Biology subjects

Ladds, G.

Publications and source records attributed to Ladds, G..

At least 19 recordsLinked to original sources

bioRxiv2026

Discovery of a pathway-selective platelet P2Y1R inverse agonist that suppresses inflammation while preserving hemostasis

The platelet P2Y1 receptor (P2Y1R) is necessary for inflammation, signalling via Rho-GTPase pathways to elicit functions that are distinct from aggregation (PLC-dependent canonical signalling pathway). Whether these distinct platelet inflammatory functions can be selectively suppressed to preserve hemostasis through the rational design of P2Y1R antagonists has not been explored. In silico molecular docking analysis examined biased nucleotide interactions within the P2Y1R binding pocket. The identified possible key amino acid residues guided rational design to synthesize compounds for pathway selective inhibition, evolving from nucleotide to non-nucleotide structures. The nucleotide analogue KMR-82-13 was predicted to engage distinct regions of the binding pocket and selectively inhibited platelet chemotaxis while preserving aggregation. These findings informed the design of a non-nucleotide compound KSN-159-27, aiming to retain key KMR-82-13-like interactions while improving drug-like properties. Docking and molecular dynamics simulation supported a stable but dynamic binding mode for KSN-159-27 within the P2Y1R pocket, consistent with pathway-selective inhibition. KSN-159-27 displayed characteristics of a pathway selective inverse agonist at P2Y1R towards G12/13-mediated pathways, but not those associated by Gq activation in P2Y1R-transfected HEK293T cells. KSN-159-27 showed functionally selective inhibition for platelet P2Y1R-mediated functions. In vivo, KSN-159-27 suppressed inflammatory cell recruitment, whilst preserving bleeding time and ADP-induced thromboembolic responses, in contrast to the neutral P2Y1R antagonist MRS2500. This first demonstration for the rational design of a pathway selective inverse agonist at platelet P2Y1Rs has significant implications for novel therapeutic strategies developed to safely target platelet activation during inflammation, in contrast to current anti-platelet drugs used in the prevention of thrombosis. Key PointsO_LIBiased inverse platelet P2Y1R agonists selectively supress inflammation whilst preserving hemostasis and the ability of platelets to aggregate. C_LIO_LIBiased inverse agonism selectively inhibited P2Y1R G12/13 (Rho-GTPAse functions) but not Gq activities (PLC functions). C_LI

pharmacology and toxicology↗

bioRxiv2025

Reducing Foetal Bovine Serum Culture Conditions does not affect GPCR Signalling

Animal use in research extends beyond the use of animal models to study physiology and disease. Many aspects of in vitro research use reagents derived from animals, most prolifically the use of foetal bovine serum (FBS) in growth media for cellular models. With the aim to reduce animal use, we investigated the effect of reduced FBS culture conditions on cell growth, as well as different stages of G protein-coupled receptor (GPCR) signalling, a wide area of research which might therefore impact many groups. We identified little differences on cell growth or GPCR signalling when reducing culture FBS percentage from 10% to 5%, using assays ranging from receptor activation to downstream transcription factors stimulation. In addition to diminishing animal use, the reduction of FBS use will also have economic and environmental benefits, which we hope will be of benefit to the wider research community.

pharmacology and toxicology↗

bioRxiv2025

Increased RAMP3 expression enhances GLP-1 mediated Glucose Stimulated Insulin Secretion in a Manner Dependent on Ca2+ Mobilisation

The targeting of the Glucagon-like peptide-1 receptor (GLP-1R) for diabetes and obesity is not a novel strategy, with recent therapeutics showing efficacy in weight loss and glycaemic control. However, they are also associated with side effects, including gastrointestinal disruptions and pancreatitis. Developing agonists with different signalling profiles, or that exert some tissue selectivity can circumvent these on-target, unwanted effects. Receptor activity-modifying proteins (RAMPs) offer the potential to do both, through modulation of agonist binding and signalling, as well as surface expression. The GLP-1R was found to interact with RAMP3, with the heterodimer able to bind agonist at the cell surface. RAMP3 expression biased the receptor towards Ca2+ mobilisation, away from the canonical cAMP-driven signalling. When examining G protein coupling, the interaction with RAMP3 reduced activation of the cognate Gs but increased secondary couplings to Gq and Gi. These increased couplings led to an elevation in glucose-stimulated insulin secretion when cells overexpressing RAMP3 were stimulated with GLP-1. A reciprocal effect was observed when looking at reduced expression of endogenous RAMP3, with a loss of sensitivity to GLP-1 in both glucose and insulin tolerance tests in a Ramp3 knockout mouse. The effects of this interaction can then inform selection of models and peptide design when targeting this receptor for therapeutic intervention. Significance StatementG Protein-Coupled Receptors, such as the Glucagon-Like Peptide-1 (GLP-1) Receptor are common drug targets, although not without side effects due to on-target, unwanted signalling outputs. This study identifies an interacting protein which alters its signalling to promote insulin secretion. This may improve the therapeutic potential of GLP-1 mimetics, enhancing effectual signalling over that associated with unwanted effects by directly targeting this receptor complex.

