Publications by authors named "Lauren T May"

52 Publications

Development of Covalent, Clickable Probes for Adenosine A and A Receptors.

J Med Chem 2021 Jun 14;64(12):8161-8178. Epub 2021 Jun 14.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, and Department of Pharmacology, Monash University, Parkville, VIC 3052, Australia.

Adenosine receptors are attractive therapeutic targets for multiple conditions, including ischemia-reperfusion injury and neuropathic pain. Adenosine receptor drug discovery efforts would be facilitated by the development of appropriate tools to assist in target validation and direct receptor visualization in different native environments. We report the development of the first bifunctional (chemoreactive and clickable) ligands for the adenosine A receptor (AR) and adenosine A receptor (AR) based on an orthosteric antagonist xanthine-based scaffold and on an existing structure-activity relationship. Bifunctional ligands were functional antagonists with nanomolar affinity and irreversible binding at the AR and AR. In-depth pharmacological profiling of these bifunctional ligands showed moderate selectivity over A and A adenosine receptors. Once bound to the receptor, ligands were successfully "clicked" with a cyanine-5 fluorophore containing the complementary "click" partner, enabling receptor detection. These bifunctional ligands are expected to aid in the understanding of AR and AR localization and trafficking in native cells and living systems.
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http://dx.doi.org/10.1021/acs.jmedchem.0c02169DOI Listing
June 2021

Inhibition of the Proliferation of Human Lung Fibroblasts by Prostacyclin Receptor Agonists is Linked to a Sustained cAMP Signal in the Nucleus.

Front Pharmacol 2021 29;12:669227. Epub 2021 Apr 29.

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

Idiopathic pulmonary fibrosis is a chronic and progressive fibrotic lung disease, and current treatments are limited by their side effects. Proliferation of human lung fibroblasts in the pulmonary interstitial tissue is a hallmark of this disease and is driven by prolonged ERK signalling in the nucleus in response to growth factors such as platelet-derived growth factor (PDGF). Agents that increase cAMP have been suggested as alternative therapies, as this second messenger can inhibit the ERK cascade. We previously examined a panel of eight Gα-cAMP-coupled G protein-coupled receptors (GPCRs) endogenously expressed in human lung fibroblasts. Although the cAMP response was important for the anti-fibrotic effects of GPCR agonists, the magnitude of the acute cAMP response was not predictive of anti-fibrotic efficacy. Here we examined the reason for this apparent disconnect by stimulating the Gα-coupled prostacyclin receptor and measuring downstream signalling at a sub-cellular level. MRE-269 and treprostinil caused sustained cAMP signalling in the nucleus and complete inhibition of PDGF-induced nuclear ERK and fibroblast proliferation. In contrast, iloprost caused a transient increase in nuclear cAMP, there was no effect of iloprost on PDGF-induced ERK in the nucleus, and this agonist was much less effective at reversing PDGF-induced proliferation. This suggests that sustained elevation of cAMP in the nucleus is necessary for efficient inhibition of PDGF-induced nuclear ERK and fibroblast proliferation. This is an important first step towards understanding of the signalling events that drive GPCR inhibition of fibrosis.
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http://dx.doi.org/10.3389/fphar.2021.669227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8116805PMC
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

Development and Application of Subtype-Selective Fluorescent Antagonists for the Study of the Human Adenosine A Receptor in Living Cells.

J Med Chem 2021 05 16;64(10):6670-6695. Epub 2021 Mar 16.

Medicinal Chemistry, Monash University, Parkville, Victoria 3052, Australia.

The adenosine A receptor (AAR) is a G-protein-coupled receptor (GPCR) that provides important therapeutic opportunities for a number of conditions including congestive heart failure, tachycardia, and neuropathic pain. The development of AAR-selective fluorescent ligands will enhance our understanding of the subcellular mechanisms underlying AAR pharmacology facilitating the development of more efficacious and selective therapies. Herein, we report the design, synthesis, and application of a novel series of AAR-selective fluorescent probes based on 8-functionalized bicyclo[2.2.2]octylxanthine and 3-functionalized 8-(adamant-1-yl) xanthine scaffolds. These fluorescent conjugates allowed quantification of kinetic and equilibrium ligand binding parameters using NanoBRET and visualization of specific receptor distribution patterns in living cells by confocal imaging and total internal reflection fluorescence (TIRF) microscopy. As such, the novel AAR-selective fluorescent antagonists described herein can be applied in conjunction with a series of fluorescence-based techniques to foster understanding of AAR molecular pharmacology and signaling in living cells.
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http://dx.doi.org/10.1021/acs.jmedchem.0c02067DOI Listing
May 2021

Biased agonism at adenosine receptors.

Cell Signal 2021 Jun 19;82:109954. Epub 2021 Feb 19.

