will remain in solution once the tag is cleaved off. “Sometimes, you cannot ‘cleave off’ the fusion partner. The proteolytic enzyme won’t reach the cleavage site because of interference from itself,” Vasanthavada explains. “On other occasions, your fusion partner will start sticking to your target protein post-cleavage.”


Riboswitch Flip Kills Bacteria

Scientists discover a novel antibacterial molecule that targets a vital RNA regulatory element.

By Ruth Williams | September 30, 2015



Part of a riboswitch


Researchers at the pharmaceutical company Merck have identified a new bacteria-killing compound with an unusual target—an RNA regulatory structure called a riboswitch. The team used its drug, described in Nature today (September 30), to successfully reduce an experimental bacterial infection in mice, suggesting that the molecule could lead to the creation of a new antibiotic. Moreover, the results indicate that riboswitches—and other RNA elements—might be hitherto unappreciated targets for antibiotics and other drugs.

“Finding an antibiotic with a new target . . . has always been one of the holy grails of antibiotics discovery,” said RNA biochemist Thomas Hermann of the University of California, San Diego, who was not involved in the work. “It looks like that’s what the Merck group has now accomplished.”

The team’s research began with the idea of finding a compound that blocks the bacterial riboflavin synthesis pathway. Riboflavin is an essential nutrient for humans and bacteria alike, but while humans must consume it as part of their diet, bacteria can either scavenge riboflavin from the environment or, if supplies are lacking, make their own. “We targeted the riboflavin pathway because it is specific to bacteria so you have a built in safety margin,” said John Howe of the Merck research laboratories in Kenilworth, New Jersey, who led the research.

The team devised a simple but “very smart phenotypic screen,” said Hermann. The researchers tested roughly 57,000 antibacterial synthetic small molecules on cultures of E. coli bacteria looking for ones whose killing ability was neutralized by the presence of riboflavin. “If the effect of that antibacterial was suppressed by riboflavin,” said Howe, “then we had a good chance that the small molecule . . . was targeting the riboflavin pathway.”

The team found one molecule that fit the criteria and called it ribocil. To investigate the molecule’s mechanism of action, they applied it to cultures of E. coli cells until colonies emerged that were resistant to its effect. The researchers then sequenced the whole genomes of each of the resistant bacterial strains to find which genes were mutated.

The majority of drugs target proteins, explained Howe, “so we assumed that the mutations would be in one of the enzymes in the riboflavin synthesis pathway.” But as it turned out, while all of the 19 resistant strains did have mutations in a gene called RibB (which produces one of the riboflavin synthesis enzymes), the mutations did not affect the protein itself. They altered a non-coding part of the messenger RNA transcript: the riboswitch.

Riboswitches are regulatory elements at the beginning of messenger RNA transcripts. They bind molecules—normally metabolites—that typically suppress the transcript’s expression. “So instead of regulating the enzyme itself, [ribocil] is regulating the production of the enzyme,” Howe said.

Indeed, through reporter assays and crystallization experiments, the team confirmed that ribocil directly interacted with the RibB riboswitch, preventing expression of the protein.

“Ninety-nine-point-nine percent of drug targets are proteins,” said Hermann, “but they were prepared for the 0.1 percent outcome, and I think that’s what I really liked about this work.”

The team went on to tweak ribocil’s chemical structure, improving its killing efficiency and prolonging its effectiveness inside the body. The researchers then showed that this enhanced version of ribocil could effectively reduce bacterial burden in mice infected with a weakened E. coli strain; the bacteria are unable to efficiently expel drugs.

Weakened E. coli were used because wild-type E. coli are adept at ejecting ribocil and other compounds before they can take effect. Finding a way to keep ribocil in the bacteria and making other improvements will be necessary before it can be used as an actual antibiotic, explained Howe.

“I’ve [got] no idea if ribocil will end up being a drug candidate,” biochemist Gerry Wright of McMaster University in Ontario, Canada, wrote in an email to The Scientist, “but the work is a proof of principle, which is very important, and it makes us look to new areas of biology as targets for antibiotics.”

J.A. Howe et al., “Selective small-molecule inhibition of an RNA structural element,” Nature,doi: 10.1038/nature15542, 2015.


riboswitchnoncoding RNAdrug developmentdisease/medicinecell & molecular biology and antibiotics


Assay Drug Dev Technol. 2015 Sep;13(7):402-14. doi: 10.1089/adt.2015.655.

