Publications by authors named "Patrick M Sexton"

245 Publications

Roles of Cholecystokinin in the Nutritional Continuum. Physiology and Potential Therapeutics.

Front Endocrinol (Lausanne) 2021 2;12:684656. Epub 2021 Jun 2.

Drug Discovery Biology theme, Monash Institute for Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia.

Cholecystokinin is a gastrointestinal peptide hormone with important roles in metabolic physiology and the maintenance of normal nutritional status, as well as potential roles in the prevention and management of obesity, currently one of the dominant causes of direct or indirect morbidity and mortality. In this review, we discuss the roles of this hormone and its receptors in maintaining nutritional homeostasis, with a particular focus on appetite control. Targeting this action led to the development of full agonists of the type 1 cholecystokinin receptor that have so far failed in clinical trials for obesity. The possible reasons for clinical failure are discussed, along with alternative pharmacologic strategies to target this receptor for prevention and management of obesity, including development of biased agonists and allosteric modulators. Cellular cholesterol is a natural modulator of the type 1 cholecystokinin receptor, with elevated levels disrupting normal stimulus-activity coupling. The molecular basis for this is discussed, along with strategies to overcome this challenge with a corrective positive allosteric modulator. There remains substantial scope for development of drugs to target the type 1 cholecystokinin receptor with these new pharmacologic strategies and such drugs may provide new approaches for treatment of obesity.
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http://dx.doi.org/10.3389/fendo.2021.684656DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8206557PMC
June 2021

Structures of the human cholecystokinin 1 (CCK1) receptor bound to Gs and Gq mimetic proteins provide insight into mechanisms of G protein selectivity.

PLoS Biol 2021 Jun 4;19(6):e3001295. Epub 2021 Jun 4.

Drug Discovery Biology Theme, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.

G protein-coupled receptors (GPCRs) are critical regulators of cellular function acting via heterotrimeric G proteins as their primary transducers with individual GPCRs capable of pleiotropic coupling to multiple G proteins. Structural features governing G protein selectivity and promiscuity are currently unclear. Here, we used cryo-electron microscopy (cryo-EM) to determine structures of the cholecystokinin (CCK) type 1 receptor (CCK1R) bound to the CCK peptide agonist, CCK-8 and 2 distinct transducer proteins, its primary transducer Gq, and the more weakly coupled Gs. As seen with other Gq/11-GPCR complexes, the Gq-α5 helix (αH5) bound to a relatively narrow pocket in the CCK1R core. Surprisingly, the backbone of the CCK1R and volume of the G protein binding pocket were essentially equivalent when Gs was bound, with the Gs αH5 displaying a conformation that arises from "unwinding" of the far carboxyl-terminal residues, compared to canonically Gs coupled receptors. Thus, integrated changes in the conformations of both the receptor and G protein are likely to play critical roles in the promiscuous coupling of individual GPCRs.
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http://dx.doi.org/10.1371/journal.pbio.3001295DOI Listing
June 2021

Evolving cryo-EM structural approaches for GPCR drug discovery.

Structure 2021 May 2. Epub 2021 May 2.

Drug Discovery Biology Theme, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville 3052, VIC, Australia; ARC Centre for Cryo-Electron Microscopy of Membrane Proteins, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville 3052, VIC, Australia. Electronic address:

G protein-coupled receptors (GPCRs) are the largest class of cell surface drug targets. Advances in stabilization of GPCR:transducer complexes, together with improvements in cryoelectron microscopy (cryo-EM) have recently been applied to structure-assisted drug design for GPCR agonists. Nonetheless, limitations in the commercial application of these approaches, including the use of nanobody 35 (Nb35) to aid complex stabilization and the high cost of 300 kV imaging, have restricted broad application of cryo-EM in drug discovery. Here, using the PF 06882961-bound GLP-1R as exemplar, we validated the formation of stable complexes with a modified Gs protein in the absence of Nb35. In parallel, we compare 200 versus 300 kV image acquisition using a Falcon 4 or K3 direct electron detector. Moreover, the 200 kV Glacios-Falcon 4 yielded a 3.2 Å map with clear density for bound drug and multiple structurally ordered waters. Our work paves the way for broader commercial application of cryo-EM for GPCR drug discovery.
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http://dx.doi.org/10.1016/j.str.2021.04.008DOI Listing
May 2021

Pharmacological Insights Into Safety and Efficacy Determinants for the Development of Adenosine Receptor Biased Agonists in the Treatment of Heart Failure.

Front Pharmacol 2021 11;12:628060. Epub 2021 Mar 11.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia.