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

Adenosine modulates many aspects of human physiology and pathophysiology through binding to the adenosine family of G protein-coupled receptors, which are comprised of four subtypes, the AR, AR, AR and AR. Modulation of adenosine receptor function by exogenous agonists, antagonists and allosteric modulators can be beneficial for a number of conditions including cardiovascular disease, Parkinson's disease, and cancer. Unfortunately, many preclinical drug candidates targeting adenosine receptors have failed in clinical trials due to limited efficacy and/or severe on-target undesired effects. To overcome the key barriers typically encountered when transitioning adenosine receptor ligands into the clinic, research efforts have focussed on exploiting the phenomenon of biased agonism. Biased agonism provides the opportunity to develop ligands that favour therapeutic signalling pathways, whilst avoiding signalling associated with on-target undesired effects. Recent studies have begun to define the structure-function relationships that underpin adenosine receptor biased agonism and establish how this phenomenon can be harnessed therapeutically. In this review we describe the recent advancements made towards achieving therapeutically relevant biased agonism at adenosine receptors.
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http://dx.doi.org/10.1016/j.cellsig.2021.109954DOI Listing
June 2021

Substituted Pyridazin-3(2 H)-ones as Highly Potent and Biased Formyl Peptide Receptor Agonists.

J Med Chem 2019 05 13;62(10):5242-5248. Epub 2019 May 13.

School of Pharmaceutical Sciences , Nanjing Tech University , Nanjing 211816 , People's Republic of China.

Herein we describe the development of a focused series of functionalized pyridazin-3(2 H)-one-based formyl peptide receptor (FPR) agonists that demonstrate high potency and biased agonism. The compounds described demonstrated biased activation of prosurvival signaling, ERK1/2 phosphorylation, through diminution of the detrimental FPR1/2-mediated intracellular calcium (Ca) mobilization. Compound 50 showed an EC of 0.083 μM for phosphorylation of ERK1/2 and an approximate 20-fold bias away from Ca mobilization at the hFPR1.
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http://dx.doi.org/10.1021/acs.jmedchem.8b01912DOI Listing
May 2019

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 adenosine A G protein-coupled receptor: Recent advances and therapeutic implications.

Pharmacol Ther 2019 06 22;198:20-33. Epub 2019 Jan 22.

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

The adenosine A receptor (AAR) is one of four adenosine receptor subtypes belonging to the Class A family of G protein-coupled receptors (GPCRs). Until recently, the AAR remained poorly characterised, in part due to its relatively low affinity for the endogenous agonist adenosine and therefore presumed minor physiological significance. However, the substantial increase in extracellular adenosine concentration, the sensitisation of the receptor and the upregulation of AAR expression under conditions of hypoxia and inflammation, suggest the AAR as an exciting therapeutic target in a variety of pathological disease states. Here we discuss the pharmacology of the AAR and outline its role in pathophysiology including ischaemia-reperfusion injury, fibrosis, inflammation and cancer.
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http://dx.doi.org/10.1016/j.pharmthera.2019.01.003DOI Listing
June 2019

Structural Basis for Binding of Allosteric Drug Leads in the Adenosine A Receptor.

Sci Rep 2018 11 15;8(1):16836. Epub 2018 Nov 15.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, VIC, 3052, Australia.

Despite intense interest in designing positive allosteric modulators (PAMs) as selective drugs of the adenosine A receptor (AAR), structural binding modes of the receptor PAMs remain unknown. Using the first X-ray structure of the AAR, we have performed all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) technique to determine binding modes of the AAR allosteric drug leads. Two prototypical PAMs, PD81723 and VCP171, were selected. Each PAM was initially placed at least 20 Å away from the receptor. Extensive GaMD simulations using the AMBER and NAMD simulation packages at different acceleration levels captured spontaneous binding of PAMs to the AAR. The simulations allowed us to identify low-energy binding modes of the PAMs at an allosteric site formed by the receptor extracellular loop 2 (ECL2), which are highly consistent with mutagenesis experimental data. Furthermore, the PAMs stabilized agonist binding in the receptor. In the absence of PAMs at the ECL2 allosteric site, the agonist sampled a significantly larger conformational space and even dissociated from the AAR alone. In summary, the GaMD simulations elucidated structural binding modes of the PAMs and provided important insights into allostery in the AAR, which will greatly facilitate the receptor structure-based drug design.
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http://dx.doi.org/10.1038/s41598-018-35266-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237911PMC
November 2018

Biased agonism and allosteric modulation of metabotropic glutamate receptor 5.

Clin Sci (Lond) 2018 11 2;132(21):2323-2338. Epub 2018 Nov 2.

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

Metabotropic glutamate receptors belong to class C G-protein-coupled receptors and consist of eight subtypes that are ubiquitously expressed throughout the central nervous system. In recent years, the metabotropic glutamate receptor subtype 5 (mGlu) has emerged as a promising target for a broad range of psychiatric and neurological disorders. Drug discovery programs targetting mGlu are primarily focused on development of allosteric modulators that interact with sites distinct from the endogenous agonist glutamate. Significant efforts have seen mGlu allosteric modulators progress into clinical trials; however, recent failures due to lack of efficacy or adverse effects indicate a need for a better understanding of the functional consequences of mGlu allosteric modulation. Biased agonism is an interrelated phenomenon to allosterism, describing how different ligands acting through the same receptor can differentially influence signaling to distinct transducers and pathways. Emerging evidence demonstrates that allosteric modulators can induce biased pharmacology at the level of intrinsic agonism as well as through differential modulation of orthosteric agonist-signaling pathways. Here, we present key considerations in the discovery and development of mGlu allosteric modulators and the opportunities and pitfalls offered by biased agonism and modulation.
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http://dx.doi.org/10.1042/CS20180374DOI Listing
November 2018

Dominant Negative G Proteins Enhance Formation and Purification of Agonist-GPCR-G Protein Complexes for Structure Determination.

