Publications by authors named "Arthur Christopoulos"

276 Publications

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

A robust method for particulate detection of a genetic tag for 3D electron microscopy.

Elife 2021 Apr 27;10. Epub 2021 Apr 27.

The University of Queensland, Institute for Molecular Bioscience, Queensland, Australia.

Genetic tags allow rapid localization of tagged proteins in cells and tissues. APEX, an ascorbate peroxidase, has proven to be one of the most versatile and robust genetic tags for ultrastructural localization by electron microscopy (EM). Here, we describe a simple method, APEX-Gold, which converts the diffuse oxidized diaminobenzidine reaction product of APEX into a silver/gold particle akin to that used for immunogold labelling. The method increases the signal-to-noise ratio for EM detection, providing unambiguous detection of the tagged protein, and creates a readily quantifiable particulate signal. We demonstrate the wide applicability of this method for detection of membrane proteins, cytoplasmic proteins, and cytoskeletal proteins. The method can be combined with different EM techniques including fast freezing and freeze substitution, focussed ion beam scanning EM, and electron tomography. Quantitation of expressed APEX-fusion proteins is achievable using membrane vesicles generated by a cell-free expression system. These membrane vesicles possess a defined quantum of signal, which can act as an internal standard for determination of the absolute density of expressed APEX-fusion proteins. Detection of fusion proteins expressed at low levels in cells from CRISPR-edited mice demonstrates the high sensitivity of the APEX-Gold method.
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http://dx.doi.org/10.7554/eLife.64630DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8104959PMC
April 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

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

Neurological, neuropsychiatric and neurodevelopmental complications of COVID-19.

Aust N Z J Psychiatry 2020 Oct 1:4867420961472. Epub 2020 Oct 1.

Department of Neurology & Neurosciences, The Central Clinical School, Alfred Hospital, Monash University, Melbourne, VIC, Australia.

Although COVID-19 is predominantly a respiratory disease, it is known to affect multiple organ systems. In this article, we highlight the impact of SARS-CoV-2 (the coronavirus causing COVID-19) on the central nervous system as there is an urgent need to understand the longitudinal impacts of COVID-19 on brain function, behaviour and cognition. Furthermore, we address the possibility of intergenerational impacts of COVID-19 on the brain, potentially via both maternal and paternal routes. Evidence from preclinical models of earlier coronaviruses has shown direct viral infiltration across the blood-brain barrier and indirect secondary effects due to other organ pathology and inflammation. In the most severely ill patients with pneumonia requiring intensive care, there appears to be additional severe inflammatory response and associated thrombophilia with widespread organ damage, including the brain. Maternal viral (and other) infections during pregnancy can affect the offspring, with greater incidence of neurodevelopmental disorders, such as autism, schizophrenia and epilepsy. Available reports suggest possible vertical transmission of SARS-CoV-2, although longitudinal cohort studies of such offspring are needed. The impact of paternal infection on the offspring and intergenerational effects should also be considered. Research targeted at mechanistic insights into all aspects of pathogenesis, including neurological, neuropsychiatric and haematological systems alongside pulmonary pathology, will be critical in informing future therapeutic approaches. With these future challenges in mind, we highlight the importance of national and international collaborative efforts to gather the required clinical and preclinical data to effectively address the possible long-term sequelae of this global pandemic, particularly with respect to the brain and mental health.
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http://dx.doi.org/10.1177/0004867420961472DOI Listing
October 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

Sustainable Pharmacy Education in the Time of COVID-19.

Am J Pharm Educ 2020 06;84(6):ajpe8088

Monash University, Faculty of Pharmacy and Pharmaceutical Sciences, Parkville, Victoria, Australia.

Pharmacy schools and colleges worldwide are facing unprecedented challenges to ensuring sustainable education during the novel coronavirus (COVID-19) pandemic. The experiences of pharmacy educators in the Asia-Pacific region in delivering emergency remote teaching, ensuring purposeful experiential placements, supporting displaced or isolated students, and communicating with faculty members, staff members, and students are discussed. The role of this pandemic in accelerating opportunities for new models of pharmacy education across the world is also discussed.
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http://dx.doi.org/10.5688/ajpe8088DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7334345PMC
June 2020

Probe dependence and biased potentiation of metabotropic glutamate receptor 5 is mediated by differential ligand interactions in the common allosteric binding site.