pharmacology and toxicology↗

bioRxiv2024

ER-mitochondria distance is a critical parameter for efficient mitochondrial Ca2+ uptake and oxidative metabolism.

IP3 receptor (IP3R)-mediated Ca2+ transfer at the mitochondria-endoplasmic reticulum (ER) contact sites (MERCS) drives mitochondrial Ca2+ uptake and oxidative metabolism and is linked to different pathologies, including Parkinsons disease (PD). The dependence of Ca2+ transfer efficiency on the ER-mitochondria distance remains unexplored. Employing molecular rulers that stabilize ER-mitochondrial distances at 5 nm resolution, and using genetically-encoded Ca2+ indicators targeting the ER lumen and the sub-mitochondrial compartments, we now show that a distance of [~]20 nm is optimal for Ca2+ transfer and mitochondrial oxidative metabolism due to enrichment of IP3R at MERCS. In human iPSC-derived astrocytes from PD patients, 20 nm MERCS were specifically reduced which correlated with a reduction of mitochondrial Ca2+ uptake. Our work determines with precision the optimal distance for Ca2+ flux between ER and mitochondria and suggests a new paradigm for fine control over mitochondrial function.

cell biology↗

bioRxiv2024

Activation of the proton-sensing GPCR, GPR65 on fibroblast-like synoviocytes contributes to inflammatory joint pain.

Inflammation is associated with localised acidosis, however, attributing physiological and pathological roles to proton-sensitive receptors is challenging due to their diversity and widespread expression. Here, agonists of the proton-sensing GPCR, GPR65, were systematically characterised. The synthetic agonist BTB09089 (BTB) recapitulated many proton-induced signalling events and demonstrated selectivity for GPR65. BTB was used to show that GPR65 activation on fibroblast-like synoviocytes (FLS), cells that line synovial joints, results in the secretion of pro-inflammatory mediators capable of recruiting immune cells and sensitising sensory neurons. Intra-articular injection of BTB resulted in GPR65-dependent sensitisation of knee-innervating neurons and nocifensive behaviours in mice. Stimulation of GPR65 on human FLS also triggered the release of inflammatory mediators and synovial fluid samples from human osteoarthritis patients were shown to activate GPR65. These results suggest a role of GPR65 in mediating cell-cell interactions that drive inflammatory joint pain in both mice and humans.

neuroscience↗

bioRxiv2024

Exploring human rare disease variants from a multidimensional perspective illuminates receptor - G protein coupling diversity

G protein-coupled receptors (GPCRs) are transmembrane proteins capable of detecting signals as diverse as odours, neurotransmitters, and hormones. Upon activation, receptor signalling converges onto four G protein subtypes to regulate intracellular responses. Therefore, variation in a single G protein gene can potentially impact the function of numerous receptors. In this work, we have performed a multidimensional study of rare disease mutations in Gs, a prototypical G protein. By integrating data from 3D structures, GPCR / G protein functional pairings, transcriptomics, biophysics, and molecular dynamics with systems pharmacology modelling, our results reveal why mutations impairing receptor / Gs coupling result in highly specific context-based signalling defects. Furthermore, we show that mutations leading to the same rare disease can alter different signal transduction steps, highlighting the importance of patient-specific treatment strategies. By closely dissecting G protein coupling, our study provides a blueprint to interrogate GPCR pathway signalling diversity in different (patho)physiological contexts.

systems biology↗

bioRxiv2024

Agonist efficacy at the B2AR is driven by agonist-induced differences in receptor affinity for the Gs protein, not ligand binding kinetics.

