The chemokine receptor CX3CR1 has been implicated as an attractive therapeutic target in several diseases, including atherosclerosis and diabetes. However, there has been a lack of non-peptide CX3CR1 inhibitors to substantiate these findings. A selective small-molecule inhibitor of CX3CR1, AZD8797, was recently reported and we present here an in-depth in vitro characterization of that molecule. In a flow adhesion assay, AZD8797 antagonized the natural ligand, fractalkine (CX3CL1), in both human whole blood (hWB) and in a B-lymphocyte cell line with IC50 values of 300 and 6 nM respectively. AZD8797 also prevented G-protein activation in a [(35)S]GTPγS (guanosine 5'-[γ-thio]triphosphate) accumulation assay. In contrast, dynamic mass redistribution (DMR) experiments revealed a weak Gαi-dependent AZD8797 agonism. Additionally, AZD8797 positively modulated the CX3CL1 response at sub-micromolar concentrations in a β-arrestin recruitment assay. In equilibrium saturation binding experiments, AZD8797 reduced the maximal binding of (125)I-CX3CL1 without affecting Kd. Kinetic experiments, determining the kon and koff of AZD8797, demonstrated that this was not an artefact of irreversible or insurmountable binding, thus a true non-competitive mechanism. Finally we show that both AZD8797 and GTPγS increase the rate with which CX3CL1 dissociates from CX3CR1 in a similar manner, indicating a connection between AZD8797 and the CX3CR1-bound G-protein. Collectively, these data show that AZD8797 is a non-competitive allosteric modulator of CX3CL1, binding CX3CR1 and effecting G-protein signalling and β-arrestin recruitment in a biased way.

The G-protein coupled receptor 55 (GPR55) is activated by cannabinoids and non-cannabinoid molecules and has been speculated to play a modulatory role in a large variety of physiological and pathological processes, including in metabolically perturbed states. We therefore generated male mice deficient in the gene coding for the cannabinoid/lysophosphatidylinositol (LPI) receptor Gpr55 and characterized them under normal dietary conditions as well as during high energy dense diet feeding followed by challenge with the CB1 receptor antagonist/GPR55 agonist rimonabant. Gpr55 deficient male mice (Gpr55 KO) were phenotypically indistinguishable from their wild type (WT) siblings for the most part. However, Gpr55 KO animals displayed an intriguing nocturnal pattern of motor activity and energy expenditure (EE). During the initial 6 hours of the night, motor activity was significantly elevated without any significant effect observed in EE. Interestingly, during the last 6 hours of the night motor activity was similar but EE was significantly decreased in the Gpr55 KO mice. No significant difference in motor activity was detected during daytime, but EE was lower in the Gpr55 KO compared to WT mice. The aforementioned patterns were not associated with alterations in energy intake, daytime core body temperature, body weight (BW) or composition, although a non-significant tendency to increased adiposity was seen in Gpr55 KO compared to WT mice. Detailed analyses of daytime activity in the Open Field paradigm unveiled lower horizontal activity and rearing time for the Gpr55 KO mice. Moreover, the Gpr55 KO mice displayed significantly faster reaction time in the tail flick test, indicative of thermal hyperalgesia. The BW-decreasing effect of rimonabant in mice on long-term cafeteria diet did not differ between Gpr55 KO and WT mice. In conclusion, Gpr55 deficiency is associated with subtle effects on diurnal/nocturnal EE and motor activity behaviours but does not appear per se critically required for overall metabolism or behaviours.

Inflammatory signals and increased prostaglandin synthesis play a role during the development of diabetes. The prostaglandin D2 (PGD2) receptor, GPR44/DP2, is highly expressed in human islets and activation of the pathway results in impaired insulin secretion. The role of GPR44 activation on islet function and survival rate during chronic hyperglycaemic conditions is not known. In this study, we investigate GPR44 inhibition by using a selective GPR44 antagonist (AZ8154) in human islets both in vitro and in vivo in diabetic mice transplanted with human islets.