Biochem Pharmacol 2020 07 8;177:114013. Epub 2020 May 8.

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

The metabotropic glutamate receptor 5 (mGlu) is a promising therapeutic target for multiple CNS disorders. Recent mGlu drug discovery has focused on targeting binding sites within the mGlu 7-transmembrane domain (7TM) that are topographically distinct from that of the endogenous ligand. mGlu primarily couples to G proteins leading to mobilization of intracellular Ca (iCa), but also activates iCa independent signaling pathways, with biased agonism/modulation operative for multiple positive allosteric modulator (PAM) and PAM-agonist chemotypes. Although several residues within the common allosteric binding pocket are key determinants of PAM activity, how these residues affect biased modulation is unknown. The current study probed the molecular basis of mGlu PAM biased modulation. Modulation of mGlu activity by four chemically distinct mGlu PAMs (VU0424465, DPFE, VU29 and VU0409551) was assessed across two distinct receptor endpoints (iCa mobilization and ERK1/2 phosphorylation) at mGlu receptors containing single-point mutations of allosteric binding pocket residues informed by computational modeling. Many mutations had differential effects on PAM affinity and cooperativity across signaling endpoints, resulting in gain or reversal of bias at the level of both affinity and functional cooperativity. Additionally, mutants had differential effects on functional cooperativity between the orthosteric ligands, DHPG and glutamate, and the PAMs, DPFE and VU29, but not VU0409551, indicating that probe dependence is linked to orthosteric agonists conferring activation states that differentially influence allosteric ligand-receptor interactions in a chemotype dependent fashion. Collectively, these data provide crucial insight into the residues that govern different activation states adopted by mGlu in order to signal via distinct intracellular pathways when co-bound by orthosteric agonists and PAMs.
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http://dx.doi.org/10.1016/j.bcp.2020.114013DOI Listing
July 2020

Differential contribution of metabotropic glutamate receptor 5 common allosteric binding site residues to biased allosteric agonism.

Biochem Pharmacol 2020 07 5;177:114011. Epub 2020 May 5.

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

Allosteric modulators of metabotropic glutamate receptor subtype 5 (mGlu) represent an attractive therapeutic strategy for multiple CNS disorders. Chemically distinct mGlu positive allosteric modulators (PAMs) that interact with a common binding site can demonstrate biased allosteric agonism relative to the orthosteric agonist, DHPG, when comparing activity in signaling assays such as IP accumulation, ERK1/2 phosphorylation (pERK1/2) and iCa mobilization. However, the structural basis for such biased agonism is not well understood. Therefore, we evaluated biased allosteric agonism mediated by four mGlu PAM-agonists from diverse chemical scaffolds (i.e., DPFE, VU0409551, VU29, and VU0424465) for three measures of mGlu activation (i.e., iCa mobilization, IP accumulation and ERK1/2 phosphorylation) at eight single-point mutations within the common allosteric binding pocket of mGlu. In particular, mGlu allosteric site mutations had differential effects on the intrinsic efficacy of mGlu PAMs for multiple signaling pathways. Specifically, a loss of agonism for iCa mobilization was evident for DPFE and VU0409551 for most mutants, whereas IP accumulation and ERK phosphorylation were retained, albeit with reduced maximal responses. Additionally, bias profiles between iCa mobilization and IP/ERK pathways remained similar to wild type for most mutants. However, W784A and A809G mutants lost bias between IP accumulation and ERK phosphorylation for VU0424465, whereas a loss of bias between iCa mobilization and ERK1/2 phosphorylation was evident for F787A, S808A and A809G mutants. These data provide further insight into the structural requirements for allosteric agonism across multiple mGlu-mediated signaling pathways. An understanding of mGlu biased agonism at a structural level may provide the foundation for rational structure-based design of biased allosteric ligands for the treatment of neurological disorders.
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http://dx.doi.org/10.1016/j.bcp.2020.114011DOI Listing
July 2020

Acetylcholine Muscarinic M Receptors as a Therapeutic Target for Alcohol Use Disorder: Converging Evidence From Humans and Rodents.