IntroductionThe {beta}2-adrenoceptor ({beta}2AR) is a class A G protein-coupled receptor (GPCR). It is therapeutically relevant in asthma, whereby {beta}2AR agonists relieve bronchoconstriction. The {beta}2AR is a prototypical GPCR for structural and biophysical studies. However, the molecular basis of agonist efficacy at the {beta}2AR is not understood. We hypothesized that the kinetics of ligand binding and GPCR-G protein interactions could play a role in ligand efficacy. We characterised the molecular pharmacology of a range of {beta}2AR agonists and examined the correlation between ligand and mini-Gs binding kinetics and efficacy. MethodsWe used a Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) based competition ligand binding assay to measure the affinity and residence times of a range of {beta}2AR agonists binding to the human {beta}2AR. TR-FRET between Lumi4-Tb3+ on the N terminus of the {beta}2AR and fluorescent CA200693 (S)-propranolol-green was measured using a PHERAstar FSX. The ability of these {beta}2AR agonists to activate the heterotrimeric Gs protein was measured using the CASE Gs protein biosensor. This assay senses a reduction in NanoBRET between the nano-luciferase (nLuc) donor on the G subunit and Venus acceptor on the G{psi}, on receptor activation, quantified using the operational model of agonism. NanoBRET was also used to measure interactions between DDM solubilised {beta}2AR-nLuc and purified Venus-mini-Gs. A large excess of unlabelled mini-Gs was used to dissociate the {beta}2AR-nLuc: Venus-mini-Gs complex. ResultsCharacterisation of the molecular pharmacology of seven {beta}2AR agonists showed a broad range of ligand binding affinities (pKi = 4.4 {+/-} 0.09 to 9.2 {+/-} 0.08) and kinetics parameters. There was no correlation between ligand residence times and their ability (log{tau} ) to activate the Gs protein (R2=0.26, p=0.29). However, there were statistically significant differences in the association rate (kon (fast)) (3.36{+/-}0.64x105 to 9.19{+/-} 0.42x105) and affinity (Kd) values of mini-Gs binding to the agonist-{beta}2AR complex (pKd =6.0 to 6.7). Both an increase in ligand driven mini-Gs kon(fast) rate and associated increase in mini-Gs pKd for the receptor, were moderately correlated with efficacy (log{tau} ) (R2 =0.58 and R2 =0.50 respectively). ConclusionsThese data support a model in which agonists of increased efficacy cause the {beta}2AR to adopt a conformation that is more likely to recruit G protein. Conversely, these data did not support a role for agonist binding kinetics in the molecular basis of efficacy.

pharmacology and toxicology↗

bioRxiv2022

Species-dependent actions of the Gαob selective adenosine A1 receptor agonist BnOCPA

We have previously reported that in rat hippocampal area CA1, the A1R-selective agonist, BnOCPA, potently inhibited excitatory synaptic transmission but did not cause membrane hyperpolarisation in CA1 pyramidal neurons, as would be expected of A1R agonists. This functional discrimination by BnOCPA may arise from its ability, in cAMP inhibition assays, to selectively activate only Gob out of the six Gi/o subtypes. This may explain why BnOCPA is a potent analgesic that does not cause sedation or cardiorespiratory depression in the rat. Since many preclinical studies are performed using mice, we have here investigated whether BnOCPAs functional discrimination extends to the mouse. While the potency of BnOCPA against the inhibition of hippocampal synaptic transmission was comparable between rats and mice, we discovered that low concentrations of BnOCPA hyperpolarised mouse CA1 neurons and reduced both their input resistance and firing rate in an A1R-dependent manner. In interleaved experiments we confirmed our previous observations in the rat that concentrations of BnOCPA equivalent to those tested in the mouse had little or no effect on membrane potential or input resistance. Using NanoBRET binding we established that BnOCPA had similar affinity at the mouse and rat A1Rs, and displayed little difference in G protein coupling, as determined using the TRUPATH assay. Thus, although the mechanism for the loss of BnOCPA functional selectivity between pre- and postsynaptic receptors in the mouse hippocampus is currently unclear, it may stem from differences in expression of the individual G proteins subunits or the coupling to murine K+ channels. Short summaryWe describe the differential actions of the selective A1R agonist BnOCPA in mouse vs rat hippocampus. In mice, BnOCPA does not show a selectivity between pre and postsynaptic A1Rs, unlike in rats. This may stem from differences in the G protein coupling to K+ channels.