Adenosine A receptors (AR) are a potential target for cardiac injury treatment due to their cardioprotective/antihypertrophic actions, but drug development has been hampered by on-target side effects such as bradycardia and altered renal hemodynamics. Biased agonism has emerged as an attractive mechanism for AR-mediated cardioprotection that is haemodynamically safe. Here we investigate the pre-clinical pharmacology, efficacy and side-effect profile of the AR agonist neladenoson, shown to be safe but ineffective in phase IIb trials for the treatment of heart failure. We compare this agent with the well-characterized, pan-adenosine receptor (AR) agonist NECA, capadenoson, and the AR biased agonist VCP746, previously shown to be safe and cardioprotective in pre-clinical models of heart failure. We show that like VCP746, neladenoson is biased away from Ca influx relative to NECA and the cAMP pathway at the AR, a profile predictive of a lack of adenosine-like side effects. Additionally, neladenoson was also biased away from the MAPK pathway at the AR. In contrast to VCP746, which displays more 'adenosine-like' signaling at the AR, neladenoson was a highly selective AR agonist, with biased, weak agonism at the AR. Together these results show that unwanted hemodynamic effects of AR agonists can be avoided by compounds biased away from Ca influx relative to cAMP, relative to NECA. The failure of neladenoson to reach primary endpoints in clinical trials suggests that AR-mediated cAMP inhibition may be a poor indicator of effectiveness in chronic heart failure. This study provides additional information that can aid future screening and/or design of improved AR agonists that are safe and efficacious in treating heart failure in patients.
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http://dx.doi.org/10.3389/fphar.2021.628060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7991592PMC
March 2021

AM833 Is a Novel Agonist of Calcitonin Family G Protein-Coupled Receptors: Pharmacological Comparison with Six Selective and Nonselective Agonists.

J Pharmacol Exp Ther 2021 Jun 16;377(3):417-440. Epub 2021 Mar 16.

Drug Discovery Biology Theme, Monash Institute of Pharmaceutical Sciences, Parkville, Victoria, Australia (M.M.F., P.K., T.T.T., G.M., C.A.H., P.Z., S.G.B.F., D.W., P.M.S.); Research and Development, Novo Nordisk, Denmark (T.K., T.R.C.); and ARC Centre for Cryo-electron Microscopy of Membrane Proteins, Monash Institute of Pharmaceutical Sciences, Parkville, Victoria, Australia (D.W., P.M.S.)

Obesity and associated comorbidities are a major health burden, and novel therapeutics to help treat obesity are urgently needed. There is increasing evidence that targeting the amylin receptors (AMYRs), heterodimers of the calcitonin G protein-coupled receptor (CTR) and receptor activity-modifying proteins, improves weight control and has the potential to act additively with other treatments such as glucagon-like peptide-1 receptor agonists. Recent data indicate that AMYR agonists, which can also independently activate the CTR, may have improved efficacy for treating obesity, even though selective activation of CTRs is not efficacious. AM833 (cagrilintide) is a novel lipidated amylin analog that is undergoing clinical trials as a nonselective AMYR and CTR agonist. In the current study, we have investigated the pharmacology of AM833 across 25 endpoints and compared this peptide with AMYR selective and nonselective lipidated analogs (AM1213 and AM1784), and the clinically used peptide agonists pramlintide (AMYR selective) and salmon CT (nonselective). We also profiled human CT and rat amylin as prototypical selective agonists of CTR and AMYRs, respectively. Our results demonstrate that AM833 has a unique pharmacological profile across diverse measures of receptor binding, activation, and regulation. SIGNIFICANCE STATEMENT: AM833 is a novel nonselective agonist of calcitonin family receptors that has demonstrated efficacy for the treatment of obesity in phase 2 clinical trials. This study demonstrates that AM833 has a unique pharmacological profile across diverse measures of receptor binding, activation, and regulation when compared with other selective and nonselective calcitonin receptor and amylin receptor agonists. The present data provide mechanistic insight into the actions of AM833.
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http://dx.doi.org/10.1124/jpet.121.000567DOI Listing
June 2021

Structure and dynamics of the CGRP receptor in apo and peptide-bound forms.

Science 2021 04 18;372(6538). Epub 2021 Feb 18.

Drug Discovery Biology Theme, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville 3052, Victoria, Australia.

G protein-coupled receptors (GPCRs) are key regulators of information transmission between cells and organs. Despite this, we have only a limited understanding of the behavior of GPCRs in the apo state and the conformational changes upon agonist binding that lead to G protein recruitment and activation. We expressed and purified unmodified apo and peptide-bound calcitonin gene-related peptide (CGRP) receptors from insect cells to determine their cryo-electron microscopy (cryo-EM) structures, and we complemented these with analysis of protein conformational dynamics using hydrogen-deuterium exchange mass spectrometry and three-dimensional variance analysis of the cryo-EM data. Together with our previously published structure of the active, Gs-bound CGRP receptor complex, our work provides insight into the mechanisms of class B1 GPCR activation.
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http://dx.doi.org/10.1126/science.abf7258DOI Listing
April 2021

Fine Tuning Muscarinic Acetylcholine Receptor Signaling Through Allostery and Bias.

Front Pharmacol 2020 29;11:606656. Epub 2021 Jan 29.

Drug Discovery Biology, Monash Institute for Pharmaceutical Research, Monash University, Parkville, VIC, Australia.

