Publications by authors named "Christopher J Langmead"

84 Publications

Translation-Focused Approaches to GPCR Drug Discovery for Cognitive Impairments Associated with Schizophrenia.

ACS Pharmacol Transl Sci 2020 Dec 28;3(6):1042-1062. Epub 2020 Oct 28.

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

There are no effective therapeutics for cognitive impairments associated with schizophrenia (CIAS), which includes deficits in executive functions (working memory and cognitive flexibility) and episodic memory. Compounds that have entered clinical trials are inadequate in terms of efficacy and/or tolerability, highlighting a clear translational bottleneck and a need for a cohesive preclinical drug development strategy. In this review we propose hippocampal-prefrontal-cortical (HPC-PFC) circuitry underlying CIAS-relevant cognitive processes across mammalian species as a target source to guide the translation-focused discovery and development of novel, procognitive agents. We highlight several G protein-coupled receptors (GPCRs) enriched within HPC-PFC circuitry as therapeutic targets of interest, including noncanonical approaches (biased agonism and allosteric modulation) to conventional clinical targets, such as dopamine and muscarinic acetylcholine receptors, along with prospective novel targets, including the orphan receptors GPR52 and GPR139. We also describe the translational limitations of popular preclinical cognition tests and suggest touchscreen-based assays that probe cognitive functions reliant on HPC-PFC circuitry and reflect tests used in the clinic, as tests of greater translational relevance. Combining pharmacological and behavioral testing strategies based in HPC-PFC circuit function creates a cohesive, translation-focused approach to preclinical drug development that may improve the translational bottleneck currently hindering the development of treatments for CIAS.
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http://dx.doi.org/10.1021/acsptsci.0c00117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7737210PMC
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

Structure-based development of a subtype-selective orexin 1 receptor antagonist.

Proc Natl Acad Sci U S A 2020 07 15;117(30):18059-18067. Epub 2020 Jul 15.

Department of Chemistry and Pharmacy, Medicinal Chemistry, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91058 Erlangen, Germany;

Orexins are neuropeptides that activate the rhodopsin-like G protein-coupled receptors OX1R and OX2R. The orexin system plays an important role in the regulation of the sleep-wake cycle and the regulation of feeding and emotions. The nonselective orexin receptor antagonist suvorexant has been the first drug on the market targeting the orexin system and is prescribed for the treatment of insomnia. Subtype-selective OX1R antagonists are valuable tools to further investigate the functions and physiological role of the OX1R in vivo and promising lead compounds for the treatment of drug addiction, anxiety, pain or obesity. Starting from the OX1R and OX2R crystal structures bound to suvorexant, we exploited a single amino acid difference in the orthosteric binding site by using molecular docking and structure-based drug design to optimize ligand interactions with the OX1R while introducing repulsive interactions with the OX2R. A newly established enantiospecific synthesis provided ligands showing up to 75-fold selectivity for the OX1R over the OX2R subtype. The structure of a new OX1R antagonist with subnanomolar affinity (JH112) was determined by crystallography in complex with the OX1R and corresponded closely to the docking-predicted geometry. JH112 exhibits high selectivity over a panel of different GPCRs, is able to cross the blood-brain barrier and acts as slowly diffusing and insurmountable antagonist for G protein activation and in particular β-arrestin-2 recruitment at OX1R. This study demonstrates the potential of structure-based drug design to develop more subtype-selective GPCR ligands with potentially reduced side effects and provides an attractive probe molecule and lead compound.
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http://dx.doi.org/10.1073/pnas.2002704117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7395530PMC
July 2020

β-Arrestin-2-Dependent Mechanism of GPR52 Signaling in Frontal Cortical Neurons.

ACS Chem Neurosci 2020 07 7;11(14):2077-2084. Epub 2020 Jul 7.

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

The orphan Gαs-coupled receptor GPR52 is expressed exclusively in the brain, predominantly in circuitry relating to symptoms of neuropsychiatric and cognitive disorders such as schizophrenia. While GPR52 agonists have displayed antipsychotic and procognitive efficacy in murine models, there remains limited evidence delineating the molecular mechanisms of these effects. Indeed, previous studies have solely reported canonical cAMP signaling and CREB phosphorylation downstream of GPR52 activation. In the present study, we demonstrated that the synthetic GPR52 agonist, 3-BTBZ, equipotently induces cAMP accumulation, ERK1/2 phosphorylation, and β-arrestin-1 and -2 recruitment in transfected HEK293T cells. In cultured frontal cortical neurons, however, 3-BTBZ-induced ERK1/2 phosphorylation was significantly more potent than cAMP signaling, with a more prolonged signaling profile than that in HEK293T cells. Furthermore, knock down of β-arrestin-2 in frontal cortical neurons abolished 3-BTBZ-induced ERK1/2 phosphorylation, but not cAMP accumulation. These results suggest a β-arrestin-2-dependent mechanism for GPR52-mediated ERK1/2 signaling, which may link to cognitive function . Finally, these findings highlight the context-dependence of GPCR signaling in recombinant cells and neurons, offering new insights into translationally relevant GPR52 signaling mechanisms.
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http://dx.doi.org/10.1021/acschemneuro.0c00199DOI Listing
July 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

Preface.