High-Content Assays for Characterizing the Viability and Morphology of 3D Cancer Spheroid Cultures.

Sirenko O1Mitlo T1Hesley J1Luke S1Owens W1Cromwell EF2.

Author information


There is an increasing interest in using three-dimensional (3D) spheroids for modeling cancer and tissue biology to accelerate translation research. Development of higher throughput assays to quantify phenotypic changes in spheroids is an active area of investigation. The goal of this study was to develop higher throughput high-content imaging and analysis methods to characterize phenotypic changes in human cancer spheroids in response to compound treatment. We optimized spheroid cell culture protocols using low adhesion U-bottom 96- and 384-well plates for three common cancer cell lines and improved the workflow with a one-step staining procedure that reduces assay time and minimizes variability. We streamlined imaging acquisition by using a maximum projection algorithm that combines cellular information from multiple slices through a 3D object into a single image, enabling efficient comparison of different spheroid phenotypes. A custom image analysis method was implemented to provide multiparametric characterization of single-cell and spheroid phenotypes. We report a number of readouts, including quantification of marker-specific cell numbers, measurement of cell viability and apoptosis, and characterization of spheroid size and shape. Assay performance was assessed using established anticancer cytostatic and cytotoxic drugs. We demonstrated concentration-response effects for different readouts and measured IC50 values, comparing 3D spheroid results to two-dimensional cell cultures. Finally, a library of 119 approved anticancer drugs was screened across a wide range of concentrations using HCT116 colon cancer spheroids. The proposed methods can increase performance and throughput of high-content assays for compound screening and evaluation of anticancer drugs with 3D cell models.


Molecules Hold the Mirror Up to Cancer

Imaging Technologies are Critical Tools for Basic Research and Translational and Clinical Applications


The Center for Biomedical Imaging in Oncology (CBIO) at the Dana-Farber Cancer Institute in Boston is a centralized cancer imaging research enterprise that was established to enable translational cancer research and drug development through the integration of preclinical and clinical imaging, access to preclinical/clinical multidisciplinary and multimodality imaging expertise, as well as drug/imaging probe development.

cancr imaging_DanaFarber_CBIO_OraganizatonalChart6613014019



  • The molecular processes behind cancer were once seen as through a glass, darkly. But now they are being reflected more clearly, thanks to advances in probe synthesis, preclinical cancer modeling, and multimodal imaging. These advances have positioned imaging as a key tool for basic research, as well as for translational and clinical applications.

To bring cancer visualization trends to light, CHI recently held a conference, Translational Imaging in Cancer Drug Development, as part of the World Preclinical Congress in Boston. This conference attracted leading imaging experts from industry and academia, including scientists and clinicians who use their expertise to accelerate cancer research. Many of the experts described how, with a little creativity, imaging modalities can be used to translate scientific discoveries into clinical applications.

Several examples of creative imaging from the conference are discussed in this article. To start, this article will highlight one investigator’s new take on a familiar technique, positron emission tomography (PET).

“Along with the scientific challenge posed by President Obama’s Precision Medicine Initiative, molecular imaging probes have substantially improved and expanded to include the noninvasive characterization of tumors and tumor microenvironments,” said Quang-Dé Nguyen, Ph.D., director of the Lurie Family Imaging Center (LFIC) at the Dana Farber Cancer Institute. “PET is becoming a method of choice for studying tumor biology in real time.”

LFIC is fully equipped to meet the creative demands of translational molecular imaging. It is an integral part of the Center for Biomedical Imaging in Oncology (CBIO), which also includes a clinical imaging research group. In addition to LFIC and CBIO, the Dana Farber Cancer Institute includes medicinal chemistry capabilities and expertise, and has recently established the Molecular Cancer Imaging Facility housing the only PET cyclotron in the state dedicated entirely to the development of novel radiotracers for cancer research.

“A unique attribute of our Cancer Center is the fully developed Mouse Hospital, mirroring every aspect of human cancer diagnostics and care,” noted Dr. Nguyen. The center uses genetically engineered mouse models that can be matched to the specific genotype of a given individual patient. Alternatively, the Center can rapidly generate xenograft mice and orthotopic murine tumor models using human tumor cells obtained from biopsies. In either case, the resulting mouse model is a faithful genetic mirror of the patient’s tumor.

