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Lesson 4 Cell Signaling And Motility: G Proteins, Signal Transduction: Curations and Articles of reference as supplemental information: #TUBiol3373
Curator: Stephen J. Williams, Ph.D.
Updated 7/15/2019
Below please find the link to the Powerpoint presentation for lesson #4 for #TUBiol3373. The lesson first competes the discussion on G Protein Coupled Receptors, including how cells terminate cell signals. Included are mechanisms of receptor desensitization. Please NOTE that desensitization mechanisms like B arrestin decoupling of G proteins and receptor endocytosis occur after REPEATED and HIGH exposures to agonist. Hydrolysis of GTP of the alpha subunit of G proteins, removal of agonist, and the action of phosphodiesterase on the second messenger (cAMP or cGMP) is what results in the downslope of the effect curve, the termination of the signal after agonist-receptor interaction.
Today’s lesson 3 explains how extracellular signals are transduced (transmitted) into the cell through receptors to produce an agonist-driven event (effect). This lesson focused on signal transduction from agonist through G proteins (GTPases), and eventually to the effectors of the signal transduction process. Agonists such as small molecules like neurotransmitters, hormones, nitric oxide were discussed however later lectures will discuss more in detail the large growth factor signalings which occur through receptor tyrosine kinases and the Ras family of G proteins as well as mechanosignaling through Rho and Rac family of G proteins.
Transducers: The Heterotrimeric G Proteins (GTPases)
An excellent review of heterotrimeric G Proteins found in the brain is given by
Cyclic AMP is an important second messenger. It forms, as shown, when the membrane enzyme adenylyl cyclase is activated (as indicated, by the alpha subunit of a G protein).
The cyclic AMP then goes on the activate specific proteins. Some ion channels, for example, are gated by cyclic AMP. But an especially important protein activated by cyclic AMP is protein kinase A, which goes on the phosphorylate certain cellular proteins. The scheme below shows how cyclic AMP activates protein kinase A.
Updated 7/15/2019
Additional New Studies on Regulation of the Beta 2 Adrenergic Receptor
We had discussed regulation of the G protein coupled beta 2 adrenergic receptor by the B-AR receptor kinase (BARK)/B arrestin system which uncouples and desensitizes the receptor from its G protein system. In an article by Xiangyu Liu in Science in 2019, the authors describe another type of allosteric modulation (this time a POSITIVE allosteric modulation) in the intracellular loop 2. See below:
Mechanism of β2AR regulation by an intracellular positive allosteric modulator
Xiangyu Liu1,*, Ali Masoudi2,*, Alem W. Kahsai2,*, Li-Yin Huang2, Biswaranjan Pani2, Dean P. Staus2, Paul J. Shim2, Kunio Hirata3,4, Rishabh K. Simhal2, Allison M. Schwalb2, Paula K. Rambarat2, Seungkirl Ahn2, Robert J. Lefkowitz2,5,6,†, Brian Kobilka1
Positive reinforcement in a GPCR
Many drug discovery efforts focus on G protein–coupled receptors (GPCRs), a class of receptors that regulate many physiological processes. An exemplar is the β2-adrenergic receptor (β2AR), which is targeted by both blockers and agonists to treat cardiovascular and respiratory diseases. Most GPCR drugs target the primary (orthosteric) ligand binding site, but binding at allosteric sites can modulate activation. Because such allosteric sites are less conserved, they could possibly be targeted more specifically. Liu et al. report the crystal structure of β2AR bound to both an orthosteric agonist and a positive allosteric modulator that increases receptor activity. The structure suggests why the modulator compound is selective for β2AR over the closely related β1AR. Furthermore, the structure reveals that the modulator acts by enhancing orthosteric agonist binding and stabilizing the active conformation of the receptor.
