Showing papers by "Michel Bouvier published in 2022"

Community guidelines for GPCR ligand bias: IUPHAR review 32

Abstract: GPCRs modulate a plethora of physiological processes and mediate the effects of one‐third of FDA‐approved drugs. Depending on which ligand activates a receptor, it can engage different intracellular transducers. This ‘biased signalling’ paradigm requires that we now characterize physiological signalling not just by receptors but by ligand–receptor pairs. Ligands eliciting biased signalling may constitute better drugs with higher efficacy and fewer adverse effects. However, ligand bias is very complex, making reproducibility and description challenging. Here, we provide guidelines and terminology for any scientists to design and report ligand bias experiments. The guidelines will aid consistency and clarity, as the basic receptor research and drug discovery communities continue to advance our understanding and exploitation of ligand bias. Scientific insight, biosensors, and analytical methods are still evolving and should benefit from and contribute to the implementation of the guidelines, together improving translation from in vitro to disease‐relevant in vivo models.

Common coupling map advances GPCR-G protein selectivity

Abstract: Two-thirds of human hormones and one-third of clinical drugs act on membrane receptors that couple to G proteins to achieve appropriate functional responses. While G protein transducers from literature are annotated in the Guide to Pharmacology database, two recent large-scale datasets now expand the receptor-G protein ‘couplome’. However, these three datasets differ in scope and reported G protein couplings giving different coverage and conclusions on GPCR-G protein signaling. Here, we report a common coupling map uncovering novel couplings supported by both large-scale studies, the selectivity/promiscuity of GPCRs and G proteins, and how the co-coupling and co-expression of G proteins compare to the families from phylogenetic relationships. The coupling map and insights on GPCR-G protein selectivity will catalyze advances in receptor research and cellular signaling towards the exploitation of G protein signaling bias in design of safer drugs.

Structure-based discovery of nonopioid analgesics acting through the α2A-adrenergic receptor