ACS Pharmacol Transl Sci 2018 Sep 26;1(1):12-20. Epub 2018 Jul 26.

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

Advances in structural biology have yielded exponential growth in G protein-coupled receptor (GPCR) structure solution. Nonetheless, the instability of fully active GPCR complexes with cognate heterotrimeric G proteins has made them elusive. Existing structures have been limited to nanobody-stabilized GPCR:Gs complexes. Here we present methods for enhanced GPCR:G protein complex stabilization via engineering G proteins with reduced nucleotide affinity, limiting Gα:Gβγ dissociation. We illustrate the application of dominant negative G proteins of Gαs and Gαi2 to the purification of stable complexes where this was not possible with wild-type G protein. Active state complexes of adenosine:A1 receptor:Gαi2βγ and calcitonin gene-related peptide (CGRP):CLR:RAMP1:Gαsβγ:Nb35 were purified to homogeneity and were stable in negative stain electron microscopy. These were suitable for structure determination by cryo-electron microscopy at 3.6 and 3.3 Å resolution, respectively. The dominant negative Gα-proteins are thus high value tools for structure determination of agonist:GPCR:G protein complexes that are critical for informed translational drug discovery.
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http://dx.doi.org/10.1021/acsptsci.8b00017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7089020PMC
September 2018

Structure of the adenosine-bound human adenosine A receptor-G complex.

Nature 2018 06 20;558(7711):559-563. Epub 2018 Jun 20.

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

The class A adenosine A receptor (AR) is a G-protein-coupled receptor that preferentially couples to inhibitory G heterotrimeric G proteins, has been implicated in numerous diseases, yet remains poorly targeted. Here we report the 3.6 Å structure of the human AR in complex with adenosine and heterotrimeric G protein determined by Volta phase plate cryo-electron microscopy. Compared to inactive AR, there is contraction at the extracellular surface in the orthosteric binding site mediated via movement of transmembrane domains 1 and 2. At the intracellular surface, the G protein engages the AR primarily via amino acids in the C terminus of the Gα α5-helix, concomitant with a 10.5 Å outward movement of the AR transmembrane domain 6. Comparison with the agonist-bound β adrenergic receptor-G-protein complex reveals distinct orientations for each G-protein subtype upon engagement with its receptor. This active AR structure provides molecular insights into receptor and G-protein selectivity.
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http://dx.doi.org/10.1038/s41586-018-0236-6DOI Listing
June 2018

New paradigms in adenosine receptor pharmacology: allostery, oligomerization and biased agonism.

Br J Pharmacol 2018 11 21;175(21):4036-4046. Epub 2018 Jun 21.

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

Adenosine receptors are a family of GPCRs containing four subtypes (A , A , A and A receptors), all of which bind the ubiquitous nucleoside adenosine. These receptors play an important role in physiology and pathophysiology and therefore represent attractive drug targets for a range of conditions. The theoretical framework surrounding drug action at adenosine receptors now extends beyond the notion of prototypical agonism and antagonism to encompass more complex pharmacological concepts. New paradigms include allostery, in which ligands bind a topographically distinct receptor site from that of the endogenous agonist, homomeric or heteromeric interactions across receptor oligomers and biased agonism, that is, ligand-dependent differential intracellular signalling. This review provides a concise overview of allostery, oligomerization and biased agonism at adenosine receptors and outlines how these paradigms may enhance future drug discovery endeavours focussed on the development of novel therapeutic agents acting at adenosine receptors.

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.14337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6177620PMC
November 2018

"Selective" Class C G Protein-Coupled Receptor Modulators Are Neutral or Biased mGlu Allosteric Ligands.

Mol Pharmacol 2018 05 7;93(5):504-514. Epub 2018 Mar 7.

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

Numerous positive and negative allosteric modulators (PAMs and NAMs) of class C G protein-coupled receptors (GPCRs) have been developed as valuable preclinical pharmacologic tools and therapeutic agents. Although many class C GPCR allosteric modulators have undergone subtype selectivity screening, most assay paradigms have failed to perform rigorous pharmacologic assessment. Using mGlu as a representative class C GPCR, we tested the hypothesis that allosteric modulator selectivity was based on cooperativity rather than affinity. Specifically, we aimed to identify ligands that bound to mGlu but exhibited neutral cooperativity with mGlu agonists. We additionally evaluated the potential for these ligands to exhibit biased pharmacology. Radioligand binding, intracellular calcium (iCa) mobilization, and inositol monophosphate (IP) accumulation assays were undertaken in human embryonic kidney cells expressing low levels of rat mGlu (HEK293A-mGlu-low) for diverse allosteric chemotypes. Numerous "non-mGlu" class C GPCR allosteric modulators incompletely displaced allosteric mGlu radioligand [H]methoxy-PEPy binding, consistent with a negative allosteric interaction. Affinity estimates for CPCCOEt (mGlu ligand), PHCCC (mGlu ligand), GS39783 (GABA ligand), AZ12216052 (mGlu ligand), and CGP7930 (GABA ligand) at mGlu were within 10-fold of their target receptor. Most class C GPCR allosteric modulators had neutral cooperativity with both orthosteric and allosteric mGlu agonists in functional assays; however, NPS2143 (calcium-sensing receptor (CaSR) NAM), cinacalcet (CaSR PAM), CGP7930, and AZ12216052 were partial mGlu agonists for IP accumulation, but not iCa mobilization. By using mGlu as a model class C GPCR, we find that for many class C GPCR allosteric modulators, subtype selectivity is driven by cooperativity and misinterpreted owing to unappreciated bias.
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http://dx.doi.org/10.1124/mol.117.111518DOI Listing
May 2018

Structure-based discovery of selective positive allosteric modulators of antagonists for the M muscarinic acetylcholine receptor.