Abstract
Drugs targeting the orthosteric, primary binding site of G protein–coupled receptors are the most common therapeutics. Allosteric binding sites, elsewhere on the receptors, are less well-defined, and so less exploited clinically. We report the crystal structure of the prototypic β2-adrenergic receptor in complex with an orthosteric agonist and compound-6FA, a positive allosteric modulator of this receptor. It binds on the receptor’s inner surface in a pocket created by intracellular loop 2 and transmembrane segments 3 and 4, stabilizing the loop in an α-helical conformation required to engage the G protein. Structural comparison explains the selectivity of the compound for β2– over the β1-adrenergic receptor. Diversity in location, mechanism, and selectivity of allosteric ligands provides potential to expand the range of receptor drugs.
Recent structures of GPCRs bound to allosteric modulators have revealed that receptor surfaces are decorated with diverse cavities and crevices that may serve as allosteric modulatory sites (1). This substantiates the notion that GPCRs are structurally plastic and can be modulated by a variety of allosteric ligands through distinct mechanisms (2-7). Most of these structures have been solved with negative allosteric modulators (NAMs), which stabilize receptors in their inactive states (1). To date, only a single structure of an active GPCR bound to a small-molecule positive allosteric modulator (PAM) has been reported, namely, the M2 muscarinic acetylcholine receptor with LY2119620 (8). Thus, mechanisms of PAMs and their potential binding sites remain largely unexplored.
Fig 1. Structure of the active state T4L-B2AR in complex with the orthosteric agonist BI-167107, nanobody 689, and compound 6FA. (A) The chemical structure of compound-6FA (Cmpd-6FA). (B) Isoproterenol (ISO) competition binding with 125I-cyanopindolol (CYP) to the β2AR reconstituted in nanodisks in the presence of vehicle (0.32% dimethylsulfoxide; DMSO), Cmpd-6, or Cmpd-6FA at 32 μM. Values were normalized to percentages of the maximal 125I-CYP binding level obtained from a one-site competition binding–log IC50 (median inhibitory concentration) curve fit. Binding curves were generated by GraphPad Prism. Points on curves represent mean ± SEM obtained from five independent experiments performed in duplicate. (C) Analysis of Cmpd-6FA interaction with the BI-167107–bound β2AR by ITC. Representative thermogram (inset) and binding isotherm, of three independent experiments, with the best titration curve fit are shown. Summary of thermodynamic parameters obtained by ITC: binding affinity (KD = 1.2 ± 0.1 μM), stoichiometry (N = 0.9 ± 0.1 sites), enthalpy (ΔH = 5.0 ± 1.2 kcal mol−1), and entropy (ΔS =13 ± 2.0 cal mol−1 deg−1). (D) Side view of T4L-β2AR bound to the orthosteric agonist BI-167107, nanobody 6B9 (Nb6B9), and Cmpd-6FA. The gray box indicates the membrane layer as defined by the OPM database. (E) Close-up view of Cmpd-6FA binding site. Covering Cmpd-6FA is 2Fo– Fc electron density contoured at 1.0 σ (green mesh).From Science 28 Jun 2019:
Vol. 364, Issue 6447, pp. 1283-1287
Fig 3. Fig. 3Mechanism of allosteric activation of the β2AR by Cmpd-6FA.
(A) Superposition of the inactive β2AR bound to the antagonist carazolol (PDB code: 2RH1) and the active β2AR bound to the agonist BI-167107, Cmpd-6FA, and Nb6B9. Close-up view of the Cmpd-6FA binding site is shown. The residues of the inactive (yellow) and active (blue) β2AR are depicted, and the hydrogen bond formed between Asp1303.49and Tyr141ICL2 in the active state is indicated by a black dashed line. (B) Topography of Cmpd-6FA binding surface on the active β2AR (left, blue) and the corresponding surface of the inactive β2AR (right, yellow) with Cmpd-6FA (orange sticks) docked on top. Molecular surfaces are of only those residues involved in interaction with Cmpd-6FA. Steric clash between Cmpd-6FA and the surface of inactive β2AR is represented by a purple asterisk. (C) Overlay of the β2AR bound to BI-167107, Nb6B9, and Cmpd-6FA with the β2AR–Gscomplex (PDB code: 3SN6). The inset shows the position of Phe139ICL2 relative to the α subunit of Gs. (D) Superposition of the active β2AR bound to the agonist BI-167107, Nb6B9, and Cmpd-6FA (blue) with the inactive β2AR bound to carazolol (yellow) (PDB code: 2RH1) as viewed from the cytoplasm. For clarity, Nb6B9 and the orthosteric ligands are omitted. The arrows indicate shifts in the intracellular ends of the TM helices 3, 5, and 6 upon activation and their relative distances.