The M and M muscarinic acetylcholine receptors (mAChRs) are highly pursued drug targets for neurological diseases, in particular for Alzheimer's disease and schizophrenia. Due to high sequence homology, selective targeting of any of the M-M mAChRs through the endogenous ligand binding site has been notoriously difficult to achieve. With the discovery of highly subtype selective mAChR positive allosteric modulators in the new millennium, selectivity through targeting an allosteric binding site has opened new avenues for drug discovery programs. However, some hurdles remain to be overcome for these promising new drug candidates to progress into the clinic. One challenge is the potential for on-target side effects, such as for the M mAChR where over-activation of the receptor by orthosteric or allosteric ligands can be detrimental. Therefore, in addition to receptor subtype selectivity, a drug candidate may need to exhibit a biased signaling profile to avoid such on-target adverse effects. Indeed, recent studies in mice suggest that allosteric modulators for the M mAChR that bias signaling toward specific pathways may be therapeutically important. This review brings together details on the signaling pathways activated by the M and M mAChRs, evidence of biased agonism at these receptors, and highlights pathways that may be important for developing new subtype selective allosteric ligands to achieve therapeutic benefit.
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http://dx.doi.org/10.3389/fphar.2020.606656DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7878563PMC
January 2021

Restoring Agonist Function at a Chemogenetically Modified M Muscarinic Acetylcholine Receptor.

ACS Chem Neurosci 2020 12 16;11(24):4270-4279. Epub 2020 Nov 16.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria 3052, Australia.

Designer receptors exclusively activated by designer drugs (DREADDs) have been successfully employed to activate signaling pathways associated with specific muscarinic acetylcholine receptor (mAChR) subtypes. The M DREADD mAChR displays minimal responsiveness to the endogenous agonist acetylcholine (ACh) but responds to clozapine--oxide (CNO), an otherwise pharmacologically inert ligand. We have previously shown that benzyl quinolone carboxylic acid (BQCA), an M mAChR positive allosteric modulator (PAM), can rescue ACh responsiveness at these receptors. However, whether this effect is chemotype specific or applies to next-generation M PAMs with distinct scaffolds is unknown. Here, we reveal that new M PAMs restore ACh function at the M DREADD while modulating ACh binding at the M wild-type mAChR. Importantly, we demonstrate that the modulation of ACh function by M PAMs is translated using transgenic M DREADD mice. Our data provide important insights into mechanisms that define allosteric ligand modulation of agonist affinity vs efficacy and how these effects play out in the regulation of responses.
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http://dx.doi.org/10.1021/acschemneuro.0c00540DOI Listing
December 2020

Differential GLP-1R Binding and Activation by Peptide and Non-peptide Agonists.

Mol Cell 2020 11 6;80(3):485-500.e7. Epub 2020 Oct 6.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia. Electronic address:

Peptide drugs targeting class B1 G-protein-coupled receptors (GPCRs) can treat multiple diseases; however, there remains substantial interest in the development of orally delivered non-peptide drugs. Here, we reveal unexpected overlap between signaling and regulation of the glucagon-like peptide-1 (GLP-1) receptor by the non-peptide agonist PF 06882961 and GLP-1 that was not observed for another compound, CHU-128. Compounds from these patent series, including PF 06882961, are currently in clinical trials for treatment of type 2 diabetes. High-resolution cryoelectron microscopy (cryo-EM) structures reveal that the binding sites for PF 06882961 and GLP-1 substantially overlap, whereas CHU-128 adopts a unique binding mode with a more open receptor conformation at the extracellular face. Structural differences involving extensive water-mediated hydrogen bond networks could be correlated to functional data to understand how PF 06882961, but not CHU-128, can closely mimic the pharmacological properties of GLP-1. These findings will facilitate rational structure-based discovery of non-peptide agonists targeting class B GPCRs.
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http://dx.doi.org/10.1016/j.molcel.2020.09.020DOI Listing
November 2020

Development of Novel 4-Arylpyridin-2-one and 6-Arylpyrimidin-4-one Positive Allosteric Modulators of the M Muscarinic Acetylcholine Receptor.

ChemMedChem 2021 Jan 25;16(1):216-233. Epub 2020 Sep 25.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, 3052, Victoria, Australia.

This study investigated the structure-activity relationships of 4-phenylpyridin-2-one and 6-phenylpyrimidin-4-one M muscarinic acetylcholine receptor (M mAChRs) positive allosteric modulators (PAMs). The presented series focuses on modifications to the core and top motif of the reported leads, MIPS1650 (1) and MIPS1780 (2). Profiling of our novel analogues showed that these modifications result in more nuanced effects on the allosteric properties compared to our previous compounds with alterations to the biaryl pendant. Further pharmacological characterisation of the selected compounds in radioligand binding, IP accumulation and β-arrestin 2 recruitment assays demonstrated that, despite primarily acting as affinity modulators, the PAMs displayed different pharmacological properties across the two cellular assays. The novel PAM 7 f is a potential lead candidate for further development of peripherally restricted M PAMs, due to its lower blood-brain-barrier (BBB) permeability and improved exposure in the periphery compared to lead 2.
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http://dx.doi.org/10.1002/cmdc.202000540DOI Listing
January 2021

Structure and dynamics of the active Gs-coupled human secretin receptor.

Nat Commun 2020 08 18;11(1):4137. Epub 2020 Aug 18.

Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, AZ, 85259, USA.