Abstract: Because nonopioid analgesics are much sought after, we computationally docked more than 301 million virtual molecules against a validated pain target, the α2A-adrenergic receptor (α2AAR), seeking new α2AAR agonists chemotypes that lack the sedation conferred by known α2AAR drugs, such as dexmedetomidine. We identified 17 ligands with potencies as low as 12 nanomolar, many with partial agonism and preferential Gi and Go signaling. Experimental structures of α2AAR complexed with two of these agonists confirmed the docking predictions and templated further optimization. Several compounds, including the initial docking hit ‘9087 [mean effective concentration (EC50) of 52 nanomolar] and two analogs, ‘7075 and PS75 (EC50 4.1 and 4.8 nanomolar), exerted on-target analgesic activity in multiple in vivo pain models without sedation. These newly discovered agonists are interesting as therapeutic leads that lack the liabilities of opioids and the sedation of dexmedetomidine. Description A path to pain relief The serious problems associated with opioid addiction have motivated the search for non-opioid pain-relief drugs. The α2A-adrenergic receptor (α2AAR) is a validated pain receptor and is targeted by dexmetadomine, a drug used in hospitals but unsuitable for broader use because it causes sedation and is not orally bioavailable. Fink et al. screened more than 300 million virtual molecules and identified agonists that bind α2AAR with reasonable affinity and are structurally unrelated to known agonists. Crystal structures of two of the compounds bound to α2AAR allowed optimization to improve potency. The optimized compounds were effective in a neuropathic pain model without causing sedation, making them promising leads for further development. —VV A high-throughput virtual screen followed by experimental testing generates therapeutic leads for non-opioid pain killers INTRODUCTION Epidemics of pain and opioid abuse underscore the need for new nonopioid therapeutics to treat pain. Many nonopioid receptors are involved in pain processing (nociception), but only a few have been validated therapeutically. Of particular interest is the α2A-adrenergic receptor (α2AAR), a G protein–coupled receptor (GPCR) whose activation in the central nervous system has pain-relieving effects. The known therapeutics targeting the α2AAR, like clonidine and dexmedetomidine, are known to be analgesic. They are also strongly sedating, which is important for the primary indication of dexmedetomidine. This, however, has restricted the use of these drugs to hospital settings and kept them from being used in broader patient populations. RATIONALE Because GPCRs, like α2AAR, can signal into the cell through multiple downstream effectors, we reasoned that agonists that were chemically dissimilar to the highly related dexmedetomidine, clonidine, and brimonidine might have different signaling and might be able to separate sedation from analgesia. We sought these chemotypes among a virtual library of more than 301 million diverse, readily accessible molecules in the ZINC15 library (http://zinc15.docking.org), few of which have been previously synthesized. We computationally docked each virtual molecule into the highly similar α2BAR binding site, prioritizing those that physically fit and that were chemically unrelated to the known drugs. RESULTS From the high-ranking docked compounds, we selected 48 for de novo synthesis and testing. Against the α2BAR used in the virtual docking screens, 30 molecules bound for a 63% hit rate, among the highest to date for docking campaigns. Seventeen further bound to α2AAR with binding constants in the low-nanomolar to low-micromolar concentration range. Several acted as full or partial agonists of α2AAR, activating the receptor. Among these was ‘9087 [mean effective concentration (EC50) of 52 nM]. Notably, the docking-derived agonists preferentially activated Gi, Go, and Gz G protein subtypes, which contrasts with known drugs, like dexmedetomidine and brimonidine, that activate a much broader set of G proteins and recruit β-arrestins. Thus, the new agonists activate a more selective set of cellular pathways than the known α2AAR drugs, something we had hoped for when prioritizing new chemotypes. The structures of two of these agonists were experimentally determined in complex with the activated state of α2AAR. These experimental ligand geometries closely corresponded to computational predictions. They also templated the optimization of the initial docking hits and led to more potent analogs, including PS75 (EC50 4.8 nM). The physical features of these agonists allowed them to reach high brain concentrations after systemic dosing. In animal behavioral assays, six of these previously uncharacterized agonists relieved pain behaviors in neuropathic, inflammatory, and acute thermal nociception assays. Gene mutation and reversal of receptor binding with an α2AR antagonist confirmed that analgesia occurred primarily through α2AAR. Crucially, when compared with dexmedetomidine, none of the new compounds caused sedation, even at substantially higher doses than required for pain relief. CONCLUSION The separation of analgesic properties from sedation of the new agonists is important for further α2AAR drug development. The newly identified agonists, especially ‘9087 and PS75, overcome the sedation liability of the previously known drugs, and several are orally bioavailable. This makes them lead molecules for the development of nonopioid pain therapeutics. Newly identified α2AAR agonists are analgesic without sedation. More than 301 million molecules were docked against the activated α2BAR. Experimental testing identified α2AAR agonists with diverse chemical scaffolds. The experimental structure of the ‘9087-α2AAR complex superposed closely to the computational prediction. The newly discovered agonists had efficacy in an in vivo neuropathic pain model (top right) without sedation, unlike dexmedetomidine (DEX) (bottom right). Gi1-activation EC50 (nanomolar) and Emax (percentage) values are shown. Single-letter abbreviations for the amino acid residues are as follows: D, Asp; F, Phe; I, Ile; S, Ser; V, Val; and Y, Tyr. ns, not significant; *P < 0.05; ****P < 0.0001; SNI, spared nerve injury.

Size-Reduced Macrocyclic Analogues of [Pyr1]-apelin-13 Showing Negative Gα12 Bias Still Produce Prolonged Cardiac Effects.