Biol Psychiatry 2020 12 29;88(12):898-909. Epub 2020 Feb 29.

Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, Victoria, Australia. Electronic address:

Background: Alcohol use disorder (AUD) is a major socioeconomic burden on society, and current pharmacotherapeutic treatment options are inadequate. Aberrant alcohol use and seeking alters frontostriatal function.

Methods: We performed genome-wide RNA sequencing and subsequent quantitative polymerase chain reaction and receptor binding validation in the caudate-putamen of human AUD samples to identify potential therapeutic targets. We then back-translated our top candidate targets into a rodent model of long-term alcohol consumption to assess concordance of molecular adaptations in the rat striatum. Finally, we adopted rat behavioral models of alcohol intake and seeking to validate a potential therapeutic target.

Results: We found that G protein-coupled receptors were the top canonical pathway differentially regulated in individuals with AUD. The M muscarinic acetylcholine receptor (mAChR) was downregulated at the gene and protein levels in the putamen, but not in the caudate, of AUD samples. We found concordant downregulation of the M mAChR, specifically on dopamine D receptor-expressing medium spiny neurons in the rat dorsolateral striatum. Systemic administration of the selective M mAChR positive allosteric modulator, VU0467154, reduced home cage and operant alcohol self-administration, motivation to obtain alcohol, and cue-induced reinstatement of alcohol seeking in rats. Local microinjections of VU0467154 in the rat dorsolateral striatum reduced alcohol self-administration and cue-induced reinstatement of alcohol seeking.

Conclusions: Collectively, these results identify the M mAChR as a potential therapeutic target for the treatment of AUD and the D receptor-positive medium spiny neurons in the dorsolateral striatum as a key site mediating the actions of M mAChR in relation to alcohol consumption and seeking.
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http://dx.doi.org/10.1016/j.biopsych.2020.02.019DOI Listing
December 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

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

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

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

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

Mu and Delta Opioid Receptors Are Coexpressed and Functionally Interact in the Enteric Nervous System of the Mouse Colon.

Cell Mol Gastroenterol Hepatol 2020 20;9(3):465-483. Epub 2019 Nov 20.

Drug Discovery Biology Theme, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia; Australian Research Council Centre of Excellence in Convergent Bio-Nano Science and Technology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville Campus, Melbourne, VIC, Australia; Department of Anatomy and Neuroscience, University of Melbourne, Parkville, VIC, Australia. Electronic address:

Background & Aims: Functional interactions between the mu opioid receptor (MOR) and delta opioid receptor (DOR) represent a potential target for novel analgesics and may drive the effects of the clinically approved drug eluxadoline for the treatment of diarrhea-predominant irritable bowel syndrome. Although the enteric nervous system (ENS) is a likely site of action, the coexpression and potential interaction between MOR and DOR in the ENS are largely undefined. In the present study, we have characterized the distribution of MOR in the mouse ENS and examined MOR-DOR interactions by using pharmacologic and cell biology techniques.

Methods: MOR and DOR expression was defined by using MORmCherry and MORmCherry-DOR-eGFP knockin mice. MOR-DOR interactions were assessed by using DOR-eGFP internalization assays and by pharmacologic analysis of neurogenic contractions of the colon.

Results: Although MOR was expressed by approximately half of all myenteric neurons, MOR-positive submucosal neurons were rarely observed. There was extensive overlap between MOR and DOR in both excitatory and inhibitory pathways involved in the coordination of intestinal motility. MOR and DOR can functionally interact, as shown through heterologous desensitization of MOR-dependent responses by DOR agonists. Functional evidence suggests that MOR and DOR may not exist as heteromers in the ENS. Pharmacologic studies show no evidence of cooperativity between MOR and DOR. DOR internalizes independently of MOR in myenteric neurons, and MOR-evoked contractions are unaffected by the sequestration of DOR.

Conclusions: Collectively, these findings demonstrate that although MOR and DOR are coexpressed in the ENS and functionally interact, they are unlikely to exist as heteromers under physiological conditions.
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http://dx.doi.org/10.1016/j.jcmgh.2019.11.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7036548PMC
May 2021

THE CONCISE GUIDE TO PHARMACOLOGY 2019/20: Introduction and Other Protein Targets.