The class B secretin GPCR (SecR) has broad physiological effects, with target potential for treatment of metabolic and cardiovascular disease. Molecular understanding of SecR binding and activation is important for its therapeutic exploitation. We combined cryo-electron microscopy, molecular dynamics, and biochemical cross-linking to determine a 2.3 Å structure, and interrogate dynamics, of secretin bound to the SecR:Gs complex. SecR exhibited a unique organization of its extracellular domain (ECD) relative to its 7-transmembrane (TM) core, forming more extended interactions than other family members. Numerous polar interactions formed between secretin and the receptor extracellular loops (ECLs) and TM helices. Cysteine-cross-linking, cryo-electron microscopy multivariate analysis and molecular dynamics simulations revealed that interactions between peptide and receptor were dynamic, and suggested a model for initial peptide engagement where early interactions between the far N-terminus of the peptide and SecR ECL2 likely occur following initial binding of the peptide C-terminus to the ECD.
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http://dx.doi.org/10.1038/s41467-020-17791-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7435274PMC
August 2020

Evaluation of biased agonism mediated by dual agonists of the GLP-1 and glucagon receptors.

Biochem Pharmacol 2020 10 17;180:114150. Epub 2020 Jul 17.

Drug Discovery Biology Theme, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville 3052, Victoria, Australia. Electronic address:

Metabolic diseases such as obesity, diabetes, and their comorbidities have converged as one of the most serious health concerns on a global scale. Selective glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) agonists are one of the major therapeutics for type 2 diabetes and obesity. Polypharmacological approaches that enable modulation of multiple metabolic targets in a single drug have emerged as a potential avenue to improve therapeutic outcomes. Among numerous peptides under development are those targeting the GLP-1R and either the glucagon receptor (GCGR), glucose-dependent insulinotropic peptide receptor (GIPR) or all 3 receptors, as dual- or tri- peptide agonists. Despite many of them entering into clinical trials, current development has been based on only a limited understanding of the spectrum of potential pharmacological properties of these ligands beyond binding selectivity. In the present study, we examined the potential for agonists that target both GLP-1R and GCGR to exhibit biased agonism, comparing activity across proximal activation of Gs protein, cAMP accumulation, pERK1/2 and β-arrestin recruitment. Three distinct dual agonists that have different relative cAMP production potency for GLP-1R versus GCGR, "peptide 15", MEDI0382 and SAR425899, and one triagonist of the GLP-1R, GCGR and GIPR were examined. We demonstrated that all novel peptides have distinct biased agonism profiles relative to either of the cognate agonists of the receptors, and to each other. This is an important feature of the pharmacology of this drug class that needs to be considered alongside selectivity, bioavailability and pharmacokinetics for rational optimization of new therapeutics.
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http://dx.doi.org/10.1016/j.bcp.2020.114150DOI Listing
October 2020

Targeting Antibiotic Resistance: From Diagnostics to Novel Antibiotics.

ACS Pharmacol Transl Sci 2020 Jun 3;3(3):371-372. Epub 2020 Jun 3.

Monash Institute of Pharmaceutical Sciences, Monash University Parkville Campus, Parkville, Melbourne 3052, Australia.

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http://dx.doi.org/10.1021/acsptsci.0c00053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7296534PMC
June 2020

In the Loop: Extrastriatal Regulation of Spiny Projection Neurons by GPR52.

ACS Chem Neurosci 2020 07 7;11(14):2066-2076. Epub 2020 Jul 7.

Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, Victoria 3010, Australia.

GPR52 is a Gα-coupled orphan receptor identified as a putative target for the treatment of schizophrenia. The unique expression and signaling profile of GPR52 in key areas of dopamine and glutamate dysregulation suggests its activation may resolve both cortical and striatal dysfunction in the disorder. GPR52 mRNA is enriched in the striatum, almost exclusively on dopamine D-expressing medium spiny neurons (MSNs), and to a lesser extent in the cortex, predominantly on D-expressing pyramidal neurons. Synthetic, small molecule GPR52 agonists are effective in preclinical models of psychosis; however, the relative contribution of cortical and striatal GPR52 is unknown. Here we show that the GPR52 agonist, 3-BTBZ, inhibits phencyclidine-induced hyperlocomotor activity to a greater degree than amphetamine-induced motor effects, suggesting a mechanism beyond functional antagonism of striatal dopamine D receptor signaling. Using DARPP-32 phosphorylation and electrophysiological recordings in either striatopallidal or striatonigral MSNs, we were surprised to find no significant effect of 3-BTBZ in striatopallidal MSNs, but GPR52-mediated effects in striatonigral MSNs, where its mRNA is absent. 3-BTBZ increases phosphorylation of T75 on DARPP-32 in striatonigral MSNs, an effect that was dependent on cortical inputs. A similar role for GPR52 in regulating extrastriatal glutamatergic drive onto striatonigral MSNs was also evident in recordings of spontaneous excitatory postsynaptic currents and was shown to be dependent on the metabotropic glutamate (mGlu) receptor subtype 1. Our results demonstrate that GPR52-mediated regulation of striatal function depends heavily on extrastriatal inputs, which may further support its utility as a novel target for the treatment of schizophrenia.
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http://dx.doi.org/10.1021/acschemneuro.0c00197DOI Listing
July 2020

Cryo-electron microscopy structure of the glucagon receptor with a dual-agonist peptide.

J Biol Chem 2020 07 5;295(28):9313-9325. Epub 2020 May 5.