Adv Pharmacol 2020 ;88:xiii-xvii

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

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http://dx.doi.org/10.1016/S1054-3589(20)30037-5DOI Listing
January 2020

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

Biol Psychiatry 2020 Dec 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

New Advances in Targeting the Resolution of Inflammation: Implications for Specialized Pro-Resolving Mediator GPCR Drug Discovery.

ACS Pharmacol Transl Sci 2020 Feb 20;3(1):88-106. Epub 2020 Jan 20.

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

Chronic inflammation is a component of numerous diseases including autoimmune, metabolic, neurodegenerative, and cancer. The discovery and characterization of specialized pro-resolving mediators (SPMs) critical to the resolution of inflammation, and their cognate G protein-coupled receptors (GPCRs) has led to a significant increase in the understanding of this physiological process. Approximately 20 ligands, including lipoxins, resolvins, maresins, and protectins, and 6 receptors (FPR2/ALX, GPR32, GPR18, chemerin, BLT, and GPR37) have been identified highlighting the complex and multilayered nature of resolution. Therapeutic efforts in targeting these receptors have proved challenging, with very few ligands apparently progressing through to preclinical or clinical development. To date, some knowledge gaps remain in the understanding of how the activation of these receptors, and their downstream signaling, results in efficient resolution via apoptosis, phagocytosis, and efferocytosis of polymorphonuclear leukocytes (mainly neutrophils) and macrophages. SPMs bind and activate multiple receptors (ligand poly-pharmacology), while most receptors are activated by multiple ligands (receptor pleiotropy). In addition, allosteric binding sites have been identified signifying the capacity of more than one ligand to bind simultaneously. These fundamental characteristics of SPM receptors enable alternative targeting strategies to be considered, including biased signaling and allosteric modulation. This review describes those ligands and receptors involved in the resolution of inflammation, and highlights the most recent clinical trial results. Furthermore, we describe alternative mechanisms by which these SPM receptors could be targeted, paving the way for the identification of new therapeutics, perhaps with greater efficacy and fidelity.
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http://dx.doi.org/10.1021/acsptsci.9b00075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7089045PMC
February 2020

Negative allosteric modulators of the human calcium-sensing receptor bind to overlapping and distinct sites within the 7-transmembrane domain.

Br J Pharmacol 2020 04 12;177(8):1917-1930. Epub 2020 Feb 12.

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

Background And Purpose: Negative allosteric modulators (NAMs) that target the calcium-sensing receptor (CaS receptor) were originally developed for the treatment of osteoporosis by stimulating the release of endogenous parathyroid hormone, but failed in human clinical trials. Several chemically and structurally distinct NAM scaffolds have been described, but it is not known how these different scaffolds interact with the CaS receptor to inhibit receptor signalling in response to agonists.

Experimental Approach: In the present study, we used a mutagenesis approach combined with analytical pharmacology and computational modelling to probe the binding sites of four distinct NAM scaffolds.

Key Results: Although all four scaffolds bind to the 7-transmembrane and/or extracellular or intracellular loops, they occupy distinct regions, as previously shown for positive allosteric modulators of the CaS receptor. Furthermore, different NAM scaffolds mediate negative allosteric modulation via distinct amino acid networks.

Conclusion And Implications: These findings aid our understanding of how different NAMs bind to and inhibit the CaS receptor. Elucidation of allosteric binding sites in the CaS receptor has implications for the discovery of novel allosteric modulators.
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http://dx.doi.org/10.1111/bph.14961DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7070164PMC
April 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

Drug-receptor kinetics and sigma-1 receptor affinity differentiate clinically evaluated histamine H receptor antagonists.

Neuropharmacology 2019 01 22;144:244-255. Epub 2018 Oct 22.

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

The histamine H receptor is a G protein-coupled receptor (GPCR) drug target that is highly expressed in the CNS, where it acts as both an auto- and hetero-receptor to regulate neurotransmission. As such, it has been considered as a relevant target in disorders as varied as Alzheimer's disease, schizophrenia, neuropathic pain and attention deficit hyperactivity disorder. A range of competitive antagonists/inverse agonists have progressed into clinical development, with pitolisant approved for the treatment of narcolepsy. Given the breadth of compounds developed and potential therapeutic indications, we assessed the comparative pharmacology of six investigational histamine H agents, including pitolisant, using native tissue and recombinant cells. Whilst all of the compounds tested displayed robust histamine H receptor inverse agonism and did not differentiate between the main H receptor splice variants, they displayed a wide range of affinities and kinetic properties, and included rapidly dissociating (pitolisant, S 38093-2, ABT-239) and slowly dissociating (GSK189254, JNJ-5207852, PF-3654746) agents. S 38093-2 had the lowest histamine H receptor affinity (pK values 5.7-6.2), seemingly at odds with previously reported, potent in vivo activity in models of cognition. We show here that at pro-cognitive and anti-hyperalgesic/anti-allodynic doses, S 38093-2 preferentially occupies the mouse sigma-1 receptor in vivo, only engaging the histamine H receptor at doses associated with wakefulness promotion and neurotransmitter (histamine, ACh) release. Furthermore, pitolisant, ABT-239 and PF-3654746 also displayed appreciable sigma-1 receptor affinity, suggesting that this property differentiates clinically evaluated histamine H receptor antagonists and may play a role in their efficacy.
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http://dx.doi.org/10.1016/j.neuropharm.2018.10.028DOI Listing
January 2019

Molecular pharmacology of GPCRs.

