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The C. elegans proteins PAQR-2 (a homolog of the human seven-transmembrane domain AdipoR1 and AdipoR2 proteins) and IGLR-2 (a homolog of the mammalian LRIG proteins characterized by a single transmembrane domain and the presence of immunoglobulin domains and leucine-rich repeats in their extracellular portion) form a complex that protects against plasma membrane rigidification by promoting the expression of fatty acid desaturases and the incorporation of polyunsaturated fatty acids into phospholipids, hence increasing membrane fluidity. In the present study, we leveraged a novel gain-of-function allele of PAQR-1, a PAQR-2 paralog, to carry out structure-function studies. We found that the transmembrane domains of PAQR-2 are responsible for its functional requirement for IGLR-2, that PAQR-1 does not require IGLR-2 but acts via the same pathway as PAQR-2, and that the divergent N-terminal cytoplasmic domains of the PAQR-1 and PAQR-2 proteins serve a regulatory function and may regulate access to the catalytic site of these proteins. We also show that overexpression of human AdipoR1 or AdipoR2 alone is sufficient to confer increased palmitic acid resistance in HEK293 cells, and thus act in a manner analogous to the PAQR-1 gain-of-function allele., Competing Interests: The authors have declared that no competing interests exist.

GPR81 is a novel drug target that is implicated in the control of glucose and lipid metabolism. The lack of potent GPR81 modulators suitable for in vivo studies has limited the pharmacological characterization of this lactate sensing receptor. We performed a high throughput screen (HTS) and identified a GPR81 agonist chemical series containing a central acyl urea scaffold linker. During SAR exploration two additional new series were evolved, one containing cyclic acyl urea bioisosteres and another a central amide bond. These three series provide different selectivity and physicochemical properties suitable for in-vivo studies., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Demonstration of target binding is a key requirement for understanding the mode of action of new therapeutics. The cellular thermal shift assay (CETSA) has been introduced as a powerful label-free method to assess target engagement in physiological environments. Here, we present the application of live-cell CETSA to different classes of integral multipass transmembrane proteins using three case studies, the first showing a large and robust stabilization of the outer mitochondrial five-pass transmembrane protein TSPO, the second being a modest stabilization of SERCA2, and the last describing an atypical compound-driven stabilization of the GPCR PAR2. Our data demonstrated that using modified protocols with detergent extraction after the heating step, CETSA can reliably be applied to several membrane proteins of different complexity. By showing examples with distinct CETSA behaviors, we aim to provide the scientific community with an overview of different scenarios to expect during CETSA experiments, especially for challenging, membrane bound targets.

Immunoassays, utilizing the affinity of antibodies to their antigens, are powerful techniques and have been widely used for quantifying analytes, such as cytokines, in biological samples in the clinic and in drug discovery. Various immunoassays have been developed to fit for different purposes. Recently, bead-based flow cytometry assays have emerged as interesting options for multiplex quantification of analytes. In this study, we compared high-throughput flow cytometry multiplex iQue QBeads PlexScreen assays with several other commonly used immunoassays, including MSD, Luminex, ELISA, HTRF, and AlphaLISA assays. Head-to-head comparisons of quantification data of the following cytokines were made: (1) IL-2, IL-4, IL-6, IL-13, IL-17A, IFNγ, KC/GRO, RANTES, and TNFα in mouse bronchoalveolar lavage fluid samples; (2) IL-10 and TNFα in supernatants from a THP-1 cell assay; (3) IL-6, IL-10, IL-12p70, and TNFα in supernatants from a human monocyte-derived dendritic cell assay; and (4) IL-2 in supernatants from a human CD4+ cell assay. The results demonstrated a good assay correlation between the iQue and the compared assays for the cytokine studied. Although overall good assay correlations were observed, our results showed that the iQue assay generated different absolute cytokine values for some cytokines in the same sample sets compared with other assays.