Br J Pharmacol 2019 12;176 Suppl 1:S1-S20

Centre for Discovery Brain Sciences, University of Edinburgh, Edinburgh, EH8 9XD, UK.

The Concise Guide to PHARMACOLOGY 2019/20 is the fourth in this series of biennial publications. The Concise Guide provides concise overviews of the key properties of nearly 1800 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide represents approximately 400 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.14747. In addition to this overview, in which are identified Other protein targets which fall outside of the subsequent categorisation, there are six areas of focus: G protein-coupled receptors, ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2019, and supersedes data presented in the 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.
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http://dx.doi.org/10.1111/bph.14747DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6844537PMC
December 2019

THE CONCISE GUIDE TO PHARMACOLOGY 2019/20: G protein-coupled receptors.

Br J Pharmacol 2019 12;176 Suppl 1:S21-S141

Centre for Discovery Brain Sciences, University of Edinburgh, Edinburgh, EH8 9XD, UK.

The Concise Guide to PHARMACOLOGY 2019/20 is the fourth in this series of biennial publications. The Concise Guide provides concise overviews of the key properties of nearly 1800 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide represents approximately 400 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.14748. G protein-coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2019, and supersedes data presented in the 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.
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http://dx.doi.org/10.1111/bph.14748DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6844580PMC
December 2019

Evaluation of Operational Models of Agonism and Allosterism at Receptors with Multiple Orthosteric Binding Sites.

Mol Pharmacol 2020 01 8;97(1):35-45. Epub 2019 Nov 8.

Drug Discovery Biology and Department of Pharmacology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Melbourne, Australia (K.J.G., J.D., A.C., K.L.); Laboratory of Molecular Neuropharmacology and Bioinformatics, Institut de Neurociències and Unitat de Bioestadística, Facultat de Medicina, Universitat Autònoma de Barcelona, Barcelona, Spain (J.G.); Instituto de Salud Carlos III, Centro de Investigación Biomédica en Red de Salud Mental, Bellaterra, Spain (J.G.); and Unitat de Neurociència Traslacional, Parc Taulí Hospital Universitari, Institut d'Investigació i Innovació Parc Taulí and Institut de Neurociències, Universitat Autònoma de Barcelona, Bellaterra, Spain (J.G.)

Current operational models of agonism and allosterism quantify ligand actions at receptors where agonist concentration-response relationships are nonhyperbolic by introduction of a transducer slope that relates receptor occupancy to response. However, for some receptors nonhyperbolic concentration-response relationships arise from multiple endogenous agonist molecules binding to a receptor in a cooperative manner. Thus, we developed operational models of agonism in systems with cooperative agonist binding and evaluated the models by simulating data describing agonist effects. The models were validated by analyzing experimental data demonstrating the effects of agonists and allosteric modulators at receptors where agonist binding follows hyperbolic (M muscarinic acetylcholine receptors) or nonhyperbolic relationships (metabotropic glutamate receptor 5 and calcium-sensing receptor). For hyperbolic agonist concentration-response relationships, no differences in estimates of ligand affinity, efficacy, or cooperativity were observed when the slope was assigned to either a transducer slope or agonist binding slope. In contrast, for receptors with nonhyperbolic agonist concentration-response relationships, estimates of ligand affinity, efficacy, or cooperativity varied depending on the assignment of the slope. The extent of this variation depended on the magnitude of the slope value and agonist efficacy, and for allosteric modulators on the magnitude of cooperativity. The modified operational models described herein are well suited to analyzing agonist and modulator interactions at receptors that bind multiple orthosteric agonists in a cooperative manner. Accounting for cooperative agonist binding is essential to accurately quantify agonist and drug actions. SIGNIFICANCE STATEMENT: Some orthosteric agonists bind to multiple sites on a receptor, but current analytical methods to characterize such interactions are limited. Herein, we develop and validate operational models of agonism and allosterism for receptors with multiple orthosteric binding sites, and demonstrate that such models are essential to accurately quantify agonist and drug actions. These findings have important implications for the discovery and development of drugs targeting receptors such as the calcium-sensing receptor, which binds at least five calcium ions.
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http://dx.doi.org/10.1124/mol.119.118091DOI Listing
January 2020

Molecular Determinants of the Intrinsic Efficacy of the Antipsychotic Aripiprazole.