Monash Institute of Pharmaceutical Sciences, Drug Discovery Biology, Monash University, Parkville, Victoria, Australia

Unimolecular dual agonists of the glucagon (GCG) receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R) are a new class of drugs that are potentially superior to GLP-1R-specific agonists for the management of metabolic disease. The dual-agonist, peptide 15 (P15), is a glutamic acid 16 analog of GCG with GLP-1 peptide substitutions between amino acids 17 and 24 that has potency equivalent to those of the cognate peptide agonists at the GCGR and GLP-1R. Here, we have used cryo-EM to solve the structure of an active P15-GCGR-G complex and compared this structure to our recently published structure of the GCGR-G complex bound to GCG. This comparison revealed that P15 has a reduced interaction with the first extracellular loop (ECL1) and the top of transmembrane segment 1 (TM1) such that there is increased mobility of the GCGR extracellular domain and at the C terminus of the peptide compared with the GCG-bound receptor. We also observed a distinct conformation of ECL3 and could infer increased mobility of the far N-terminal His-1 residue in the P15-bound structure. These regions of conformational variance in the two peptide-bound GCGR structures were also regions that were distinct between GCGR structures and previously published peptide-bound structures of the GLP-1R, suggesting that greater conformational dynamics may contribute to the increased efficacy of P15 in activation of the GLP-1R compared with GCG. The variable domains in this receptor have previously been implicated in biased agonism at the GLP-1R and could result in altered signaling of P15 at the GCGR compared with GCG.
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http://dx.doi.org/10.1074/jbc.RA120.013793DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7363120PMC
July 2020

Pharmacological characterization of mono-, dual- and tri-peptidic agonists at GIP and GLP-1 receptors.

Biochem Pharmacol 2020 07 29;177:114001. Epub 2020 Apr 29.

Drug Discovery Biology Theme, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, 3052 Victoria, Australia. Electronic address:

Glucose-dependent insulinotropic peptide (GIP) is an incretin hormone with physiological roles in adipose tissue, the central nervous system and bone metabolism. While selective ligands for GIP receptor (GIPR) have not been advanced for disease treatment, dual and triple agonists of GIPR, in conjunction with that of glucagon-like peptide-1 (GLP-1) and glucagon receptors, are currently in clinical trials, with an expectation of enhanced efficacy beyond that of GLP-1 receptor (GLP-1R) agonist monotherapy for diabetic patients. Consequently, it is important to understand the pharmacological behavior of such drugs. In this study, we have explored signaling pathway specificity and the potential for biased agonism of mono-, dual- and tri-agonists of GIPR using human embryonic kidney 293 (HEK293) cells recombinantly expressing human GIPR or GLP-1R. Compared to GIP(1-42), the GIPR mono-agonists Pro3GIP and Lys3GIP are biased towards ERK1/2 phosphorylation (pERK1/2) relative to cAMP accumulation at GIPR, whereas the triple agonist at GLP-1R/GCGR/GIPR is biased towards pERK1/2 relative to β-arrestin2 recruitment. Moreover, the dual GIPR/GLP-1R agonist, LY3298176, is biased towards pERK1/2 relative to cAMP accumulation at both GIPR and GLP-1R compared to their respective endogenous ligands. These data reveal novel pharmacological properties of potential therapeutic agents that may impact on diversity in clinical responses.
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http://dx.doi.org/10.1016/j.bcp.2020.114001DOI Listing
July 2020

Structure and Dynamics of Adrenomedullin Receptors AM and AM Reveal Key Mechanisms in the Control of Receptor Phenotype by Receptor Activity-Modifying Proteins.

ACS Pharmacol Transl Sci 2020 Apr 20;3(2):263-284. Epub 2020 Mar 20.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville 3052, Victoria, Australia.

Adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) receptors are critically important for metabolism, vascular tone, and inflammatory response. AM receptors are also required for normal lymphatic and blood vascular development and angiogenesis. They play a pivotal role in embryo implantation and fertility and can provide protection against hypoxic and oxidative stress. CGRP and AM receptors are heterodimers of the calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1) (CGRPR), as well as RAMP2 or RAMP3 (AMR and AMR, respectively). However, the mechanistic basis for RAMP modulation of CLR phenotype is unclear. In this study, we report the cryo-EM structure of the AMR in complex with AM and Gs at a global resolution of 3.0 Å, and structures of the AMR in complex with either AM or intermedin/adrenomedullin 2 (AM2) and Gs at 2.4 and 2.3 Å, respectively. The structures reveal distinctions in the primary orientation of the extracellular domains (ECDs) relative to the receptor core and distinct positioning of extracellular loop 3 (ECL3) that are receptor-dependent. Analysis of dynamic data present in the cryo-EM micrographs revealed additional distinctions in the extent of mobility of the ECDs. Chimeric exchange of the linker region of the RAMPs connecting the TM helix and the ECD supports a role for this segment in controlling receptor phenotype. Moreover, a subset of the motions of the ECD appeared coordinated with motions of the G protein relative to the receptor core, suggesting that receptor ECD dynamics could influence G protein interactions. This work provides fundamental advances in our understanding of GPCR function and how this can be allosterically modulated by accessory proteins.
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http://dx.doi.org/10.1021/acsptsci.9b00080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7155201PMC
April 2020

Molecular Mechanisms of Class B GPCR Activation: Insights from Adrenomedullin Receptors.

ACS Pharmacol Transl Sci 2020 Apr 26;3(2):246-262. Epub 2020 Feb 26.