Br J Pharmacol 2018 11;175(21):4005-4008

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

Linked Articles: This article is part of a themed section on Molecular Pharmacology of GPCRs. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.21/issuetoc.
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http://dx.doi.org/10.1111/bph.14474DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6177621PMC
November 2018

Comparative genotypic and phenotypic analysis of human peripheral blood monocytes and surrogate monocyte-like cell lines commonly used in metabolic disease research.

PLoS One 2018 10;13(5):e0197177. Epub 2018 May 10.

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

Monocyte-like cell lines (MCLCs), including THP-1, HL-60 and U-937 cells, are used routinely as surrogates for isolated human peripheral blood mononuclear cells (PBMCs). To systematically evaluate these immortalised cells and PBMCs as model systems to study inflammation relevant to the pathogenesis of type II diabetes and immuno-metabolism, we compared mRNA expression of inflammation-relevant genes, cell surface expression of cluster of differentiation (CD) markers, and chemotactic responses to inflammatory stimuli. Messenger RNA expression analysis suggested most genes were present at similar levels across all undifferentiated cells, though notably, IDO1, which encodes for indoleamine 2,3-dioxygenase and catabolises tryptophan to kynureninase (shown to be elevated in serum from diabetic patients), was not expressed in any PMA-treated MCLC, but present in GM-CSF-treated PBMCs. There was little overall difference in the pattern of expression of CD markers across all cells, though absolute expression levels varied considerably and the correlation between MCLCs and PBMCs was improved upon MCLC differentiation. Functionally, THP-1 and PBMCs migrated in response to chemoattractants in a transwell assay, with varying sensitivity to MCP-1, MIP-1α and LTB-4. However, despite similar gene and CD expression profiles, U-937 cells were functionally impaired as no migration was observed to any chemoattractant. Our analysis reveals that the MCLCs examined only partly replicate the genotypic and phenotypic properties of human PBMCs. To overcome such issues a universal differentiation protocol should be implemented for these cell lines, similar to those already used with isolated monocytes. Although not perfect, in our hands the THP-1 cells represent the closest, simplified surrogate model of PBMCs for study of inflammatory cell migration.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0197177PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5944989PMC
July 2018

Divergent effects of strontium and calcium-sensing receptor positive allosteric modulators (calcimimetics) on human osteoclast activity.

Br J Pharmacol 2018 11 3;175(21):4095-4108. Epub 2018 Jun 3.

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

Background And Purpose: Strontium ranelate, a drug approved and until recently used for the treatment of osteoporosis, mediates its effects on bone at least in part via the calcium-sensing (CaS) receptor. However, it is not known whether bone-targeted CaS receptor positive allosteric modulators (PAMs; calcimimetics) represent an alternative (or adjunctive) therapy to strontium (Sr ).

Experimental Approach: We assessed three structurally distinct calcimimetics [cinacalcet, AC-265347 and a benzothiazole tri-substituted urea (BTU-compound 13)], alone and in combination with extracellular calcium (Ca ) or Sr , in G protein-dependent signalling assays and trafficking experiments in HEK293 cells and their effects on cell differentiation, tartrate-resistant acid phosphatase (TRAP) activity and hydroxyapatite resorption assays in human blood-derived osteoclasts.

Key Results: Sr activated CaS receptor-dependent signalling in HEK293 cells in a similar manner to Ca , and inhibited the maturation, TRAP expression and hydroxyapatite resorption capacity of human osteoclasts. Calcimimetics potentiated Ca - and Sr -mediated CaS receptor signalling in HEK293 cells with distinct biased profiles, and only cinacalcet chaperoned an endoplasmic reticulum-retained CaS mutant receptor to the cell surface in HEK293 cells, indicative of a conformational state different from that engendered by AC-265347 and BTU-compound 13. Intriguingly, only cinacalcet modulated human osteoclast function, reducing TRAP activity and profoundly inhibiting resorption.

Conclusion And Implications: Although AC-265347 and BTU-compound 13 potentiated Ca - and Sr -induced CaS receptor activation, they neither replicated nor potentiated the ability of Sr to inhibit human osteoclast function. In contrast, the FDA-approved calcimimetic, cinacalcet, inhibited osteoclast TRAP activity and hydroxyapatite resorption, which may contribute to its clinical effects on bone mineral density LINKED ARTICLES: This article is part of a themed section on Molecular Pharmacology of GPCRs. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.21/issuetoc.
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http://dx.doi.org/10.1111/bph.14344DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6177624PMC
November 2018

Bitopic Binding Mode of an M Muscarinic Acetylcholine Receptor Agonist Associated with Adverse Clinical Trial Outcomes.

Mol Pharmacol 2018 06 25;93(6):645-656. Epub 2018 Apr 25.