Proc Natl Acad Sci U S A 2018 03 16;115(10):E2419-E2428. Epub 2018 Feb 16.

Department of Pharmacology, University of California San Diego School of Medicine, La Jolla, CA 92093;

Subtype-selective antagonists for muscarinic acetylcholine receptors (mAChRs) have long been elusive, owing to the highly conserved orthosteric binding site. However, allosteric sites of these receptors are less conserved, motivating the search for allosteric ligands that modulate agonists or antagonists to confer subtype selectivity. Accordingly, a 4.6 million-molecule library was docked against the structure of the prototypical M mAChR, seeking molecules that specifically stabilized antagonist binding. This led us to identify a positive allosteric modulator (PAM) that potentiated the antagonist -methyl scopolamine (NMS). Structure-based optimization led to compound '628, which enhanced binding of NMS, and the drug scopolamine itself, with a cooperativity factor (α) of 5.5 and a of 1.1 μM, while sparing the endogenous agonist acetylcholine. NMR spectral changes determined for methionine residues reflected changes in the allosteric network. Moreover, '628 slowed the dissociation rate of NMS from the M mAChR by 50-fold, an effect not observed at the other four mAChR subtypes. The specific PAM effect of '628 on NMS antagonism was conserved in functional assays, including agonist stimulation of [S]GTPγS binding and ERK 1/2 phosphorylation. Importantly, the selective allostery between '628 and NMS was retained in membranes from adult rat hypothalamus and in neonatal rat cardiomyocytes, supporting the physiological relevance of this PAM/antagonist approach. This study supports the feasibility of discovering PAMs that confer subtype selectivity to antagonists; molecules like '628 can convert an armamentarium of potent but nonselective GPCR antagonist drugs into subtype-selective reagents, thus reducing their off-target effects.
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http://dx.doi.org/10.1073/pnas.1718037115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5877965PMC
March 2018

A Structure-Activity Relationship Study of Bitopic N-Substituted Adenosine Derivatives as Biased Adenosine A Receptor Agonists.

J Med Chem 2018 03 23;61(5):2087-2103. Epub 2018 Feb 23.

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

The adenosine A receptor (AAR) is a potential novel therapeutic target for myocardial ischemia-reperfusion injury. However, to date, clinical translation of prototypical AAR agonists has been hindered due to dose limiting adverse effects. Recently, we demonstrated that the biased bitopic agonist 1, consisting of an adenosine pharmacophore linked to an allosteric moiety, could stimulate cardioprotective AAR signaling in the absence of unwanted bradycardia. Therefore, this study aimed to investigate the structure-activity relationship of compound 1 biased agonism. A series of novel derivatives of 1 were synthesized and pharmacologically profiled. Modifications were made to the orthosteric adenosine pharmacophore, linker, and allosteric 2-amino-3-benzoylthiophene pharmacophore to probe the structure-activity relationships, particularly in terms of biased signaling, as well as AAR activity and subtype selectivity. Collectively, our findings demonstrate that the allosteric moiety, particularly the 4-(trifluoromethyl)phenyl substituent of the thiophene scaffold, is important in conferring bitopic ligand bias at the AAR.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00047DOI Listing
March 2018

α-Adrenoceptors activate mTOR signalling and glucose uptake in cardiomyocytes.

Biochem Pharmacol 2018 02 24;148:27-40. Epub 2017 Nov 24.

Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, SE-106 91 Stockholm, Sweden. Electronic address:

The capacity of G protein-coupled receptors to modulate mechanistic target of rapamycin (mTOR) activity is a newly emerging paradigm with the potential to link cell surface receptors with cell survival. Cardiomyocyte viability is linked to signalling pathways involving Akt and mTOR, as well as increased glucose uptake and utilization. Our aim was to determine whether the α-adrenoceptor (AR) couples to these protective pathways, and increased glucose uptake. We characterised α-AR signalling in CHO-K1 cells co-expressing the human α-AR and GLUT4 (CHOαGLUT4myc) and in neonatal rat ventricular cardiomyocytes (NRVM), and measured glucose uptake, intracellular Ca mobilization, and phosphorylation of mTOR, Akt, 5' adenosine monophosphate-activated kinase (AMPK) and S6 ribosomal protein (S6rp). In both systems, noradrenaline and the α-AR selective agonist A61603 stimulated glucose uptake by parallel pathways involving mTOR and AMPK, whereas another α-AR agonist oxymetazoline increased glucose uptake predominantly by mTOR. All agonists promoted phosphorylation of mTOR at Ser2448 and Ser2481, indicating activation of both mTORC1 and mTORC2, but did not increase Akt phosphorylation. In CHOαGLUT4myc cells, siRNA directed against rictor but not raptor suppressed α-AR mediated glucose uptake. We have thus identified mTORC2 as a key component in glucose uptake stimulated by α-AR agonists. Our findings identify a novel link between the α-AR, mTORC2 and glucose uptake, that have been implicated separately in cardiomyocyte survival. Our studies provide an improved framework for examining the utility of α-AR selective agonists as tools in the treatment of cardiac dysfunction.
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http://dx.doi.org/10.1016/j.bcp.2017.11.016DOI Listing
February 2018

Characterisation of endogenous A and A receptor-mediated cyclic AMP responses in HEK 293 cells using the GloSensor™ biosensor: Evidence for an allosteric mechanism of action for the A-selective antagonist PSB 603.