Allosteric sites may not face the same evolutionary pressure as do orthosteric sites, and thus are more divergent across subtypes within a receptor family (24–26). Therefore, allosteric sites may provide a greater source of specificity for targeting GPCRs.
D. M. Thal, A. Glukhova, P. M. Sexton, A. Christopoulos, Structural insights into G-protein-coupled receptor allostery. Nature 559, 45–53 (2018). doi:10.1038/s41586-018-0259-zpmid:29973731CrossRefPubMedGoogle Scholar
D. Wacker, R. C. Stevens, B. L. Roth, How Ligands Illuminate GPCR Molecular Pharmacology. Cell 170, 414–427 (2017).
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Additional information on Nitric Oxide as a Cellular Signal
Nitric oxide is actually a free radical and can react with other free radicals, resulting in a very short half life (only a few seconds) and so in the body is produced locally to its site of action (i.e. in endothelial cells surrounding the vascular smooth muscle, in nerve cells). In the late 1970s, Dr. Robert Furchgott observed that acetylcholine released a substance that produced vascular relaxation, but only when the endothelium was intact. This observation opened this field of research and eventually led to his receiving a Nobel prize. Initially, Furchgott called this substance endothelium-derived relaxing factor (EDRF), but by the mid-1980s he and others identified this substance as being NO.
Nitric oxide is implicated in many pathologic processes as well. Nitric oxide post translational modifications have been attributed to nitric oxide’s role in pathology however, although the general mechanism by which nitric oxide exerts its physiological effects is by stimulation of soluble guanylate cyclase to produce cGMP, these post translational modifications can act as a cellular signal as well. For more information of NO pathologic effects and how NO induced post translational modifications can act as a cellular signal see the following:
Intravenous drug for the treatment of Acute Heart Failure (AHF) by Trevena, Inc. (Trevena) – Leader in the Discovery of G-protein coupled receptor (GPCR) biased ligands
Reporter: Aviva Lev-Ari, PhD, RN
Cardiovascular biotech Trevena files for a $86 million IPO
10/9/13
Trevena, a clinical-stage biotech developing a treatment for acute heart failure, filed on Wednesday with the SEC to raise up to $86 million in an initial public offering. The King of Prussia, PA-based company, which was founded in 2007 and booked $1 million in grant and collaboration revenue for the 12 months ended June 30, 2013, plans to list on the NASDAQ under the symbol TRVN. Trevena initially filed confidentially on September 6, 2013. Barclays and Jefferies are the joint bookrunners on the deal. No pricing terms were disclosed.
Trevena a clinical stage biopharmaceutical company that discovers, develops and intends to commercialize therapeutics that use a novel approach to target G protein coupled receptors, or GPCRs.
Heart Biotech Trevena, Inc. Files for a $86 Million IPO
10/10/2013 9:19:18 AM
Trevena, Inc. (Nasdaq: TRVN) filed a registration with the U.S. Securities and Exchange Commission for an Initial Public Offering of its Common Stock. The proposed maximum offering price is $86.25 million. The company plans to list on the Nasdaq Global Market under the ticker, “TRVN.” The offering is being made via Barclays, Jefferies, Canaccord Genuity, JMP Securities, and Needham & Company.