The Centre for Translational Pharmacology, Institute of Molecular, Cell, and Systems Biology, College of Medical, Veterinary, and Life Sciences, University of Glasgow, Glasgow, Scotland (S.J.B., C.M., K.J.T., L.D., S.M.B., A.B.T.); Eli Lilly & Co. Neuroscience, Windlesham, Surrey, United Kingdom (C.B., A.J.M., H.E.S., M.D.C., L.M.B.); Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia (P.M.S., A.C., C.J.L.); and Eli Lilly & Co. Neuroscience, Indianapolis, Indiana (C.C.F.)

The realization of the therapeutic potential of targeting the M muscarinic acetylcholine receptor (mAChR) for the treatment of cognitive decline in Alzheimer's disease has prompted the discovery of M mAChR ligands showing efficacy in alleviating cognitive dysfunction in both rodents and humans. Among these is GSK1034702 (7-fluoro-5-methyl-3-[1-(oxan-4-yl)piperidin-4-yl]-1-benzimidazol-2-one), described previously as a potent M receptor allosteric agonist, which showed procognitive effects in rodents and improved immediate memory in a clinical nicotine withdrawal test but induced significant side effects. Here we provide evidence using ligand binding, chemical biology and functional assays to establish that rather than the allosteric mechanism claimed, GSK1034702 interacts in a bitopic manner at the M mAChR such that it can concomitantly span both the orthosteric and an allosteric binding site. The bitopic nature of GSK1034702, together with the intrinsic agonist activity and a lack of muscarinic receptor subtype selectivity reported here, all likely contribute to the adverse effects of this molecule in clinical trials. Although they impart beneficial effects on learning and memory, we conclude that these properties are undesirable in a clinical candidate due to the likelihood of adverse side effects. Rather, our data support the notion that "pure" positive allosteric modulators showing selectivity for the M mAChR with low levels of intrinsic activity would be preferable to provide clinical efficacy with low adverse responses.
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http://dx.doi.org/10.1124/mol.118.111872DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5963591PMC
June 2018

Structure-Activity Relationships of Pan-Gα Coupled Muscarinic Acetylcholine Receptor Positive Allosteric Modulators.

ACS Chem Neurosci 2018 07 30;9(7):1818-1828. Epub 2018 Apr 30.

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

Recent years have seen a large increase in the discovery of allosteric ligands targeting muscarinic acetylcholine receptors (mAChRs). One of the challenges in screening such compounds is to understand their mechanisms of action and define appropriate parameter estimates for affinity, cooperativity and efficacy. Herein we describe the mechanisms of action and structure-activity relationships for a series of "pan-G-coupled" muscarinic acetylcholine (ACh) receptor (mAChR) positive allosteric modulators (PAMs). Using a combination of radioligand binding, functional inositol phosphate accumulation assays, receptor alkylation and operational data analysis, we show that most compounds in the series derive their variable potency and selectivity from differential cooperativity at the M, M and M mAChRs. None of the PAMs showed greater than 10-fold subtype selectivity for the agonist-free receptor, but VU6007705, VU6007678, and VU6008555 displayed markedly increased cooperativity compared to the parent molecule and M mAChR-preferring PAM, ML380 (αβ > 100), in the presence of ACh. Most of the activity of these PAMs derives from their ability to potentiate ACh binding affinity at mAChRs, though VU6007678 was notable for also potentiating ACh signaling efficacy and robust allosteric agonist activity. These data provide key insights for the future design of more potent and subtype-selective mAChR PAMs.
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http://dx.doi.org/10.1021/acschemneuro.8b00136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6106869PMC
July 2018

Discovery and Optimization of Potent and CNS Penetrant M-Preferring Positive Allosteric Modulators Derived from a Novel, Chiral N-(Indanyl)piperidine Amide Scaffold.

ACS Chem Neurosci 2018 07 26;9(7):1572-1581. Epub 2018 Apr 26.

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

The pharmacology of the M muscarinic acetylcholine receptor (mAChR) is the least understood of the five mAChR subtypes due to a historic lack of selective small molecule tools. To address this shortcoming, we have continued the optimization effort around the prototypical M positive allosteric modulator (PAM) ML380 and have discovered and optimized a new series of M PAMs based on a chiral N-(indanyl)piperidine amide core with robust SAR, human and rat M PAM EC values <100 nM and rat brain/plasma K values of ∼0.40. Interestingly, unlike M and M PAMs with unprecedented mAChR subtype selectivity, this series of M PAMs displayed varying degrees of PAM activity at the other two natively G-coupled mAChRs, M and M, yet were inactive at M and M.
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http://dx.doi.org/10.1021/acschemneuro.8b00126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6104635PMC
July 2018

Muscarinic M receptors modulate ethanol seeking in rats.

Neuropsychopharmacology 2018 06 5;43(7):1510-1517. Epub 2018 Feb 5.

The Florey Institute of Neuroscience and Mental Health, Parkville, VIC, 3052, Australia.