Biochem Pharmacol 2018 01 26;147:55-66. Epub 2017 Oct 26.

Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University of Nottingham, Nottingham NG7 2UH, UK; Centre of Membrane Proteins and Receptors (COMPARE), University of Birmingham and University of Nottingham, Midlands, UK. Electronic address:

Endogenous adenosine A receptors (AAR) mediate cAMP accumulation in HEK 293 cells. Here we have used a biosensor to investigate the mechanism of action of the AAR antagonist PSB 603 in HEK 293 cells. The A agonist CGS 21680 elicited a small response in these cells (circa 20% of that obtained with NECA), suggesting that they also contain a small population of A receptors. The responses to NECA and adenosine were antagonised by PSB 603, but not by the selective AAR antagonist SCH 58261. In contrast, CGS 21680 responses were not antagonised by high concentrations of PSB 603, but were sensitive to inhibition by SCH 58261. Analysis of the effect of increasing concentrations of PSB 603 on the response to NECA indicated a non-competitive mode of action yielding a marked reduction in the NECA E with no significant effect on EC values. Kinetics analysis of the effect of PSB 603 on the AAR-mediated NECA responses confirmed a saturable effect that was consistent with an allosteric mode of antagonism. The possibility that PSB 603 acts as a negative allosteric modulator of AAR suggests new approaches to the development of therapeutic agents to treat conditions where adenosine levels are high.
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http://dx.doi.org/10.1016/j.bcp.2017.10.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5770336PMC
January 2018

A kinetic view of GPCR allostery and biased agonism.

Nat Chem Biol 2017 Aug;13(9):929-937

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

G-protein-coupled receptors (GPCRs) are one of the most tractable classes of drug targets. These dynamic proteins can adopt multiple active states that are linked to distinct functional outcomes. Such states can be differentially stabilized by ligands interacting with the endogenous agonist-binding orthosteric site and/or by ligands acting via spatially distinct allosteric sites, leading to the phenomena of 'biased agonism' or 'biased modulation'. These paradigms are having a major impact on modern drug discovery, but it is becoming increasingly apparent that 'kinetic context', at the level of both ligand-receptor and receptor-signal pathway kinetics, can have a profound impact on the observation and quantification of these phenomena. The concept of kinetic context thus represents an important new consideration that should be routinely incorporated into contemporary chemical biology and drug discovery studies of GPCR bias and allostery.
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http://dx.doi.org/10.1038/nchembio.2431DOI Listing
August 2017

Targeting Adenosine Receptors for the Treatment of Cardiac Fibrosis.

Front Pharmacol 2017 5;8:243. Epub 2017 May 5.

Monash Institute of Pharmaceutical Sciences, Monash University, ParkvilleVIC, Australia.

Adenosine is a ubiquitous molecule with key regulatory and cytoprotective mechanisms at times of metabolic imbalance in the body. Among a plethora of physiological actions, adenosine has an important role in attenuating ischaemia-reperfusion injury and modulating the ensuing fibrosis and tissue remodeling following myocardial damage. Adenosine exerts these actions through interaction with four adenosine G protein-coupled receptors expressed in the heart. The adenosine A receptor (AAR) is the most abundant adenosine receptor (AR) in cardiac fibroblasts and is largely responsible for the influence of adenosine on cardiac fibrosis. and studies demonstrate that acute AAR stimulation can decrease fibrosis through the inhibition of fibroblast proliferation and reduction in collagen synthesis. However, in contrast, there is also evidence that chronic AAR antagonism reduces tissue fibrosis. This review explores the opposing pro- and anti-fibrotic activity attributed to the activation of cardiac ARs and investigates the therapeutic potential of targeting ARs for the treatment of cardiac fibrosis.
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http://dx.doi.org/10.3389/fphar.2017.00243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5418340PMC
May 2017

Capadenoson, a clinically trialed partial adenosine A receptor agonist, can stimulate adenosine A receptor biased agonism.

Biochem Pharmacol 2017 07 23;135:79-89. Epub 2017 Mar 23.