neuroscience↗

bioRxiv2022

Cancer-Associated Mutations Enhance The Sensitivity Of The Trupath GαQ/11 System

G protein-coupled receptors (GPCRs) are the largest family of cell surface receptors and are a common drug target. They can be stabilised in different conformational states by ligands to activate multiple transducers and effectors leading to a variety of cellular responses. The potential of agonists to activate select pathways has important implications for drug discovery. Thus, there is a clear need to profile the initial GPCR signal transduction event, activation of G proteins, to enhance understanding of receptor coupling and guide drug design. The BRET-based biosensor suite, TRUPATH, was recently developed to enable quantification of the activation profiles of all non-visual G proteins (excluding Golf and G14) and has since been utilised in numerous studies. However, it fails to detect Gq/11 activation for a number of GPCRs previously reported to display promiscuous secondary coupling to Gq/11. Here we report modifications to the Gq and G11 biosensors in the switch I region that prevent intrinsic GTPase activity (R183C/Q). Except for the PAC1R, substitution with cancer-associated mutations, Cys or Gln, significantly increased sensitivity to allow detection of robust, reliable, and representative Gq/11 responses to Class B1 GPCRs. We also demonstrate the utility of these modified biosensors for promiscuously coupled class A GPCR that have primary Gs-coupling. Thus, we propose that modification to Gq/11 may also be necessary in other biosensor systems to enable detection of Gq/11 activation.

pharmacology and toxicology↗

bioRxiv2021

RAMPs regulate signalling bias and internalisation of the GIPR

Gastric inhibitory polypeptide (GIP) receptor is a class B1 GPCR, that responds to GIP and physiologically potentiates glucose-stimulated insulin secretion. Like most class B1 GPCRs, GIPR has been shown to interact with RAMPs, yet the effects of RAMPs on its signalling and trafficking remain poorly understood. We demonstrate that RAMPs modulate G protein activation and GIPR internalisation profiles. RAMP3 reduced GIPR Gs activation and cAMP production but retained GIPR at the cell surface, and this was associated with prolonged ERK1/2 phosphorylation and {beta}-arrestin association. By contrast, RAMP1/2 reduced Gq/11/15 activation of the GIPR. Through knockout mice studies, we show that RAMP1 is important to the normal physiological functioning of GIPR to regulate blood glucose levels. Thus, RAMPs act on G protein/{beta}-arrestin complexes, having both acute and chronic effects on GIPR function, while this study also raises the possibility of a more general role of RAMP3 to enhance GPCR plasma membrane localisation.

pharmacology and toxicology↗

bioRxiv2020

CGRP-receptor family reveals endogenous GPCR agonist bias and its significance in primary human cardiovascular cells

Agonist bias at G protein-coupled receptors has attracted considerable interest, although its relevance for physiologically-produced agonists is not always clear. Here, using primary human cells and gene editing techniques, we demonstrate for the first time, endogenous agonist bias with physiological consequences for the calcitonin-like receptor (CLR). We reveal that by switching the accessory protein: receptor activity-modifying protein (RAMP) associated with CLR we can re-route the physiological pathways activated by the stimulating peptide agonists. These results have revealed a unique role in calcium-mediated nitric oxide signalling for the little-understood peptide adrenomedullin 2 and distinct pro-proliferative effects of calcitonin-gene related peptide (CGRP) and adrenomedullin in cardiovascular cells. This work reveals that CLR-based agonist bias occurs naturally in human cells and has a fundamental purpose for its existence. We anticipate this will be a starting point for more studies into RAMP function in native environments and its importance in endogenous GPCR signalling.

pharmacology and toxicology↗

bioRxiv2020

Deciphering the Agonist Binding Mechanism to the Adenosine A1 Receptor

Despite being amongst the most characterized G protein-coupled receptors (GPCRs), adenosine receptors (ARs) have always been a difficult target in drug design. To date, no agonist other than the natural effector and the diagnostic regadenoson has been approved for human use. Recently, the structure of the adenosine A1 receptor (A1R) was determined in the active, Gi protein complexed state; this has important repercussions for structure-based drug design. Here, we employed supervised molecular dynamics simulations and mutagenesis experiments to extend the structural knowledge of the binding of selective agonists to A1R. Our results identify new residues involved in the association and dissociation pathway, suggest the binding mode of N6-cyclopentyladenosine (CPA) related ligands, and highlight the dramatic effect that chemical modifications can have on the overall binding mechanism.