School of Biological Sciences, University of Auckland, Auckland, 1010, New Zealand.

Adrenomedullin (AM) is a 52 amino acid peptide that plays a regulatory role in the vasculature. Receptors for AM comprise the class B G protein-coupled receptor, the calcitonin-like receptor (CLR), in complex with one of three receptor activity-modifying proteins (RAMPs). The C-terminus of AM is involved in binding to the extracellular domain of the receptor, while the N-terminus is proposed to interact with the juxtamembranous portion of the receptor to activate signaling. There is currently limited information on the molecular determinants involved in AM signaling, thus we set out to define the importance of the AM N-terminus through five signaling pathways (cAMP production, ERK phosphorylation, CREB phosphorylation, Akt phosphorylation, and IP production). We characterized the three CLR:RAMP complexes through the five pathways, finding that each had a distinct repertoire of intracellular signaling pathways that it is able to regulate. We then performed an alanine scan of AM from residues 15-31 and found that most residues could be substituted with only small effects on signaling, and that most substitutions affected signaling through all receptors and pathways in a similar manner. We identify F18, T20, L26, and I30 as being critical for AM function, while also identifying an analogue (AM G19A) which has unique signaling properties relative to the unmodified AM. We interpret our findings in the context of new structural information, highlighting the complementary nature of structural biology and functional assays.
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http://dx.doi.org/10.1021/acsptsci.9b00083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7155197PMC
April 2020

Deconvoluting the Molecular Control of Binding and Signaling at the Amylin 3 Receptor: RAMP3 Alters Signal Propagation through Extracellular Loops of the Calcitonin Receptor.

ACS Pharmacol Transl Sci 2019 Jun 18;2(3):183-197. Epub 2019 Mar 18.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville 3052, Victoria, Australia.

Amylin is coexpressed with insulin in pancreatic islet β-cells and has potent effects on gastric emptying and food intake. The effect of amylin on satiation has been postulated to involve AMY receptors (AMYR) that are heteromers of the calcitonin receptor (CTR) and receptor activity-modifying protein 3 (RAMP3). Understanding the molecular control of signaling through the AMYR is thus important for peptide drug targeting of this receptor. We have previously used alanine scanning mutagenesis to study the contribution of the extracellular surface of the CTR to binding and signaling initiated by calcitonin (CT) and related peptides (Dal Maso, E., . (2019) The molecular control of calcitonin receptor signaling. , 31-51). That work revealed ligand- and pathway-specific effects of mutation, with extracellular loops (ECLs) 2 and 3 particularly important in the distinct propagation of signaling mediated by individual peptides. In the current study, we have used equivalent alanine scanning of ECL2 and ECL3 of the CTR in the context of coexpression with RAMP3 to form AMYRs, to examine functional affinity and efficacy of peptides in cAMP accumulation and extracellular signal-regulated kinase (ERK) phosphorylation (pERK). The effect of mutation was determined on representatives of the three major distinct classes of CT peptide, salmon CT (sCT), human CT (hCT), and porcine CT (pCT), as well as rat amylin (rAmy) or human α-CGRP (calcitonin gene-related peptide, hCGRP) whose potency is enhanced by RAMP interaction. We demonstrate that the dynamic nature of CTR ECL2 and ECL3 in propagation of signaling is fundamentally altered when complexed with RAMP3 to form the AMYR, despite only having predicted direct interactions with ECL2. Moreover, the work shows that the role of these loops in receptor signaling is highly peptide dependent, illustrating that even subtle changes to peptide sequence may change signaling output downstream of the receptor.
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http://dx.doi.org/10.1021/acsptsci.9b00010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7088965PMC
June 2019

in 2019.

Authors:
Patrick M Sexton

ACS Pharmacol Transl Sci 2019 Feb 8;2(1). Epub 2019 Feb 8.

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http://dx.doi.org/10.1021/acsptsci.9b00005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7088877PMC
February 2019

Rational development of a high-affinity secretin receptor antagonist.

Biochem Pharmacol 2020 07 23;177:113929. Epub 2020 Mar 23.

Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Scottsdale, AZ 85259, United States. Electronic address:

The secretin receptor is a prototypic class B GPCR with substantial and broad pharmacologic importance. The aim of this project was to develop a high affinity selective antagonist as a new and important pharmacologic tool and to aid stabilization of this receptor in an inactive conformation for ultimate structural characterization. Amino-terminal truncation of the natural 27-residue ligand reduced biological activity, but also markedly reduced binding affinity. This was rationally and experimentally overcome with lactam stabilization of helical structure and with replacement of residues with natural and unnatural amino acids. A key new step in this effort was the replacement of peptide residue Leu with L-cyclohexylalanine (Cha) to enhance potential hydrophobic interactions with receptor residues Leu, Val, and Phe that were predicted from molecular modeling. Alanine-replacement mutagenesis of these residues markedly affected ligand binding and biological activity. The optimal antagonist ligand, (Y,c[E,K],I,Cha,R)sec(6-27), exhibited high binding affinity (4 nM), similar to natural secretin, and exhibited no demonstrable biological activity to stimulate cAMP accumulation, intracellular calcium mobilization, or β-arrestin-2 translocation. It acts as an orthosteric competitive antagonist, predicted to bind within the peptide-binding groove in the receptor extracellular domain. The analogous peptide that was one residue longer, retaining Thr, exhibited partial agonist activity, while further truncation of even a single residue (Phe) reduced binding affinity. This sec(6-27)-based peptide will be an important new tool for pharmacological and structural studies.
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http://dx.doi.org/10.1016/j.bcp.2020.113929DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7299832PMC
July 2020

Structural basis of G and G recognition by the human glucagon receptor.