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

The adenosine A receptor (AAR) has been identified as an important therapeutic target in cardiovascular disease, however in vitro and in vivo targeting has been limited by the paucity of pharmacological tools, particularly potent agonists. Interestingly, 2-((6-amino-3,5-dicyano-4-(4-(cyclopropylmethoxy)phenyl)-2-pyridinyl)thio)acetamide (BAY60-6583), a potent and subtype-selective AAR agonist, has the same core structure as 2-amino-6-[[2-(4-chlorophenyl)-1,3-thiazol-4-yl]methylsulfanyl]-4-[4-(2-hydroxyethoxy)phenyl]pyridine-3,5-dicarbonitril (capadenoson). Capadenoson, currently classified as an adenosine A receptor (AAR) partial agonist, has undergone two Phase IIa clinical trials, initially in patients with atrial fibrillation and subsequently in patients with stable angina. Capadenoson has also been shown to decrease cardiac remodeling in an animal model of advanced heart failure and a capadenoson derivative, neladenoson bialanate, recently entered clinical development for the treatment of chronic heart failure. The therapeutic effects of capadenoson are currently thought to be mediated through the AAR. However, the ability of capadenoson to stimulate additional adenosine receptor subtypes, in particular the AAR, has not been rigorously assessed. In this study, we demonstrate that capadenoson does indeed have significant AAR activity in physiologically relevant cells, cardiac fibroblasts and cardiomyocytes, which endogenously express the AAR. Relative to the non-selective adenosine receptor agonist NECA, capadenoson was a biased AAR agonist with a preference for cAMP signal transduction over other downstream mediators in cells with recombinant and endogenous AAR expression. These findings suggest the reclassification of capadenoson as a dual AAR/AAR agonist. Furthermore, a potential AAR contribution should be an important consideration for the future clinical development of capadenoson-like therapeutics, as the AAR can promote cardioprotection and modulate cardiac fibrosis in heart disease.
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http://dx.doi.org/10.1016/j.bcp.2017.03.014DOI Listing
July 2017

Structure of the Adenosine A Receptor Reveals the Basis for Subtype Selectivity.

Cell 2017 02;168(5):867-877.e13

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

The adenosine A receptor (A-AR) is a G-protein-coupled receptor that plays a vital role in cardiac, renal, and neuronal processes but remains poorly targeted by current drugs. We determined a 3.2 Å crystal structure of the A-AR bound to the selective covalent antagonist, DU172, and identified striking differences to the previously solved adenosine A receptor (A-AR) structure. Mutational and computational analysis of A-AR revealed a distinct conformation of the second extracellular loop and a wider extracellular cavity with a secondary binding pocket that can accommodate orthosteric and allosteric ligands. We propose that conformational differences in these regions, rather than amino-acid divergence, underlie drug selectivity between these adenosine receptor subtypes. Our findings provide a molecular basis for AR subtype selectivity with implications for understanding the mechanisms governing allosteric modulation of these receptors, allowing the design of more selective agents for the treatment of ischemia-reperfusion injury, renal pathologies, and neuropathic pain.
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http://dx.doi.org/10.1016/j.cell.2017.01.042DOI Listing
February 2017

Small-molecule-biased formyl peptide receptor agonist compound 17b protects against myocardial ischaemia-reperfusion injury in mice.

Nat Commun 2017 02 7;8:14232. Epub 2017 Feb 7.

Baker IDI Heart and Diabetes Institute, 75 Commercial Road, Melbourne, Victoria 3004, Australia.

Effective treatment for managing myocardial infarction (MI) remains an urgent, unmet clinical need. Formyl peptide receptors (FPR) regulate inflammation, a major contributing mechanism to cardiac injury following MI. Here we demonstrate that FPR1/FPR2-biased agonism may represent a novel therapeutic strategy for the treatment of MI. The small-molecule FPR1/FPR2 agonist, Compound 17b (Cmpd17b), exhibits a distinct signalling fingerprint to the conventional FPR1/FPR2 agonist, Compound-43 (Cmpd43). In Chinese hamster ovary (CHO) cells stably transfected with human FPR1 or FPR2, Compd17b is biased away from potentially detrimental FPR1/2-mediated calcium mobilization, but retains the pro-survival signalling, ERK1/2 and Akt phosphorylation, relative to Compd43. The pathological importance of the biased agonism of Cmpd17b is demonstrable as superior cardioprotection in both in vitro (cardiomyocytes and cardiofibroblasts) and MI injury in mice in vivo. These findings reveal new insights for development of small molecule FPR agonists with an improved cardioprotective profile for treating MI.
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http://dx.doi.org/10.1038/ncomms14232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5309721PMC
February 2017

Novel Irreversible Agonists Acting at the A Adenosine Receptor.

J Med Chem 2016 12 13;59(24):11182-11194. Epub 2016 Dec 13.

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

The A adenosine receptor (AAR) is an important G protein-coupled receptor that regulates a range of physiological functions. Herein we report the discovery of novel irreversible agonists acting at the AAR, which have the potential to serve as useful research tools for studying receptor structure and function. A series of novel adenosine derivatives bearing electrophilic substituents was synthesized, and four compounds, 8b, 15a, 15b, and 15d, were shown to possess similar potency and efficacy to the reference high efficacy agonist, NECA, in an assay of ERK1/2 phosphorylation assay. Insensitivity to antagonist addition in a real-time, label-free, xCELLigence assay was subsequently used to identify compounds that likely mediated their agonism through an irreversible interaction with the AAR. Of these compounds, 15b and 15d were more directly validated as irreversible agonists of the AAR using membrane-based [H]DPCPX and [S]GTPγS binding experiments.
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http://dx.doi.org/10.1021/acs.jmedchem.6b01561DOI Listing
December 2016

Extracellular Loop 2 of the Adenosine A1 Receptor Has a Key Role in Orthosteric Ligand Affinity and Agonist Efficacy.