pharmacology and toxicology↗

bioRxiv2020

A Multisite Model of Allosterism for the Adenosine A1 Receptor

Despite being a target for about one-third of approved drugs, G protein-coupled receptors (GPCRs) still represent a tremendous reservoir for therapeutic strategies against countless diseases. For example, several cardiovascular and central nervous systems conditions could benefit from clinical agents that activate the adenosine 1 receptor (A1R), however, the pursuit of A1R agonists for clinical use are usually impeded by both on- and off-target side effects. One of the possible strategies to overcome this issue is the development of positive allosteric modulators (PAMs) capable of selectively enhancing the effect of a specific receptor subtype and triggering functional selectivity (a phenomenon also referred to as bias). Intriguingly, besides enforcing the effect of agonists upon binding to an allosteric site, most of the A1R PAMs display intrinsic partial agonism and orthosteric competition with antagonists. To rationalize this behaviour, we simulated the binding of the prototypical PAMs PD81723 and VCP171, the antagonist 13B, and the bitopic agonist VCP746. We propose that a single PAM can bind several A1R sites rather than a unique allosteric pocket, reconciling the structure-activity relationship and the mutagenesis results.

biophysics↗

bioRxiv2020

Non-opioid analgesia based on Gα signalling bias

The development of therapeutic agonists for G protein-coupled receptors (GPCRs) is hampered by the propensity of GPCRs to couple to multiple intracellular signalling pathways. This promiscuous coupling leads to numerous downstream cellular effects, some of which are therapeutically undesirable. This is especially the case for adenosine A1 receptors (A1Rs) whose clinical potential is undermined by the sedation and cardiorespiratory depression caused by conventional agonists. We have discovered that the A1R-selective agonist, BnOCPA, is a potent and powerful analgesic but does not cause sedation, bradycardia, hypotension or respiratory depression. This unprecedented discrimination between native A1Rs arises from BnOCPAs unique and exquisitely selective activation of Gob among the six Gi/o subtypes, and in the absence of {beta}-arrestin recruitment. BnOCPA thus demonstrates a highly-specific G-selective activation of the native A1R, sheds new light on GPCR signalling, and reveals new possibilities for the development of novel therapeutics based on the far-reaching concept of selective G agonism. Short summaryWe describe the selective activation of an adenosine A1 receptor-mediated intracellular pathway that provides potent analgesia in the absence of sedation or cardiorespiratory depression, paving the way for novel medicines based on the far-reaching concept of selective G agonism.

physiology↗

bioRxiv2019

GLP-1R is downregulated in beta cells of NOD mice and T1D patients

Glucagon-like peptide 1 (GLP-1) is produced by L cells in the small intestine in response to ingested glucose and increases insulin release from pancreatic beta cells by activation of its cognate receptor (GLP-1R). Stimulation of this receptor also contributes to increased beta cell survival and regeneration. We have found that pancreatic beta cells from Non Obese Diabetic (NOD) mice express significantly lower levels of GLP-1R than C57BL/6 mice, leaving the NOD beta cells with an impaired response to GLP-1 stimulation. The lower expression appears to be caused by accelerated degradation of GLP-1R in the beta cells, a process that can be reversed by inhibiting trafficking to the lysosome. Importantly, our results appear to translate to the human disease since we also observed significantly lower expression of the GLP-1R in pancreatic islets from donors with type 1 diabetes. These results suggest that beta cell physiology may play a role in susceptibility to autoimmune inflammation.

physiology↗

bioRxiv2019

Promiscuous receptor activation mediates glucagonostatic effects of GLP-1(9-36) and GLP-1(7-36)