Despite the cost to both individual and society, alcohol use disorders (AUDs) remain a major health risk within society, and both relapse and heavy drinking are still poorly controlled with current medications. Here we demonstrate for the first time that a centrally active and selective negative allosteric modulator for the rat M muscarinic acetylcholine receptor (mAChR), ML375, decreases ethanol self-administration and attenuates cue-induced reinstatement of ethanol seeking in ethanol-preferring (iP) rats. Importantly, ML375 did not affect sucrose self-administration or general locomotor activity indicative of a selective effect on ethanol seeking. Based on the expression profile of M mAChRs in the brain and the distinct roles different aspects of the dorsal striatum have on long-term and short-term ethanol use, we studied whether intra-striatal microinjection of ML375 modulated ethanol intake in rats. We show in iP rats with an extensive history of ethanol intake that intra-dorsolateral (DL), but not intra-dorsomedial, striatal injections of ML375 reduced ethanol self-administration to a similar extent as the nicotinic acetylcholine receptor ligand varenicline, which has preclinical and clinical efficacy in reducing the reinforcing effects of ethanol. These data implicate the DL striatum as a locus for the effects of cholinergic-acting drugs on ethanol seeking in rats with a history of long-term ethanol use. Accordingly, we demonstrate in rats that selectively targeting the M mAChR can modulate both voluntary ethanol intake and cue-induced ethanol seeking and thereby provide direct evidence that the M mAChR is a potential novel target for pharmacotherapies aimed at treating AUDs.
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http://dx.doi.org/10.1038/s41386-017-0007-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5983544PMC
June 2018

G Protein-Coupled Receptors Targeting Insulin Resistance, Obesity, and Type 2 Diabetes Mellitus.

Pharmacol Rev 2018 01;70(1):39-67

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia (D.M.R., R.J.S., P.M.S., C.J.L.); and Institut de Recherches Servier, Pôle d'Innovation Thérapeutique Métabolisme, Suresnes, France (P.D.)

G protein-coupled receptors (GPCRs) continue to be important discovery targets for the treatment of type 2 diabetes mellitus (T2DM). Many GPCRs are directly involved in the development of insulin resistance and -cell dysfunction, and in the etiology of inflammation that can lead to obesity-induced T2DM. This review summarizes the current literature describing a number of well-validated GPCR targets, but also outlines several new and promising targets for drug discovery. We highlight the importance of understanding the role of these receptors in the disease pathology, and their basic pharmacology, which will pave the way to the development of novel pharmacological probes that will enable these targets to fulfill their promise for the treatment of these metabolic disorders.
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http://dx.doi.org/10.1124/pr.117.014373DOI Listing
January 2018

Systematic Testing of Belief-Propagation Estimates for Absolute Free Energies in Atomistic Peptides and Proteins.

J Chem Theory Comput 2018 Jan 22;14(1):426-443. Epub 2017 Dec 22.

Department of Biomedical Engineering, Oregon Health and Science University , Portland, Oregon 97239, United States.

Motivated by the extremely high computing costs associated with estimates of free energies for biological systems using molecular simulations, we further the exploration of existing "belief propagation" (BP) algorithms for fixed-backbone peptide and protein systems. The precalculation of pairwise interactions among discretized libraries of side-chain conformations, along with representation of protein side chains as nodes in a graphical model, enables direct application of the BP approach, which requires only ∼1 s of single-processor run time after the precalculation stage. We use a "loopy BP" algorithm, which can be seen as an approximate generalization of the transfer-matrix approach to highly connected (i.e., loopy) graphs, and it has previously been applied to protein calculations. We examine the application of loopy BP to several peptides as well as the binding site of the T4 lysozyme L99A mutant. The present study reports on (i) the comparison of the approximate BP results with estimates from unbiased estimators based on the Amber99SB force field; (ii) investigation of the effects of varying library size on BP predictions; and (iii) a theoretical discussion of the discretization effects that can arise in BP calculations. The data suggest that, despite their approximate nature, BP free-energy estimates are highly accurate-indeed, they never fall outside confidence intervals from unbiased estimators for the systems where independent results could be obtained. Furthermore, we find that libraries of sufficiently fine discretization (which diminish library-size sensitivity) can be obtained with standard computing resources in most cases. Altogether, the extremely low computing times and accurate results suggest the BP approach warrants further study.
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http://dx.doi.org/10.1021/acs.jctc.7b00775DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5933972PMC
January 2018

G protein-coupled receptors as anabolic drug targets in osteoporosis.

Pharmacol Ther 2018 04 7;184:1-12. Epub 2017 Nov 7.

Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, VIC 3052, Australia. Electronic address:

Osteoporosis is a progressive bone disorder characterised by imbalance between bone building (anabolism) and resorption (catabolism). Most therapeutics target inhibition of osteoclast-mediated bone resorption, but more recent attention in early drug discovery has focussed on anabolic targets in osteoblasts or their precursors. Two marketed agents that display anabolic properties, strontium ranelate and teriparatide, mediate their actions via the G protein-coupled calcium-sensing and parathyroid hormone-1 receptors, respectively. This review explores their activity, the potential for improved therapeutics targeting these receptors and other putative anabolic GPCR targets, including Smoothened, Wnt/Frizzled, relaxin family peptide, adenosine, cannabinoid, prostaglandin and sphingosine-1-phosphate receptors.
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http://dx.doi.org/10.1016/j.pharmthera.2017.10.015DOI Listing
April 2018

High throughput, quantitative analysis of human osteoclast differentiation and activity.