Dr. Nguyen’s team deploys PET imaging to inform patient treatment in co-clinical trials. Once a patient’s genotype is identified, an appropriate mouse model is selected, sometimes in combination with additional mutations. The mouse is treated with a desired therapy, and functional and molecular outcomes can be rapidly detected by PET imaging. Mouse-derived data can then inform the design of the clinical trial and be fully integrated with clinical data.

In a seminal study, lung tumors carrying several combinations of cancer mutations were simultaneously tested in genetically engineered mouse models and in patients with lung cancer enrolled in a clinical trial to assess response to a combination therapy with a novel drug compared to standard of care. The radiolabeled glucose analog was used to visualize the lung tumors by PET in both mice and patients.

Remarkably, within 24 hours after therapy initiation, preclinical PET imaging demonstrated treatment response to the combined regimen for some but not all the mutations. This information helped identify the resistant mutation in patients being considered for enrollment in the clinical trial and allowed enrichment of the patient population by selecting patients carrying those mutations that had showed metabolic response in the preclinical setting.


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Metabolic Genomics and Pharmaceutics, Vol. 1 of BioMed Series D available on Amazon Kindle

Metabolic Genomics and Pharmaceutics, Vol. 1 of BioMed Series D available on Amazon Kindle

Reporter: Stephen S Williams, PhD


Leaders in Pharmaceutical Business Intelligence would like to announce the First volume of their BioMedical E-Book Series D:

Metabolic Genomics & Pharmaceutics, Vol. I

SACHS FLYER 2014 Metabolomics SeriesDindividualred-page2

which is now available on Amazon Kindle at


This e-Book is a comprehensive review of recent Original Research on  METABOLOMICS and related opportunities for Targeted Therapy written by Experts, Authors, Writers. This is the first volume of the Series D: e-Books on BioMedicine – Metabolomics, Immunology, Infectious Diseases.  It is written for comprehension at the third year medical student level, or as a reference for licensing board exams, but it is also written for the education of a first time baccalaureate degree reader in the biological sciences.  Hopefully, it can be read with great interest by the undergraduate student who is undecided in the choice of a career. The results of Original Research are gaining value added for the e-Reader by the Methodology of Curation. The e-Book’s articles have been published on the Open Access Online Scientific Journal, since April 2012.  All new articles on this subject, will continue to be incorporated, as published with periodical updates.

We invite e-Readers to write an Article Reviews on Amazon for this e-Book on Amazon.

All forthcoming BioMed e-Book Titles can be viewed at:


Leaders in Pharmaceutical Business Intelligence, launched in April 2012 an Open Access Online Scientific Journal is a scientific, medical and business multi expert authoring environment in several domains of  life sciences, pharmaceutical, healthcare & medicine industries. The venture operates as an online scientific intellectual exchange at their website http://pharmaceuticalintelligence.com and for curation and reporting on frontiers in biomedical, biological sciences, healthcare economics, pharmacology, pharmaceuticals & medicine. In addition the venture publishes a Medical E-book Series available on Amazon’s Kindle platform.

Analyzing and sharing the vast and rapidly expanding volume of scientific knowledge has never been so crucial to innovation in the medical field. WE are addressing need of overcoming this scientific information overload by:

  • delivering curation and summary interpretations of latest findings and innovations on an open-access, Web 2.0 platform with future goals of providing primarily concept-driven search in the near future
  • providing a social platform for scientists and clinicians to enter into discussion using social media
  • compiling recent discoveries and issues in yearly-updated Medical E-book Series on Amazon’s mobile Kindle platform

This curation offers better organization and visibility to the critical information useful for the next innovations in academic, clinical, and industrial research by providing these hybrid networks.

Table of Contents for Metabolic Genomics & Pharmaceutics, Vol. I

Chapter 1: Metabolic Pathways

Chapter 2: Lipid Metabolism

Chapter 3: Cell Signaling

Chapter 4: Protein Synthesis and Degradation

Chapter 5: Sub-cellular Structure

Chapter 6: Proteomics

Chapter 7: Metabolomics

Chapter 8:  Impairments in Pathological States: Endocrine Disorders; Stress

                   Hypermetabolism and Cancer

Chapter 9: Genomic Expression in Health and Disease 






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