Abstract: We previously reported a series of macrocyclic analogues of [Pyr1]-apelin-13 (Ape13) with increased plasma stability and potent APJ agonist properties. Based on the most promising compound in this series, we synthesized and then evaluated novel macrocyclic compounds of Ape13 to identify agonists with specific pharmacological profiles. These efforts led to the development of analogues 39 and 40, which possess reduced molecular weight (MW 1020 Da vs Ape13, 1534 Da). Interestingly, compound 39 (Ki 0.6 nM), which does not activate the Gα12 signaling pathway while maintaining potency and efficacy similar to Ape13 to activate Gαi1 (EC50 0.8 nM) and β-arrestin2 recruitment (EC50 31 nM), still exerts cardiac actions. In addition, analogue 40 (Ki 5.6 nM), exhibiting a favorable Gα12-biased signaling and an increased in vivo half-life (t1/2 3.7 h vs <1 min of Ape13), produces a sustained cardiac response up to 6 h after a single subcutaneous bolus injection.

Discovery of Two Novel Antiplatelet Clinical Candidates (BMS-986120 and BMS-986141) That Antagonize Protease-Activated Receptor 4.

Abstract: Protease-activated receptor 4 (PAR4) is a G-protein coupled receptor that is expressed on human platelets and activated by the coagulation enzyme thrombin. PAR4 plays a key role in blood coagulation, and its importance in pathological thrombosis has been increasingly recognized in recent years. Herein, we describe the optimization of a series of imidazothiadiazole PAR4 antagonists to a first-in-class clinical candidate, BMS-986120 (43), and a backup clinical candidate, BMS-986141 (49). Both compounds demonstrated excellent antithrombotic efficacy and minimal bleeding time prolongation in monkey models relative to the clinically important antiplatelet agent clopidogrel and provide a potential opportunity to improve the standard of care in the treatment of arterial thrombosis.

New oral protease-activated receptor 4 antagonist BMS-986120: tolerability, pharmacokinetics, pharmacodynamics, and gene variant effects in humans

Abstract: Abstract BMS-986120 is a novel first-in-class oral protease-activated receptor 4 (PAR4) antagonist exhibiting robust antithrombotic activity that has shown low bleeding risk in monkeys. We sought to assess pharmacokinetics, pharmacodynamics, and tolerability of BMS-986120 in healthy participants and platelet responses to BMS-986120 in participants carrying PAR4 A120T variants. Phase I, randomized, double-blind, placebo-controlled single-ascending-dose (SAD; N = 56) and multiple-ascending-dose (MAD; N = 32) studies were conducted. Exposure was approximately dose-proportional: maximum concentrations 27.3 and 1536 ng/mL, areas under the curve (AUC) to infinity of 164 and 15,603 h*ng/mL, and half-lives of 44.7 and 84.1 hours for 3.0 and 180 mg, respectively. The accumulation index suggested an ~2-fold AUC increase at steady state. Single doses of 75 and 180 mg BMS-986120 produced ≥80% inhibition of 12.5 μM PAR4 agonist peptide (AP)-induced platelet aggregation through at least 24 hours postdose, and doses ≥10 mg for ~7 days inhibited aggregation completely through 24 hours. No differences in PAR4-mediated platelet response were seen between AA120 versus TT120 PAR4 variants. In cells expressing A120 or T120 PAR4 proteins, no differences in half-maximal effective concentration in receptor activation by PAR4-AP were observed. BMS-986120 was well tolerated with dose-proportional pharmacokinetics and concentration-dependent pharmacodynamics in healthy participants over a wide dose range. ClinicalTrials.gov ID: NCT02208882.

Structural Elements Directing G Proteins and β-Arrestin Interactions with the Human Melatonin Type 2 Receptor Revealed by Natural Variants

Abstract: G protein-coupled receptors (GPCRs) can engage distinct subsets of signaling pathways, but the structural determinants of this functional selectivity remain elusive. The naturally occurring genetic variants of GPCRs, selectively affecting different pathways, offer an opportunity to explore this phenomenon. We previously identified 40 coding variants of the MTNR1B gene encoding the melatonin MT2 receptor (MT2). These mutations differently impact the β-arrestin 2 recruitment, ERK activation, cAMP production, and Gαi1 and Gαz activation. In this study, we combined functional clustering and structural modeling to delineate the molecular features controlling the MT2 functional selectivity. Using non-negative matrix factorization, we analyzed the signaling signatures of the 40 MT2 variants yielding eight clusters defined by unique signaling features and localized in distinct domains of MT2. Using computational homology modeling, we describe how specific mutations can selectively affect the subsets of signaling pathways and offer a proof of principle that natural variants can be used to explore and understand the GPCR functional selectivity.