ACS Chem Biol 2019 08 5;14(8):1780-1792. Epub 2019 Aug 5.

Division of Pharmacology, Physiology and Neuroscience, School of Life Sciences, Queen's Medical Centre , University of Nottingham , Nottingham NG7 2UH , U.K.

Partial agonists of the dopamine D receptor (DR) have been developed to treat the symptoms of schizophrenia without causing the side effects elicited by antagonists. The receptor-ligand interactions that determine the intrinsic efficacy of such drugs, however, are poorly understood. Aripiprazole has an extended structure comprising a phenylpiperazine primary pharmacophore and a 1,2,3,4-tetrahydroquinolin-2-one secondary pharmacophore. We combined site-directed mutagenesis, analytical pharmacology, ligand fragments, and molecular dynamics simulations to identify the DR-aripiprazole interactions that contribute to affinity and efficacy. We reveal that an interaction between the secondary pharmacophore of aripiprazole and a secondary binding pocket defined by residues at the extracellular portions of transmembrane segments 1, 2, and 7 determines the intrinsic efficacy of aripiprazole. Our findings reveal a hitherto unappreciated mechanism for fine-tuning the intrinsic efficacy of DR agonists.
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http://dx.doi.org/10.1021/acschembio.9b00342DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7365685PMC
August 2019

Cryptic pocket formation underlies allosteric modulator selectivity at muscarinic GPCRs.

Nat Commun 2019 07 23;10(1):3289. Epub 2019 Jul 23.

Departments of Computer Science, Molecular and Cellular Physiology, and Structural Biology, and Institute for Computational and Mathematical Engineering, Stanford University, Stanford, CA, 94305, USA.

Allosteric modulators are highly desirable as drugs, particularly for G-protein-coupled receptor (GPCR) targets, because allosteric drugs can achieve selectivity between closely related receptors. The mechanisms by which allosteric modulators achieve selectivity remain elusive, however, particularly given recent structures that reveal similar allosteric binding sites across receptors. Here we show that positive allosteric modulators (PAMs) of the M1 muscarinic acetylcholine receptor (mAChR) achieve exquisite selectivity by occupying a dynamic pocket absent in existing crystal structures. This cryptic pocket forms far more frequently in molecular dynamics simulations of the M1 mAChR than in those of other mAChRs. These observations reconcile mutagenesis data that previously appeared contradictory. Further mutagenesis experiments validate our prediction that preventing cryptic pocket opening decreases the affinity of M1-selective PAMs. Our findings suggest opportunities for the design of subtype-specific drugs exploiting cryptic pockets that open in certain receptors but not in other receptors with nearly identical static structures.
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http://dx.doi.org/10.1038/s41467-019-11062-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6650467PMC
July 2019

DREADD Agonist 21 Is an Effective Agonist for Muscarinic-Based DREADDs and .

ACS Pharmacol Transl Sci 2018 Sep 27;1(1):61-72. Epub 2018 Jul 27.

Centre for Translational Pharmacology, Institute of Molecular, Cell, and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, Scotland G12 8QQ, United Kingdom.

Chemogenetic tools such as designer receptors exclusively activated by designer drugs (DREADDs) are routinely used to modulate neuronal and non-neuronal signaling and activity in a relatively noninvasive manner. The first generation of DREADDs were templated from the human muscarinic acetylcholine receptor family and are relatively insensitive to the endogenous agonist acetylcholine but instead are activated by clozapine--oxide (CNO). Despite the undisputed success of CNO as an activator of muscarinic DREADDs, it has been known for some time that CNO is subject to a low rate of metabolic conversion to clozapine, raising the need for alternative chemical actuators of muscarinic-based DREADDs. Here we show that DREADD agonist 21 (C21) (11-(1-piperazinyl)-5-dibenzo[,][1,4]diazepine) is a potent and selective agonist at both excitatory (hM3Dq) and inhibitory (hM4Di) DREADDs and has excellent bioavailability, pharmacokinetic properties, and brain penetrability. We also show that C21-induced activation of hM3Dq and hM4Di can modulate bidirectional feeding in defined circuits in mice. These results indicate that C21 represents an alternative to CNO for studies where metabolic conversion of CNO to clozapine is a concern.
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http://dx.doi.org/10.1021/acsptsci.8b00012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6407913PMC
September 2018

Kinetic and system bias as drivers of metabotropic glutamate receptor 5 allosteric modulator pharmacology.