Anal Biochem 2017 Feb 14;519:51-56. Epub 2016 Dec 14.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia. Electronic address:

Osteoclasts are multinuclear cells that degrade bone under both physiological and pathophysiological conditions. Osteoclasts are therefore a major target of osteoporosis therapeutics aimed at preserving bone. Consequently, analytical methods for osteoclast activity are useful for the development of novel biomarkers and/or pharmacological agents for the treatment of osteoporosis. The nucleation state of an osteoclast is indicative of its maturation and activity. To date, activity is routinely measured at the population level with only approximate consideration of the nucleation state (an 'osteoclast population' is typically defined as cells with ≥3 nuclei). Using a fluorescent substrate for tartrate-resistant acid phosphatase (TRAP), a routinely used marker of osteoclast activity, we developed a multi-labelled imaging method for quantitative measurement of osteoclast TRAP activity at the single cell level. Automated image analysis enables interrogation of large osteoclast populations in a high throughput manner using open source software. Using this methodology, we investigated the effects of receptor activator of nuclear factor kappa-B ligand (RANK-L) on osteoclast maturation and activity and demonstrated that TRAP activity directly correlates with osteoclast maturity (i.e. nuclei number). This method can be applied to high throughput screening of osteoclast-targeting compounds to determine changes in maturation and activity.
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http://dx.doi.org/10.1016/j.ab.2016.12.010DOI Listing
February 2017

Isoform-Specific Biased Agonism of Histamine H3 Receptor Agonists.

Mol Pharmacol 2017 Feb 18;91(2):87-99. Epub 2016 Nov 18.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia (D.M.R., A.E.C., N.A.D., S.B., R.B., R.J.S., W.N.C., P.M.S., A.C., C.J.L.); and Institut de Recherches Internationales Servier, Suresnes, France (C.M.C., E.M.)

The human histamine H receptor (hHR) is subject to extensive gene splicing that gives rise to a large number of functional and nonfunctional isoforms. Despite the general acceptance that G protein-coupled receptors can adopt different ligand-induced conformations that give rise to biased signaling, this has not been studied for the HR; further, it is unknown whether splice variants of the same receptor engender the same or differential biased signaling. Herein, we profiled the pharmacology of histamine receptor agonists at the two most abundant hHR splice variants (hHR and hHR) across seven signaling endpoints. Both isoforms engender biased signaling, notably for 4-[3-(benzyloxy)propyl]-1H-imidazole (proxyfan) [e.g., strong bias toward phosphorylation of glycogen synthase kinase 3β (GSK3β) via the full-length receptor] and its congener 3-(1H-imidazol-4-yl)propyl-(4-iodophenyl)-methyl ether (iodoproxyfan), which are strongly consistent with the former's designation as a "protean" agonist. The 80 amino acid IL3 deleted isoform hHR is more permissive in its signaling than hHR: 2-(1H-imidazol-5-yl)ethyl imidothiocarbamate (imetit), proxyfan, and iodoproxyfan were all markedly biased away from calcium signaling, and principal component analysis of the full data set revealed divergent profiles for all five agonists. However, most interesting was the identification of differential biased signaling between the two isoforms. Strikingly, hHR was completely unable to stimulate GSK3β phosphorylation, an endpoint robustly activated by the full-length receptor. To the best of our knowledge, this is the first quantitative example of differential biased signaling via isoforms of the same G protein-coupled receptor that are simultaneously expressed in vivo and gives rise to the possibility of selective pharmacological targeting of individual receptor splice variants.
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http://dx.doi.org/10.1124/mol.116.106153DOI Listing
February 2017

HIV-1 Capsid Function Is Regulated by Dynamics: Quantitative Atomic-Resolution Insights by Integrating Magic-Angle-Spinning NMR, QM/MM, and MD.

J Am Chem Soc 2016 Oct 18;138(42):14066-14075. Epub 2016 Oct 18.

Department of Chemistry and Biochemistry, University of Delaware , Newark, Delaware 19716, United States.

HIV-1 CA capsid protein possesses intrinsic conformational flexibility, which is essential for its assembly into conical capsids and interactions with host factors. CA is dynamic in the assembled capsid, and residues in functionally important regions of the protein undergo motions spanning many decades of time scales. Chemical shift anisotropy (CSA) tensors, recorded in magic-angle-spinning NMR experiments, provide direct residue-specific probes of motions on nano- to microsecond time scales. We combined NMR, MD, and density-functional-theory calculations, to gain quantitative understanding of internal backbone dynamics in CA assemblies, and we found that the dynamically averaged N CSA tensors calculated by this joined protocol are in remarkable agreement with experiment. Thus, quantitative atomic-level understanding of the relationships between CSA tensors, local backbone structure, and motions in CA assemblies is achieved, demonstrating the power of integrating NMR experimental data and theory for characterizing atomic-resolution dynamics in biological systems.
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http://dx.doi.org/10.1021/jacs.6b08744DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5380593PMC
October 2016

Positive Allosteric Modulation of the Muscarinic M1 Receptor Improves Efficacy of Antipsychotics in Mouse Glutamatergic Deficit Models of Behavior.