KING OF PRUSSIA, Pa., July 8, 2013 /PRNewswire/ — Trevena, Inc. (Trevena), a clinical stage pharmaceutical company and the leader in the discovery of G-protein coupled receptor (GPCR) biased ligands, today announced the electronic publication of Trevena’s manuscript, “First Clinical Experience with TRV027: Pharmacokinetics and Pharmacodynamics in Healthy Volunteers.” The manuscript can be viewed online at the Journal of Clinical Pharmacology’s website (http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1552-4604/ earlyview).
TRV027 is an experimental intravenous drug now in mid-stage clinical trials for the treatment of acute heart failure (AHF). The printed manuscript will appear in a future print issue of the journal. David G. Soergel, M.D., Trevena’s Senior Vice President of Clinical Development, Jonathan D. Violin, PhD, Director of Biology and Co-Founder of Trevena, and Michael W. Lark, PhD, Trevena’s Chief Scientific Officer and Senior Vice President of Research at Trevena, were among the publication’s authors.
The manuscript summarizes results from the first-in-human clinical study in which the compound was administered to healthy human subjects. In this study, the tolerability, pharmacokinetics and pharmacodynamics of multiple doses of TRV027 were explored. TRV027 was safe and well-tolerated, with a usefully short half-life and dose-proportional increases in systemic exposure. The compound showed a decrease in mean arterial pressure in subjects that had an elevation in their renin angiotensin aldosterone system, a common characteristic in AHF patients. TRV027’s activity, observed in the study, is consistent with its mechanism of action and previously published preclinical findings.
“In this phase 1 study, we successfully translated the unique activity profile of TRV027 from preclinical species into humans. These data supported our decision to progress TRV027 into Phase 2 studies in heart failure patients,” commented Dr. Soergel.
About TRV027 and AHF
TRV027 is a novel beta-arrestin biased ligand of the angiotensin II type 1 receptor (AT1R) that combines the proven benefits of angiotensin blockade with new beta-arrestin-mediated biology to preserve cardiac and renal function. TRV027 is being developed by Trevena under a recently announced collaborative licensing option agreement with Forest Laboratories Inc. For more details, please find a copy of the May 9, 2013 press release on the Trevena website, under the “News” tab (http://www.trevenainc.com/).
In March 2013, Trevena also presented the results of a Phase 2a study on the hemodynamic effects of TRV027 in patients with advanced systolic heart failure as a poster at the annual American College of Cardiology meeting. Completion of the ascending dose-titration Phase 2a study was announced in October 2012, in which the safety, tolerability, pharmacokinetics, and invasive hemodynamics of TRV027 (formally TRV120027) was measured (NCT01187836). The drug was generally well-tolerated and produced a beneficial set of hemodynamic effects in the study. A phase 2b clinical trial of TRV027 is expected to begin later this year.
The American Heart Association estimated that AHF hospitalization costs the U.S. healthcare system more than $20 billion each year in direct spending. AHF is already the leading reason for hospitalization of individuals over 65 years old in the United States, with over 1 million hospital admissions per year. AHF is also the most costly diagnosis for Medicare in the nation. Despite the significance of this problem, current therapies are not producing meaningful improvements in patient outcomes. AHF incidence is increasing globally, and both heart failure mortality and hospital re-admission following an AHF event remain extremely high.
About Trevena
Trevena, Inc. is dedicated to discovering and developing the next generation of GPCR targeted medicines. GPCRs are the targets for at least one-third of modern medicinal products, and remain the predominant class of targets under clinical evaluation. Trevena’s expertise lies in engineering “biased ligands” that activate only the beneficial signaling pathways downstream of a GPCR to unlock new biology and avoid drug adverse effects. In addition to TRV027, Trevena’s pipeline currently includes a clinical stage mu-opioid biased ligand for post-operative pain, and discovery-stage programs for chronic pain and Parkinson’s disease.
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