From a Cone Snail Toxin to a Competitive MC4R Antagonist.

Abstract: The melanocortin 4 receptor (MC4R) plays a role in energy homeostasis and represents a target for treating energy balance disorders. For decades, synthetic ligands have been derived from MC4R endogenous agonists and antagonists, such as setmelanotide used to treat rare forms of genetic obesity. Recently, animal venoms have demonstrated their capacity to provide melanocortin ligands with toxins from a scorpion and a spider. Here, we described a cone snail toxin, N-CTX-Ltg1a, with a nanomolar affinity for hMC4R but unrelated to any known toxins or melanocortin ligands. We then derived from the conotoxin the linear peptide HT1-0, a competitive antagonist of Gs, G15, and β-arrestin2 pathways with a low nanomolar affinity for hMC4R. Similar to endogenous ligands, HT1-0 needs hydrophobic and basic residues to bind hMC4R. Altogether, it represents the first venom-derived peptide of high affinity on MC4R and paves the way for the development of new MC4R antagonists.

Michaelis-Menten Quantification of Ligand Signaling Bias Applied to the Promiscuous Vasopressin V2 Receptor

Abstract: Activation of G protein-coupled receptors by agonists may result in the activation of one or more G proteins and recruitment of arrestins. The extent of the activation of each of these pathways depends on the intrinsic efficacy of the ligand. Quantification of intrinsic efficacy relative to a reference compound is essential for the development of novel compounds. In the operational model, changes in efficacy can be compensated by changes in the “functional” affinity, resulting in poorly defined values. To separate the effects of ligand affinity from the intrinsic activity of the receptor, we developed a Michaelis-Menten based quantification of G protein activation bias that uses experimentally measured ligand affinities and provides a single measure of ligand efficacy. We used it to evaluate the signaling of a promiscuous model receptor, the Vasopressin V2 receptor (V2R). Using BRET-based biosensors, we show that the V2R engages many different G proteins across all G protein subfamilies in response to its primary endogenous agonist, arginine vasopressin, including Gs and members of the Gi/o and G12/13 families. These signaling pathways are also activated by the synthetic peptide desmopressin, oxytocin, and the nonmammalian hormone vasotocin. We compared bias quantification using the operational model with Michaelis-Menten based quantification; the latter accurately quantified ligand efficacies despite large difference in ligand affinities. Together, these results showed that the V2R is promiscuous in its ability to engage several G proteins and that its’ signaling profile is biased by small structural changes in the ligand. SIGNIFICANCE STATEMENT By modelling the G protein activation as Michaelis-Menten reaction, we developed a novel way of quantifying signalling bias. V2R activates, or at least engages, G proteins from all G protein subfamilies, including Gi2, Gz, Gq, G12, and G13. Their relative activation may explain its Gs-independent signalling.

Molecular Mechanisms of Desensitization Underlying the Differential Effects of Formyl Peptide Receptor 2 Agonists on Cardiac Structure-Function Post Myocardial Infarction.