J Pharmacol Exp Ther 2016 Nov 14;359(2):354-365. Epub 2016 Sep 14.

Drug Discovery Biology (K.H.C.C., D.T.M, C.J.L, P.M.S, J.R.L, A.C.), Centre for Drug Candidate Optimization (D.M.S., R.T.P.), and Medicinal Chemistry (S.N.M, P.J.S.), Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Australia; and Melbourne Neuropsychiatry Centre, Department of Psychiatry and Centre for Neural Engineering, University of Melbourne, Melbourne, Australia (C.P.)

Current antipsychotics are effective in treating the positive symptoms associated with schizophrenia, but they remain suboptimal in targeting cognitive dysfunction. Recent studies have suggested that positive allosteric modulation of the M muscarinic acetylcholine receptor (mAChR) may provide a novel means of improving cognition. However, very little is known about the potential of combination therapies in extending coverage across schizophrenic symptom domains. This study investigated the effect of the M mAChR positive allosteric modulator BQCA [1-(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid], alone or in combination with haloperidol (a first-generation antipsychotic), clozapine (a second-generation atypical antipsychotic), or aripiprazole (a third-generation atypical antipsychotic), in reversing deficits in sensorimotor gating and spatial memory induced by the N-methyl-d-aspartate receptor antagonist, MK-801 [(5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine]. Sensorimotor gating and spatial memory induction are two models that represent aspects of schizophrenia modeled in rodents. In prepulse inhibition (an operational measure of sensorimotor gating), BQCA alone had minimal effects but exhibited different levels of efficacy in reversing MK-801-induced prepulse inhibition disruptions when combined with a subeffective dose of each of the three (currently prescribed) antipsychotics. Furthermore, the combined effect of BQCA and clozapine was absent in M mice. Interestingly, although BQCA alone had no effect in reversing MK-801-induced memory impairments in a Y-maze spatial test, we observed a reversal upon the combination of BQCA with atypical antipsychotics, but not with haloperidol. These findings provide proof of concept that a judicious combination of existing antipsychotics with a selective M mAChR positive allosteric modulator can extend antipsychotic efficacy in glutamatergic deficit models of behavior.
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http://dx.doi.org/10.1124/jpet.116.235788DOI Listing
November 2016

Molecular Mechanisms of Action of M5 Muscarinic Acetylcholine Receptor Allosteric Modulators.

Mol Pharmacol 2016 10 26;90(4):427-36. Epub 2016 Jul 26.

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

Recently, the first subtype-selective allosteric modulators of the M5 muscarinic acetylcholine receptor (mAChR) have been described, but their molecular mechanisms of action remain unknown. Using radioligand-binding and functional assays of inositol phosphate (IP) accumulation and Ca(2+) mobilization in a recombinant cell line stably expressing the human M5 mAChR, we investigated the effects of the positive allosteric modulator (PAM), ML380, and negative allosteric modulator, ML375. In functional assays, ML380 caused robust enhancements in the potency of the full agonists, acetylcholine (ACh), carbachol, and oxotremorine-M, while significantly increasing the maximal response to the partial agonist, pilocarpine. ML380 also demonstrated direct allosteric agonist activity. In contrast, ML375 displayed negative cooperativity with each of the agonists in a manner that varied with the pathway investigated and progressively reduced the maximal pilocarpine response. Radioligand-binding affinity cooperativity estimates were consistent with values derived from functional assays in some instances but not others, suggesting additional allosteric effects on orthosteric ligand efficacy. For ML375 this was confirmed in IP assays performed after reduction of receptor reserve by the alkylating agent, phenoxybenzamine, as it reduced the maximal ACh response. In contrast, ML380 enhanced only ACh potency after receptor alkylation, with no effect on maximal response, consistent with studies of the M1 mAChR with the prototypical PAM, BQZ12. Interaction studies between ML380 and ML375 also indicated that they most likely used an overlapping allosteric site. Our findings indicate that novel small-molecule modulators of the M5 mAChR display mixed mechanisms of action compared with previously characterized modulators of other mAChRs.
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http://dx.doi.org/10.1124/mol.116.104182DOI Listing
October 2016

Murine GPRC6A Mediates Cellular Responses to L-Amino Acids, but Not Osteocalcin Variants.

PLoS One 2016 19;11(1):e0146846. Epub 2016 Jan 19.

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

Phenotyping of Gprc6a KO mice has shown that this promiscuous class C G protein coupled receptor is variously involved in regulation of metabolism, inflammation and endocrine function. Such effects are described as mediated by extracellular calcium, L-amino acids, the bone-derived peptide osteocalcin (OCN) and the male hormone testosterone, introducing the concept of a bone-energy-metabolism-reproduction functional crosstalk mediated by GPRC6A. However, whilst the calcium and L-amino acid-sensing properties of GPRC6A are well established, verification of activity of osteocalcin at both human and mouse GPRC6A in vitro has proven somewhat elusive. This study characterises the in vitro pharmacology of mouse GPRC6A in response to its putative ligands in both recombinant and endogenous GPRC6A-expressing cells. Using cell signalling, and glucagon-like peptide (GLP)-1 and insulin release assays, our results confirm that basic L-amino acids act as agonists of the murine GPRC6A receptor in both recombinant cells and immortalised entero-endocrine and pancreatic β-cells. In contrast, our studies do not support a role for OCN as a direct ligand for mouse GPRC6A, suggesting that the reported in vivo effects of OCN that require GPRC6A may be indirect, rather than via direct activation of the receptor.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0146846PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4718634PMC
July 2016

Automated parameter estimation for biological models using Bayesian statistical model checking.