Science 2020 03;367(6484):1346-1352

CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.

Class B G protein-coupled receptors, an important class of therapeutic targets, signal mainly through the G class of heterotrimeric G proteins, although they do display some promiscuity in G protein binding. Using cryo-electron microscopy, we determined the structures of the human glucagon receptor (GCGR) bound to glucagon and distinct classes of heterotrimeric G proteins, G or G These two structures adopt a similar open binding cavity to accommodate G and G The G binding selectivity of GCGR is explained by a larger interaction interface, but there are specific interactions that affect G more than G binding. Conformational differences in the receptor intracellular loops were found to be key selectivity determinants. These distinctions in transducer engagement were supported by mutagenesis and functional studies.
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http://dx.doi.org/10.1126/science.aaz5346DOI Listing
March 2020

Biased M1-muscarinic-receptor-mutant mice inform the design of next-generation drugs.

Nat Chem Biol 2020 03 20;16(3):240-249. Epub 2020 Feb 20.

The Centre for Translational Pharmacology, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK.

Cholinesterase inhibitors, the current frontline symptomatic treatment for Alzheimer's disease (AD), are associated with low efficacy and adverse effects. M1 muscarinic acetylcholine receptors (M1 mAChRs) represent a potential alternate therapeutic target; however, drug discovery programs focused on this G protein-coupled receptor (GPCR) have failed, largely due to cholinergic adverse responses. Employing novel chemogenetic and phosphorylation-deficient, G protein-biased, mouse models, paired with a toolbox of probe molecules, we establish previously unappreciated pharmacologically targetable M1 mAChR neurological processes, including anxiety-like behaviors and hyper-locomotion. By mapping the upstream signaling pathways regulating these responses, we determine the importance of receptor phosphorylation-dependent signaling in driving clinically relevant outcomes and in controlling adverse effects including 'epileptic-like' seizures. We conclude that M1 mAChR ligands that promote receptor phosphorylation-dependent signaling would protect against cholinergic adverse effects in addition to driving beneficial responses such as learning and memory and anxiolytic behavior relevant for the treatment of AD.
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http://dx.doi.org/10.1038/s41589-019-0453-9DOI Listing
March 2020

Molecular Basis for Hormone Recognition and Activation of Corticotropin-Releasing Factor Receptors.

Mol Cell 2020 02 30;77(3):669-680.e4. Epub 2020 Jan 30.

The CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China; University of Chinese Academy of Sciences, Beijing 100049, China. Electronic address:

Corticotropin-releasing factor (CRF) and the three related peptides urocortins 1-3 (UCN1-UCN3) are endocrine hormones that control the stress responses by activating CRF1R and CRF2R, two members of class B G-protein-coupled receptors (GPCRs). Here, we present two cryoelectron microscopy (cryo-EM) structures of UCN1-bound CRF1R and CRF2R with the stimulatory G protein. In both structures, UCN1 adopts a single straight helix with its N terminus dipped into the receptor transmembrane bundle. Although the peptide-binding residues in CRF1R and CRF2R are different from other members of class B GPCRs, the residues involved in receptor activation and G protein coupling are conserved. In addition, both structures reveal bound cholesterol molecules to the receptor transmembrane helices. Our structures define the basis of ligand-binding specificity in the CRF receptor-hormone system, establish a common mechanism of class B GPCR activation and G protein coupling, and provide a paradigm for studying membrane protein-lipid interactions for class B GPCRs.
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http://dx.doi.org/10.1016/j.molcel.2020.01.013DOI Listing
February 2020

Toward a Structural Understanding of Class B GPCR Peptide Binding and Activation.

Mol Cell 2020 02 30;77(3):656-668.e5. Epub 2020 Jan 30.

Drug Discovery Biology and Department of Pharmacology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville 3052, VIC, Australia; School of Pharmacy, Fudan University, Shanghai 201203, China. Electronic address:

Class B G protein-coupled receptors (GPCRs) are important therapeutic targets for major diseases. Here, we present structures of peptide and Gs-bound pituitary adenylate cyclase-activating peptide, PAC1 receptor, and corticotropin-releasing factor (CRF), (CRF1) receptor. Together with recently solved structures, these provide coverage of the major class B GPCR subfamilies. Diverse orientations of the extracellular domain to the receptor core in different receptors are at least partially dependent on evolutionary conservation in the structure and nature of peptide interactions. Differences in peptide interactions to the receptor core also influence the interlinked TM2-TM1-TM6/ECL3/TM7 domain, and this is likely important in their diverse signaling. However, common conformational reorganization of ECL2, linked to reorganization of ICL2, modulates G protein contacts. Comparison between receptors reveals ICL2 as a key domain forming dynamic G protein interactions in a receptor- and ligand-specific manner. This work advances our understanding of class B GPCR activation and Gs coupling.
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http://dx.doi.org/10.1016/j.molcel.2020.01.012DOI Listing
February 2020

Activation of the GLP-1 receptor by a non-peptidic agonist.