Protease-activated receptors (PARs) are a family of G-protein-coupled receptors (GPCRs) that are irreversibly activated by proteolytic cleavage of the N terminus, which unmasks a tethered peptide ligand that binds and activates the transmembrane receptor domain, eliciting a cellular cascade in response to inflammatory signals and other stimuli. PARs are implicated in a wide range of diseases, such as cancer and inflammation. PARs have been the subject of major pharmaceutical research efforts but the discovery of small-molecule antagonists that effectively bind them has proved challenging. The only marketed drug targeting a PAR is vorapaxar, a selective antagonist of PAR1 used to prevent thrombosis. The structure of PAR1 in complex with vorapaxar has been reported previously. Despite sequence homology across the PAR isoforms, discovery of PAR2 antagonists has been less successful, although GB88 has been described as a weak antagonist. Here we report crystal structures of PAR2 in complex with two distinct antagonists and a blocking antibody. The antagonist AZ8838 binds in a fully occluded pocket near the extracellular surface. Functional and binding studies reveal that AZ8838 exhibits slow binding kinetics, which is an attractive feature for a PAR2 antagonist competing against a tethered ligand. Antagonist AZ3451 binds to a remote allosteric site outside the helical bundle. We propose that antagonist binding prevents structural rearrangements required for receptor activation and signalling. We also show that a blocking antibody antigen-binding fragment binds to the extracellular surface of PAR2, preventing access of the tethered ligand to the peptide-binding site. These structures provide a basis for the development of selective PAR2 antagonists for a range of therapeutic uses.

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., (© 2016 Authors.)

Background: The endocannabinoid system functions through two well characterized receptor systems, the CB1 and CB2 receptors. Work by a number of groups in recent years has provided evidence that the system is more complicated and additional receptor types should exist to explain ligand activity in a number of physiological processes., Experimental Approach: Cells transfected with the human cDNA for GPR55 were tested for their ability to bind and to mediate GTPgammaS binding by cannabinoid ligands. Using an antibody and peptide blocking approach, the nature of the G-protein coupling was determined and further demonstrated by measuring activity of downstream signalling pathways., Key Results: We demonstrate that GPR55 binds to and is activated by the cannabinoid ligand CP55940. In addition endocannabinoids including anandamide and virodhamine activate GTPgammaS binding via GPR55 with nM potencies. Ligands such as cannabidiol and abnormal cannabidiol which exhibit no CB1 or CB2 activity and are believed to function at a novel cannabinoid receptor, also showed activity at GPR55. GPR55 couples to Galpha13 and can mediate activation of rhoA, cdc42 and rac1., Conclusions: These data suggest that GPR55 is a novel cannabinoid receptor, and its ligand profile with respect to CB1 and CB2 described here will permit delineation of its physiological function(s).

Aim: To assess if adenosine is a direct growth hormone secretagogue receptor (GHSR) agonist by investigating the mechanism behind adenosine induced calcium release in human embryonic kidney 293s (HEK) cells expressing GHSR., Methods: Calcium mobilization, cyclic adenosine monophosphate (cAMP) and IP(3) experiments were performed using HEK cells stably expressing GHSR and/or adenosine A(2B) receptor (A(2B)R)., Results: Adenosine has been widely reported as a GHSR agonist. In our hands, adenosine and forskolin stimulated calcium release from IP(3) controlled stores in HEK-GHSR cells but not in non-transfected HEK cells. This release was not accompanied by increased IP(3) levels. The calcium release was both cholera toxin and U73122 sensitive, indicating the involvement of both Galpha(s)/adenylyl cyclase and Galpha(q/11)/phospholipase C pathways. Importantly, the GHSR inverse agonist [D-Arg(1) D-Phe(5) D-Trp(7,9) Leu(11)]-Substance P (SP-analogue) blocked the adenosine stimulated calcium release, demonstrating that GHSR is involved. Assessment of the GHSR-dependent calcium release using adenosine receptor agonists and antagonists resulted in a rank order of potencies resembling the profile of A(2B)R. A(2B)R over-expression in HEK-GHSR cells enhanced potency and efficacy of the adenosine induced calcium release without increasing IP(3) production. Moreover, A(2B)R over-expression in HEK cells potentiated NECA-induced cAMP production. However, GHSR expression had no effect on intracellular cAMP production., Conclusion: In HEK-GHSR cells adenosine activates endogenously expressed A(2B)R resulting in calcium mobilization. We hypothesize that the responsible mechanism is cAMP-dependent sensitization of IP(3) receptors for the high basal level of IP(3) caused by GHSR constitutive activity. Altogether, our results demonstrate that adenosine is not a direct GHSR agonist.