BMC Bioinformatics 2015 7;16 Suppl 17:S8. Epub 2015 Dec 7.

Background: Probabilistic models have gained widespread acceptance in the systems biology community as a useful way to represent complex biological systems. Such models are developed using existing knowledge of the structure and dynamics of the system, experimental observations, and inferences drawn from statistical analysis of empirical data. A key bottleneck in building such models is that some system variables cannot be measured experimentally. These variables are incorporated into the model as numerical parameters. Determining values of these parameters that justify existing experiments and provide reliable predictions when model simulations are performed is a key research problem.

Results: Using an agent-based model of the dynamics of acute inflammation, we demonstrate a novel parameter estimation algorithm by discovering the amount and schedule of doses of bacterial lipopolysaccharide that guarantee a set of observed clinical outcomes with high probability. We synthesized values of twenty-eight unknown parameters such that the parameterized model instantiated with these parameter values satisfies four specifications describing the dynamic behavior of the model.

Conclusions: We have developed a new algorithmic technique for discovering parameters in complex stochastic models of biological systems given behavioral specifications written in a formal mathematical logic. Our algorithm uses Bayesian model checking, sequential hypothesis testing, and stochastic optimization to automatically synthesize parameters of probabilistic biological models.
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http://dx.doi.org/10.1186/1471-2105-16-S17-S8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4674867PMC
August 2016

Dynamic allostery governs cyclophilin A-HIV capsid interplay.

Proc Natl Acad Sci U S A 2015 Nov 9;112(47):14617-22. Epub 2015 Nov 9.

Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716; Pittsburgh Center for HIV Protein Interactions, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260;

Host factor protein Cyclophilin A (CypA) regulates HIV-1 viral infectivity through direct interactions with the viral capsid, by an unknown mechanism. CypA can either promote or inhibit viral infection, depending on host cell type and HIV-1 capsid (CA) protein sequence. We have examined the role of conformational dynamics on the nanosecond to millisecond timescale in HIV-1 CA assemblies in the escape from CypA dependence, by magic-angle spinning (MAS) NMR and molecular dynamics (MD). Through the analysis of backbone (1)H-(15)N and (1)H-(13)C dipolar tensors and peak intensities from 3D MAS NMR spectra of wild-type and the A92E and G94D CypA escape mutants, we demonstrate that assembled CA is dynamic, particularly in loop regions. The CypA loop in assembled wild-type CA from two strains exhibits unprecedented mobility on the nanosecond to microsecond timescales, and the experimental NMR dipolar order parameters are in quantitative agreement with those calculated from MD trajectories. Remarkably, the CypA loop dynamics of wild-type CA HXB2 assembly is significantly attenuated upon CypA binding, and the dynamics profiles of the A92E and G94D CypA escape mutants closely resemble that of wild-type CA assembly in complex with CypA. These results suggest that CypA loop dynamics is a determining factor in HIV-1's escape from CypA dependence.
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http://dx.doi.org/10.1073/pnas.1516920112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4664340PMC
November 2015

Label-Free Kinetics: Exploiting Functional Hemi-Equilibrium to Derive Rate Constants for Muscarinic Receptor Antagonists.

Mol Pharmacol 2015 Oct 4;88(4):779-90. Epub 2015 Aug 4.

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

Drug receptor kinetics is as a key component in drug discovery, development, and efficacy; however, determining kinetic parameters has historically required direct radiolabeling or competition with a labeled tracer. Here we present a simple approach to determining the kinetics of competitive antagonists of G protein-coupled receptors by exploiting the phenomenon of hemi-equilibrium, the state of partial re-equilibration of agonist, antagonist, and receptor in some functional assays. Using functional [Ca(2+)]i-flux and extracellular kinases 1 and 2 phosphorylation assays that have short incubation times and therefore are prone to hemi-equilibrium "behaviors," we investigated a wide range of structurally and physicochemically distinct muscarinic acetylcholine receptor antagonists. Using a combined operational and hemi-equilibrium model of antagonism to both simulate and analyze data, we derived estimates of association and dissociation rates for the test set of antagonists, identifying both rapidly dissociating (4-DAMP, himbacine) and slowly dissociating (tiotropium, glycopyrrolate) ligands. The results demonstrate the importance of assay incubation time and the degree of receptor reserve in applying the analytical model. There was an excellent correlation between estimates of antagonist pK(B), k(on), and k(off) from functional assays and those determined by competition kinetics using whole-cell [(3)H]N-methylscopolamine binding, validating this approach as a rapid and simple method to functionally profile receptor kinetics of competitive antagonists in the absence of a labeled tracer.
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http://dx.doi.org/10.1124/mol.115.100545DOI Listing
October 2015