The receptors mediating the hemodynamic responses to cannabinoids are not clearly defined due to the multifarious pharmacology of many commonly used cannabinoid ligands. While both CB1 and TRPV1 receptors are implicated, G protein-coupled receptor 55 (GPR55) may also mediate some of the hemodynamic effects of several atypical cannabinoid ligands. The present studies attempted to unravel the pharmacology underlying the in vivo hemodynamic responses to ACEA (CB1 agonist), O-1602 (GPR55 agonist), AM251 (CB1 antagonist), and cannabidiol (CBD; GPR55 antagonist). Agonist and antagonist profiles of each ligand were determined by ligand-induced GTPγS binding in membrane preparations expressing rat and mouse CB1 and GPR55 receptors. Blood pressure responses to ACEA and O-1602 were recorded in anesthetized and conscious mice (wild type, CB1 (-/-) and GPR55(-/-)) and rats in the absence and presence of AM251 and CBD. ACEA demonstrated GTPγS activation at both receptors, while O-1602 only activated GPR55. AM251 exhibited antagonist activity at CB1 and agonist activity at GPR55, while CBD demonstrated selective antagonist activity at GPR55. The depressor response to ACEA was blocked by AM251 and attenuated by CBD, while O-1602 did not induce a depressor response. AM251 caused a depressor response that was absent in GPR55(-/-) mice but enhanced by CBD, while CBD caused a small vasodepressor response that persisted in GPR55(-/-) mice. Our findings show that assessment of the pharmacological profile of receptor activation by cannabinoid ligands in in vitro studies alongside in vivo functional studies is essential to understand the role of cannabinoids in hemodynamic control.

GPR44 (DP2, PTGDR2, CRTh2) is the receptor for the pro-inflammatory mediator prostaglandin D2 (PGD2) and it is enriched in human islets. In rodent islets, PGD2 is produced in response to glucose, suggesting that the PGD2-GPR44/DP2 axis may play a role in human islet function during hyperglycemia. Consequently, the aim of this work was to elucidate the insulinotropic role of GPR44 antagonism in vitro in human beta-cells and in type 2 diabetes (T2DM) patients.

Plasminogen binding inhibitors (PBIs) reduce the risk of bleeding in hemorrhagic conditions. However, generic PBIs are also associated with an increased risk of seizures, an adverse effect linked to unwanted activities towards inhibitory neuronal receptors. Development of novel PBIs serve to remove compounds with such properties, but progress is limited by a lack of higher throughput methods with human translatability. Herein we apply human induced pluripotent stem cell (hiPSC) derived neurons in combination with dynamic mass redistribution (DMR) technology to demonstrate robust and reproducible modulation of both GABAA and glycine receptors. These cells respond to GABA (EC50 0.33 ± 0.18 μM), glycine (EC50 11.0 ± 3.7 μM) and additional ligands in line with previous reports from patch clamp technologies. Additionally, we identify and characterize a competitive antagonistic behavior of the prototype inhibitor and drug tranexamic acid (TXA). Finally, we demonstrate proof of concept for effective counter-screening of lead series compounds towards unwanted GABAA receptor activities. No activity was observed for a previously identified PBI candidate drug, AZD6564, whereas a discontinued analog, AZ13267257, could be characterized as a potent GABAA receptor agonist.

The phospholipid l-α-lysophosphatidylinositol (LPI), an endogenous ligand for GPR55, is elevated in patients with acute coronary syndrome, and a GPR55 antagonist cannabidiol (CBD) reduces experimental ischemia/reperfusion (I/R) injury. While LPI activates multiple signaling pathways, little is known about which ones are important in cardiomyocytes. In this study we explored whether activation of the Rho kinase/ROCK/p38 MAPK pathway is responsible for LPI-induced extension of I/R injury. Using a high-throughput screening method (dynamic mass redistribution; DMR), mouse- and human-induced pluripotent stem cell (iPSC) cardiomyocytes exposed to LPI were shown to exhibit a rapid, sustained, and concentration-dependent (1 nmol L-1-30 μmol L-1) cellular response. Y-27632 (ROCK inhibitor; 10 & 50 μmol L-1) and CBD (1 μmol L-1) both abolished the DMR response to LPI (10 μmol L-1). In murine iPSC cardiomyocytes, LPI-induced ROCK and p38 MAPK phosphorylation, both of which were prevented by Y-27632 and CBD, but did not induce JNK activation or cleavage of caspase-3. In hearts isolated from wild type (WT) mice subjected to 30 minutes global I/R, LPI (10 μmol L-1) administered via the coronary circulation increased infarct size when applied prior to ischemia onset, but not when given at the time of reperfusion. The exacerbation of tissue injury by LPI was not seen in hearts from GPR55-/- mice or in the presence of Y-27632, confirming that injury is mediated via the GPR55/ROCK/p38 MAPK pathway. These findings suggest that raised levels of LPI in the vicinity of a developing infarct may worsen the outcome of AMI.