The incretin hormone glucagon-like peptide 1(7-36) (GLP-1(7-36)) stimulates insulin and inhibits glucagon secretion. The mechanisms by which GLP-1 suppresses glucagon release are unclear as glucagon-secreting -cells express GLP-1 receptors (GLP-1Rs) at very low levels. Here, we examine the underlying mechanisms. We find that both GLP-1(7-36) and its degradation product GLP-1(9-36) inhibit glucagon secretion at physiological (pM) concentrations. Whereas the effect of GLP-1(7-36) is sensitive to PKA inhibition, GLP-1(9-36) exerts its effect by a PKA-independent mechanism sensitive to pretreatment with pertussis. The glucagonostatic effects of both GLP-1(7-36) and (9-36) are retained in islets from Glp1r knockout mice but only GLP-1(9-36) remains glucagonostatic in the presence of the DPP-4 (the peptidase catalyzing the formation of GLP-1(9-36)) inhibitor sitagliptin. Glucagon receptor (GCGR) antagonism specifically prevents the inhibitory effects of GLP-1(9-36) whilst not affecting that of GLP-1(7-36). We conclude that GLP-1(7-36) and GLP-1(9-36) regulate glucagon secretion via interaction with GLP-1R and GCGR, respectively. HighlightsO_LIGLP-1(7-36) and GLP-1(9-36) inhibit glucagon secretion from alpha-cells C_LIO_LIGLP-1(7-36) and (9-36) retain glucagonostatic effect in Glp1r-/- islets C_LIO_LIGLP-1(7-36) and (9-36) activate distinct signal transduction mechanisms C_LIO_LIGLP-1(7-36) acts via GLP-1R and GLP-1(9-36) via GCGR C_LI

molecular biology↗

bioRxiv2019

Elevated intracellular cAMP concentration mediates growth suppression in glioma cells

Supressed levels of intracellular cAMP have been associated with malignancy. Thus, elevating cAMP through activation of adenylyl cyclase (AC) or by inhibition of phosphodiesterase (PDE) may be therapeutically beneficial. Here, we demonstrate that elevated cAMP levels suppress growth in C6 cells (a model of glioma) through treatment with forskolin, an AC activator, or a range of small molecule PDE inhibitors with differing selectivity profiles. Forskolin suppressed cell growth in a protein kinase A (PKA)-dependent manner by inducing a G2/M phase cell cycle arrest. In contrast, trequinsin (a non-selective PDE2/3/7 inhibitor), not only inhibited cell growth via PKA, but also stimulated (independent of PKA) caspase-3/-7 and induced an aneuploidy phenotype. Interestingly, a cocktail of individual PDE 2,3,7 inhibitors suppressed cell growth in a manner analogous to forskolin but not trequinsin. Finally, we demonstrate that concomitant targeting of both AC and PDEs synergistically elevated intracellular cAMP levels thereby potentiating their antiproliferative actions.

pharmacology and toxicology↗

bioRxiv2019

Pharmacological Characterisation of Novel Adenosine Receptor A3R Antagonists

Background and PurposeThe adenosine A3 receptor (A3R) belongs to a family of four adenosine receptor (AR) subtypes which all play distinct roles throughout the body. A3R antagonists have been described as potential treatments for numerous diseases including asthma. Given the similarity between ARs orthosteric binding sites, obtaining highly selective antagonists is a challenging but critical task. Experimental approach39 potential A3R, antagonists were screened using agonist-induced inhibition of cAMP. Positive hits were assessed for AR subtype selectivity through cAMP accumulation assays. The antagonist affinity was determined using Schild analysis (pA2 values) and fluorescent ligand binding. Further, a likely binding pose of the most potent antagonist (K18) was determined through molecular dynamic (MD) simulations and consistent calculated binding free energy differences between K18 and congeners, using a homology model of A3R, combined with mutagenesis studies. Key ResultsWe demonstrate that K18, which contains a 3-(dichlorophenyl)-isoxazole group connected through carbonyloxycarboximidamide fragment with a 1,3-thiazole ring, is a specific A3R (<1 {micro}M) competitive antagonist. Structure-activity relationship investigations revealed that loss of the 3-(dichlorophenyl)-isoxazole group significantly attenuated K18 antagonistic potency. Mutagenic studies supported by MD simulations identified the residues important for binding in the A3R orthosteric site. Finally, we introduce a model that enables estimates of the equilibrium binding affinity for rapidly disassociating compounds from real-time fluorescent ligand-binding studies. Conclusions and ImplicationsThese results demonstrate the pharmacological characterisation of a selective competitive A3R antagonist and the description of its orthosteric binding mode. Our findings may provide new insight for drug discovery. What is already knownO_LIThe search for AR subtype specific compounds often leads to ones with multiple subtype binding C_LI What this study addsO_LIThis study demonstrates the pharmacological characterisation of a selective competitive A3R antagonist C_LIO_LIMD simulations identified the residues important for binding in the A3R orthosteric site C_LI Clinical significanceO_LIThis study offers insight into A3R antagonists that may provide new opportunities for drug discovery C_LI

pharmacology and toxicology↗