Neuropharmacology 2019 05 11;149:83-96. Epub 2019 Feb 11.

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

Allosteric modulators of the metabotropic glutamate receptor subtype 5 (mGlu) have been proposed as potential therapies for various CNS disorders. These ligands bind to sites distinct from the orthosteric (or endogenous) ligand, often with improved subtype selectivity and spatio-temporal control over receptor responses. We recently revealed that mGlu allosteric agonists and positive allosteric modulators exhibit biased agonism and/or modulation. To establish whether negative allosteric modulators (NAMs) engender similar bias, we rigorously characterized the pharmacology of eight diverse mGlu NAMs. Radioligand inhibition binding studies revealed novel modes of interaction with mGlu for select NAMs, with biphasic or incomplete inhibition of the radiolabeled NAM, [H]methoxy-PEPy. We assessed mGlu-mediated intracellular Ca (iCa) mobilization and inositol phosphate (IP) accumulation in HEK293A cells stably expressing low levels of mGlu (HEK293A-rat mGlu-low) and mouse embryonic cortical neurons. The apparent affinity of acetylenic NAMs, MPEP, MTEP and dipraglurant, was dependent on the signaling pathway measured, agonist used, and cell type (HEK293A-rat mGlu-low versus mouse cortical neurons). In contrast, the acetylenic partial NAM, M-5MPEP, and structurally distinct NAMs (VU0366248, VU0366058, fenobam), had similar affinity estimates irrespective of the assay or cellular background. Biased modulation was evident for VU0366248 in mouse cortical neurons where it was a NAM for DHPG-mediated iCa mobilization, but neutral with DHPG in IP accumulation assays. Overall, this study highlights the inherent complexity in mGlu NAM pharmacology that we hypothesize may influence interpretation when translating into preclinical models and beyond in the design and development of novel therapeutics for neuropsychiatric and neurological disorders.
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http://dx.doi.org/10.1016/j.neuropharm.2019.02.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6420870PMC
May 2019

The Molecular Control of Calcitonin Receptor Signaling.

ACS Pharmacol Transl Sci 2019 Feb 11;2(1):31-51. Epub 2019 Jan 11.

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

The calcitonin receptor (CTR) is a class B G protein-coupled receptor (GPCR) that responds to the peptide hormone calcitonin (CT). CTs are clinically approved for the treatment of bone diseases. We previously reported a 4.1 Å structure of the activated CTR bound to salmon CT (sCT) and heterotrimeric Gs protein by cryo-electron microscopy (Liang, Y.-L., . Phase-plate cryo- EM structure of a class B GPCR-G protein complex. , , 118-123). In the current study, we have reprocessed the electron micrographs to yield a 3.3 Å map of the complex. This has allowed us to model extracellular loops (ECLs) 2 and 3, and the peptide N-terminus that previously could not be resolved. We have also performed alanine scanning mutagenesis of ECL1 and the upper segment of transmembrane helix 1 (TM1) and its extension into the receptor extracellular domain (TM1 stalk), with effects on peptide binding and function assessed by cAMP accumulation and ERK1/2 phosphorylation. These data were combined with previously published alanine scanning mutagenesis of ECL2 and ECL3 and the new structural information to provide a comprehensive 3D map of the molecular surface of the CTR that controls binding and signaling of distinct CT and related peptides. The work highlights distinctions in how different, related, class B receptors may be activated. The new mutational data on the TM1 stalk and ECL1 have also provided critical insights into the divergent control of cAMP versus pERK signaling and, collectively with previous mutagenesis data, offer evidence that the conformations linked to these different signaling pathways are, in many ways, mutually exclusive. This study furthers our understanding of the complex nature of signaling elicited by GPCRs and, in particular, that of the therapeutically important class B subfamily.
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http://dx.doi.org/10.1021/acsptsci.8b00056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7088896PMC
February 2019