Abstract: Formyl peptide receptor 2 (FPR2) plays an integral role in the transition of macrophages from a pro-inflammatory program to one that is pro-resolving. FPR2-mediated stimulation of resolution post myocardial infarction has demonstrated efficacy in rodent models and is hypothesized to reduce progression into heart failure. FPR2 agonists that promote long-lasting receptor internalization can lead to persistent desensitization and diminished therapeutic benefits. In vitro signaling profiles and propensities for receptor desensitization of two clinically studied FPR2 agonists, namely, BMS-986235 and ACT-389949, were evaluated. In contrast to BMS-986235, pre-stimulation with ACT-389949 led to a decrease in its potency to inhibit cAMP production. Moreover, ACT-389949 displayed greater efficacy for β-arrestin recruitment, while efficacy of Gi activation was similar for both agonists. Following agonist-promoted FPR2 internalization, effective recycling to the plasma membrane was observed only with BMS-986235. Use of G protein-coupled receptor kinase (GRK) knock-out cells revealed a differential impact of GRK2 versus GRK5/6 on β-arrestin recruitment and Gi activation promoted by the two FPR2 agonists. In vivo, decreases of granulocytes in circulation were greatly diminished in mice treated with ACT-389949 but not for BMS-986235. With short-term dosing, both compounds induced a pro-resolution polarization state in cardiac monocyte/macrophages post myocardial infarction. By contrast, with long-term dosing, only BMS-986235 preserved the infarct wall thickness and increased left ventricular ejection fraction in a rat model of myocardial infarction. Altogether, the study shows that differences in the desensitization profiles induced by ACT-389949 and BMS-986235 at the molecular level may explain their distinct inflammatory/pro-resolving activities in vivo.

Visualization of real-time receptor endocytosis in dopamine neurons enabled by NTSR1-Venus knock-in mice

Abstract: Dopamine (DA) neurons are primarily concentrated in substantia nigra (SN) and ventral tegmental area (VTA). A subset of these neurons expresses the neurotensin receptor NTSR1 and its putative ligand neurotensin (Nts). NTSR1, a G protein-coupled receptor (GPCR), which classically activates Gαq/calcium signaling, is a potential route for modulating DA activity. Drug development efforts have been hampered by the receptor’s complex pharmacology and a lack of understanding about its endogenous location and signaling responses. Therefore, we have generated NTSR1-Venus knock-in (KI) mice to study NTSR1 receptors in their physiological context. In primary hippocampal neurons, we show that these animals express functional receptors that respond to agonists by increasing intracellular calcium release and trafficking to endosomes. Moreover, systemic agonist administration attenuates locomotion in KIs as it does in control animals. Mapping receptor protein expression at regional and cellular levels, located NTSR1-Venus on the soma and dendrites of dopaminergic SN/VTA neurons. Direct monitoring of receptor endocytosis, as a proxy for activation, enabled profiling of NTSR1 agonists in neurons, as well as acute SN/VTA containing brain slices. Taken together, NTSR1-Venus animals express traceable receptors that will improve understanding of NTSR1 and DA activities and more broadly how GPCRs act in vivo.

Comment replacer le citoyen dans la gouvernance des finances publiques ?

Abstract: La place du citoyen dans le processus de décision du système financier public est un sujet crucial. Il n’est pas nouveau, mais il se pose aujourd’hui, certes dans le cadre d’un environnement marqué par le Covid-19, mais plus encore dans celui d’une métamorphose de l’État. Celui-ci, tout au long de ces quarante dernières années, s’est transformé en profondeur sous les effets de crises économiques. Des crises qui ont donné lieu en France et ailleurs à des réformes du droit et de la gestion budgétaire lesquels ont modifié le rapport citoyen/gouvernance financière publique. Il n’en demeure pas moins que malgré les professions de foi, voire les législations allant dans le sens d’une démocratisation du dispositif, le rôle du citoyen dans le processus de décision budgétaire est marqué par une ambiguïté ; laquelle tient à ce que ce processus se trouve inadapté à la complexité des sociétés contemporaines. Par ailleurs, c’est le consentement à l’impôt autrement dit le cœur du modèle financier public, voire de la démocratie, qui est victime d’un délitement. En réponse, la légitimité du modèle fiscal doit être réhabilitée en donnant un sens à l’impôt correspondant aux attentes des citoyens.