Nature 2020 01 8;577(7790):432-436. Epub 2020 Jan 8.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia.

Class B G-protein-coupled receptors are major targets for the treatment of chronic diseases, including diabetes and obesity. Structures of active receptors reveal peptide agonists engage deep within the receptor core, leading to an outward movement of extracellular loop 3 and the tops of transmembrane helices 6 and 7, an inward movement of transmembrane helix 1, reorganization of extracellular loop 2 and outward movement of the intracellular side of transmembrane helix 6, resulting in G-protein interaction and activation. Here we solved the structure of a non-peptide agonist, TT-OAD2, bound to the glucagon-like peptide-1 (GLP-1) receptor. Our structure identified an unpredicted non-peptide agonist-binding pocket in which reorganization of extracellular loop 3 and transmembrane helices 6 and 7 manifests independently of direct ligand interaction within the deep transmembrane domain pocket. TT-OAD2 exhibits biased agonism, and kinetics of G-protein activation and signalling that are distinct from peptide agonists. Within the structure, TT-OAD2 protrudes beyond the receptor core to interact with the lipid or detergent, providing an explanation for the distinct activation kinetics that may contribute to the clinical efficacy of this compound series. This work alters our understanding of the events that drive the activation of class B receptors.
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http://dx.doi.org/10.1038/s41586-019-1902-zDOI Listing
January 2020

Crystal structure of the M muscarinic acetylcholine receptor.

Proc Natl Acad Sci U S A 2019 12 26;116(51):26001-26007. Epub 2019 Nov 26.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC 3052, Australia;

The human M muscarinic acetylcholine receptor (mAChR) has recently emerged as an exciting therapeutic target for treating a range of disorders, including drug addiction. However, a lack of structural information for this receptor subtype has limited further drug development and validation. Here we report a high-resolution crystal structure of the human M mAChR bound to the clinically used inverse agonist, tiotropium. This structure allowed for a comparison across all 5 mAChR family members that revealed important differences in both orthosteric and allosteric sites that could inform the rational design of selective ligands. These structural studies, together with chimeric swaps between the extracellular regions of the M and M mAChRs, provided structural insight into kinetic selectivity, where ligands show differential residency times between related family members. Collectively, our study provides important insights into the nature of orthosteric and allosteric ligand interaction across the mAChR family that could be exploited for the design of selective drugs.
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http://dx.doi.org/10.1073/pnas.1914446116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6926013PMC
December 2019

Automatic local resolution-based sharpening of cryo-EM maps.

Bioinformatics 2020 02;36(3):765-772

Biocomputing Unit, National Center for Biotechnology (CSIC), Darwin 3, Campus Univ. Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain.

Motivation: Recent technological advances and computational developments have allowed the reconstruction of Cryo-Electron Microscopy (cryo-EM) maps at near-atomic resolution. On a typical workflow and once the cryo-EM map has been calculated, a sharpening process is usually performed to enhance map visualization, a step that has proven very important in the key task of structural modeling. However, sharpening approaches, in general, neglects the local quality of the map, which is clearly suboptimal.

Results: Here, a new method for local sharpening of cryo-EM density maps is proposed. The algorithm, named LocalDeblur, is based on a local resolution-guided Wiener restoration approach of the original map. The method is fully automatic and, from the user point of view, virtually parameter-free, without requiring either a starting model or introducing any additional structure factor correction or boosting. Results clearly show a significant impact on map interpretability, greatly helping modeling. In particular, this local sharpening approach is especially suitable for maps that present a broad resolution range, as is often the case for membrane proteins or macromolecules with high flexibility, all of them otherwise very suitable and interesting specimens for cryo-EM. To our knowledge, and leaving out the use of local filters, it represents the first application of local resolution in cryo-EM sharpening.

Availability And Implementation: The source code (LocalDeblur) can be found at https://github.com/I2PC/xmipp and can be run using Scipion (http://scipion.cnb.csic.es) (release numbers greater than or equal 1.2.1).

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btz671DOI Listing
February 2020

Use of Backbone Modification To Enlarge the Spatiotemporal Diversity of Parathyroid Hormone Receptor-1 Signaling via Biased Agonism.

J Am Chem Soc 2019 09 9;141(37):14486-14490. Epub 2019 Sep 9.

Department of Chemistry , University of Wisconsin-Madison , Madison , Wisconsin 53706 , United States.

The type-1 parathyroid hormone receptor (PTHR1), which regulates calcium homeostasis and tissue development, has two native agonists, parathyroid hormone (PTH) and PTH-related protein (PTHrP). PTH forms a complex with the PTHR1 that is rapidly internalized and induces prolonged cAMP production from endosomes. In contrast, PTHrP induces only transient cAMP production, which primarily arises from receptors on the cell surface. We show that backbone modification of PTH(1-34)-NH and abaloparatide (a PTHrP derivative) with a single homologous β-amino acid residue can generate biased agonists that induce prolonged cAMP production from receptors at the cell surface. This unique spatiotemporal profile could be useful for distinguishing effects associated with the duration of cAMP production from effects associated with the site of cAMP production.
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http://dx.doi.org/10.1021/jacs.9b04179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6930011PMC
September 2019