Mol Pharmacol 2016 Dec 28;90(6):703-714. Epub 2016 Sep 28.

Monash Institute of Pharmaceutical Sciences (A.T.N.N., J.-A.B., T.T., L.L.M, K.J.G, P.J.W, P.M.S, A.C., L.T.M), Monash e-Research Centre (T.D.N), and Department of Pharmacology (A.T.N.N, J.-A.B., K.J.G., P.M.S., A.C., L.T.M), Monash University, Parkville, Victoria, Australia

The adenosine A G protein-coupled receptor (AAR) is an important therapeutic target implicated in a wide range of cardiovascular and neuronal disorders. Although it is well established that the AAR orthosteric site is located within the receptor's transmembrane (TM) bundle, prior studies have implicated extracellular loop 2 (ECL2) as having a significant role in contributing to orthosteric ligand affinity and signaling for various G protein-coupled receptors (GPCRs). We thus performed extensive alanine scanning mutagenesis of AAR-ECL2 to explore the role of this domain on AAR orthosteric ligand pharmacology. Using quantitative analytical approaches and molecular modeling, we identified ECL2 residues that interact either directly or indirectly with orthosteric agonists and antagonists. Discrete mutations proximal to a conserved ECL2-TM3 disulfide bond selectively affected orthosteric ligand affinity, whereas a cluster of five residues near the TM4-ECL2 juncture influenced orthosteric agonist efficacy. A combination of ligand docking, molecular dynamics simulations, and mutagenesis results suggested that the orthosteric agonist 5'-N-ethylcarboxamidoadenosine binds transiently to an extracellular vestibule formed by ECL2 and the top of TM5 and TM7, prior to entry into the canonical TM bundle orthosteric site. Collectively, this study highlights a key role for ECL2 in AAR orthosteric ligand binding and receptor activation.
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http://dx.doi.org/10.1124/mol.116.105007DOI Listing
December 2016

Role of the Second Extracellular Loop of the Adenosine A1 Receptor on Allosteric Modulator Binding, Signaling, and Cooperativity.

Mol Pharmacol 2016 Dec 28;90(6):715-725. Epub 2016 Sep 28.

Monash Institute of Pharmaceutical Sciences (A.T.N.N., E.A.V., T.T., L.A., P.J.S., P.J.W., P.M.S., K.J.G., L.T.M., A.C.), Monash e-Research Centre (T.D.N.), and Department of Pharmacology (A.T.N.N., E.A.V., P.M.S., K.J.G., L.T.M., A.C), Monash University, Victoria, Australia

Allosteric modulation of adenosine A1 receptors (A1ARs) offers a novel therapeutic approach for the treatment of numerous central and peripheral disorders; however, despite decades of research, there is a relative paucity of structural information regarding the A1AR allosteric site and mechanisms governing cooperativity with orthosteric ligands. We combined alanine-scanning mutagenesis of the A1AR second extracellular loop (ECL2) with radioligand binding and functional interaction assays to quantify effects on allosteric ligand affinity, cooperativity, and efficacy. Docking and molecular dynamics (MD) simulations were performed using an A1AR homology model based on an agonist-bound A2AAR structure. Substitution of E172ECL2 for alanine reduced the affinity of the allosteric modulators PD81723 and VCP171 for the unoccupied A1AR. Residues involved in cooperativity with the orthosteric agonist NECA were different in PD81723 and VCP171; positive cooperativity between PD81723 and NECA was reduced on alanine substitution of a number of ECL2 residues, including E170ECL2 and K173ECL2, whereas mutation of W146ECL2 and W156ECL2 decreased VCP171 cooperativity with NECA. Molecular modeling localized a likely allosteric pocket for both modulators to an extracellular vestibule that overlaps with a region used by orthosteric ligands as they transit into the canonical A1AR orthosteric site. MD simulations confirmed a key interaction between E172ECL2 and both modulators. Bound PD81723 is flanked by another residue, E170ECL2, which forms hydrogen bonds with adjacent K168ECL2 and K173ECL2. Collectively, our data suggest E172ECL2 is a key allosteric ligand-binding determinant, whereas hydrogen-bonding networks within the extracellular vestibule may facilitate the transmission of cooperativity between orthosteric and allosteric sites.
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http://dx.doi.org/10.1124/mol.116.105015DOI Listing
December 2016

The hybrid molecule, VCP746, is a potent adenosine A2B receptor agonist that stimulates anti-fibrotic signalling.

Biochem Pharmacol 2016 Oct 9;117:46-56. Epub 2016 Aug 9.

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

We have recently described the rationally-designed adenosine receptor agonist, 4-(5-amino-4-benzoyl-3-(3-(trifluoromethyl)phenyl)thiophen-2-yl)-N-(6-(9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxylmethyl)tetrahydro-furan-2-yl)-9H-purin-6-ylamino)hexyl)benzamide (VCP746), a hybrid molecule consisting of an adenosine moiety linked to an adenosine A1 receptor (A1AR) allosteric modulator moiety. At the A1AR, VCP746 mediated cardioprotection in the absence of haemodynamic side effects such as bradycardia. The current study has now identified VCP746 as an important pharmacological tool for the adenosine A2B receptor (A2BAR). The binding and function of VCP746 at the A2BAR was rigorously characterised in a heterologous expression system, in addition to examination of its anti-fibrotic signalling in cardiac- and renal-derived cells. In FlpInCHO cells stably expressing the human A2BAR, VCP746 was a high affinity, high potency A2BAR agonist that stimulated Gs- and Gq-mediated signal transduction, with an apparent lack of system bias relative to prototypical A2BAR agonists. The distinct agonist profile may result from an atypical binding mode of VCP746 at the A2BAR, which was consistent with a bivalent mechanism of receptor interaction. In isolated neonatal rat cardiac fibroblasts (NCF), VCP746 stimulated potent inhibition of both TGF-β1- and angiotensin II-mediated collagen synthesis. Similar attenuation of TGF-β1-mediated collagen synthesis was observed in renal mesangial cells (RMC). The anti-fibrotic signalling mediated by VCP746 in NCF and RMC was selectively reversed in the presence of an A2BAR antagonist. Thus, we believe, VCP746 represents an important tool to further investigate the role of the A2BAR in cardiac (patho)physiology.
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http://dx.doi.org/10.1016/j.bcp.2016.08.007DOI Listing
October 2016

Sarcolemmal dependence of cardiac protection and stress-resistance: roles in aged or diseased hearts.

Br J Pharmacol 2016 10 9;173(20):2966-91. Epub 2016 Sep 9.

Menzies Health Institute Queensland, Griffith University, Gold Coast, Australia.

Disruption of the sarcolemmal membrane is a defining feature of oncotic death in cardiac ischaemia-reperfusion (I-R), and its molecular makeup not only fundamentally governs this process but also affects multiple determinants of both myocardial I-R injury and responsiveness to cardioprotective stimuli. Beyond the influences of membrane lipids on the cytoprotective (and death) receptors intimately embedded within this bilayer, myocardial ionic homeostasis, substrate metabolism, intercellular communication and electrical conduction are all sensitive to sarcolemmal makeup, and critical to outcomes from I-R. As will be outlined in this review, these crucial sarcolemmal dependencies may underlie not only the negative effects of age and common co-morbidities on myocardial ischaemic tolerance but also the on-going challenge of implementing efficacious cardioprotection in patients suffering accidental or surgically induced I-R. We review evidence for the involvement of sarcolemmal makeup changes in the impairment of stress-resistance and cardioprotection observed with ageing and highly prevalent co-morbid conditions including diabetes and hypercholesterolaemia. A greater understanding of membrane changes with age/disease, and the inter-dependences of ischaemic tolerance and cardioprotection on sarcolemmal makeup, can facilitate the development of strategies to preserve membrane integrity and cell viability, and advance the challenging goal of implementing efficacious 'cardioprotection' in clinically relevant patient cohorts. Linked Articles This article is part of a themed section on Molecular Pharmacology of G Protein-Coupled Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.20/issuetoc.
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http://dx.doi.org/10.1111/bph.13552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6400434PMC
October 2016

VCP746, a novel A1 adenosine receptor biased agonist, reduces hypertrophy in a rat neonatal cardiac myocyte model.

Clin Exp Pharmacol Physiol 2016 10;43(10):976-82

Centre of Cardiovascular Research and Education in Therapeutics, Department of Epidemiology and Preventive Medicine, Monash University, Melbourne, Vic., Australia.

VCP746 is a novel A1 adenosine receptor (A1 AR) biased agonist previously shown to be cytoprotective with no effect on heart rate. The aim of this study was to investigate the potential anti-hypertrophic effect of VCP746 in neonatal rat cardiac myocytes (NCM). NCM hypertrophy was stimulated with interleukin (IL)-1β (10 ng/mL), tumour necrosis factor (TNF)-α (10 ng/mL) or Ang II (100 nmol/L) and was assessed by (3) H-leucine incorporation assay. VCP746 significantly inhibited IL-1β-, TNF-α- and Ang II-stimulated NCM hypertrophy as determined by (3) H-leucine incorporation. The anti-hypertrophic effect of VCP746 was also more potent than that of the prototypical A1 AR agonist, N(6) -cyclopentyladenosine (CPA). Further investigation with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay showed that neither CPA nor VCP746 had any effect on cell viability, confirming that the reduction in (3) H-leucine incorporation mediated by CPA and VCP746 was not due to a reduction in cell viability. IL-1β, TNF-α and Ang II were also shown to increase the mRNA expression of hypertrophy biomarkers, ANP, β-MHC and α-SKA in NCM. Treatment with VCP746 at concentrations as low as 1 nmol/L suppressed mRNA expression of ANP, β-MHC and α-SKA stimulated by IL-1β, TNF-α or Ang II, demonstrating the broad mechanistic basis of the potent anti-hypertrophic effect of VCP746. This study has shown that the novel A1 AR agonist, VCP746, is able to attenuate cardiac myocyte hypertrophy. As such, VCP746 is potentially useful as a pharmacological agent in attenuating cardiac remodelling, especially in the post-myocardial infarction setting, given its previously established cytoprotective properties.
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http://dx.doi.org/10.1111/1440-1681.12616DOI Listing
October 2016
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