Allosteric ligands for the pharmacologically dark receptors GPR68 and GPR65

At least 120 non-olfactory G protein-coupled receptors in the human genome are ”orphans” for which endogenous ligands are unknown, and many have no selective ligands, hindering elucidation of their biological functions and clinical relevance. Among these is GPR68, a proton receptor that lacks small molecule modulators for probing its biology. Yeast-based screens against GPR68 identified the benzodiazepine drug lorazepam as a non-selective GPR68 positive allosteric modulator. Over 3000 GPR68 homology models were refined to recognize lorazepam in a putative allosteric site. Docking 3.1 million molecules predicted new GPR68 modulators many of which were confirmed in functional assays. One potent GPR68 modulator—ogerin– suppressed recall in fear conditioning in wild-type, but not in GPR68 knockout mice. The same approach led to the discovery of allosteric agonists and negative allosteric modulators for GPR65. Combining physical and structure-based screening may be broadly useful for ligand discovery for understudied and orphan GPCRs

G protein-coupled receptor 68 increases the number of B lymphocytes

G protein-coupled receptor 68 (GPR68) is a proton sensor that is activated upon binding to extracellular protons. We have previously found that GPR68 induces a proapoptotic pathway in bone marrow (BM) cells from the patients with myelodysplastic syndromes (MDS) after treated with lenalidomide. However, the function of GPR68 in normal hematopoietic cells remains unclear. With genetic loss of function approach, we found reduced frequency and number of B lymphocytes in the peripheral blood (PB) of whole body Gpr68-/- mice compared to control littermates upon aging. During hematopoietic regeneration, such as in response to fluorouracil (5-FU), we also found reduced frequency and number of B lymphocytes in Gpr68-/- mice compared to wild type mice. Mechanism studies revealed that Gpr68 expression was upregulated in B lymphocytes of BM during aging and in hematopoietic progenitor cells after treatment with 5-FU. In addition, activation of Gpr68 by its activators increased the frequency and number of B lymphocytes. Our studies indicate that Gpr68 expression is upregulated in hematopoietic cells upon aging and during hematopoietic regeneration that ends up with increased number of B lymphocytes

Fentanyl-related designer drugs W-18 and W-15 lack appreciable opioid activity in vitro and in vivo

W-18 (4-chloro-N-[1-[2-(4-nitrophenyl)ethyl]-2-piperidinylidene]-benzenesulfonamide) and W-15 (4-chloro-N-[1-(2-phenylethyl)-2-piperidinylidene]-benzenesulfonamide) represent two emerging drugs of abuse chemically related to the potent opioid agonist fentanyl (N-(1-(2-phenylethyl)-4-piperidinyl)-N-phenylpropanamide). Here, we describe the comprehensive pharmacological profiles of W-18 and W-15, as examination of their structural features predicted that they might lack opioid activity. We found W-18 and W-15 to be without detectible activity at μ, δ, κ, and nociception opioid receptors in a variety of assays. We also tested W-18 and W-15 for activity as allosteric modulators at opioid receptors and found them devoid of significant positive or negative allosteric modulatory activity. Comprehensive profiling at essentially all the druggable GPCRs in the human genome using the PRESTO-Tango platform revealed no significant activity. Weak activity at the sigma receptors and the peripheral benzodiazepine receptor was found for W-18 (Ki = 271 nM). W-18 showed no activity in either the radiant heat tail-flick or the writhing assays and also did not induce classical opioid behaviors. W-18 is extensively metabolized, but its metabolites also lack opioid activity. Thus, although W-18 and W-15 have been suggested to be potent opioid agonists, our results reveal no significant activity at these or other known targets for psychoactive drugs

Extracellular acidification stimulates GPR68 mediated IL-8 production in human pancreatic β cells.

International audienceAcute or chronic metabolic complications such as diabetic ketoacidosis are often associated with extracellular acidification and pancreatic β-cell dysfunction. However, the mechanisms by which human β-cells sense and respond to acidic pH remain elusive. In this study, using the recently developed human β-cell line EndoC-βH2, we demonstrate that β-cells respond to extracellular acidification through GPR68, which is the predominant proton sensing receptor of human β-cells. Using gain- and loss-of-function studies, we provide evidence that the β-cell enriched transcription factor RFX6 is a major regulator of GPR68. Further, we show that acidic pH stimulates the production and secretion of the chemokine IL-8 by β-cells through NF-кB activation. Blocking of GPR68 or NF-кB activity severely attenuated acidification induced IL-8 production. Thus, we provide mechanistic insights into GPR68 mediated β-cell response to acidic microenvironment, which could be a new target to protect β-cell against acidosis induced inflammation

Cytokine Regulation in Human CD4 T Cells by the Aryl Hydrocarbon Receptor and Gq-Coupled Receptors

Th17 cells contribute to host defense on mucosal surfaces but also provoke autoimmune diseases when directed against self-antigens. Identifying therapeutic targets that regulate Th17 cell differentiation and/or cytokine production has considerable value. Here, we study the aryl hydrocarbon receptor (AhR)-dependent transcriptome in human CD4 T cells treated with Th17-inducing cytokines. We show that the AhR reciprocally regulates IL-17 and IL-22 production in human CD4 T cells. Global gene expression analysis revealed that AhR ligation decreased IL21 expression, correlating with delayed upregulation of RORC during culture with Th17-inducing cytokines. Several of the AhR-dependent genes have known roles in cellular assembly, organization, development, growth and proliferation. We further show that expression of GPR15, GPR55 and GPR68 positively correlates with IL-22 production in the presence of the AhR agonist FICZ. Activation of GPR68 with the lorazepam derivative ogerin resulted in suppression of IL-22 and IL-10 secretion by T cells, with no effect on IL-17. Under neutral Th0 conditions, ogerin and the Gq/11 receptor inhibitor YM254890 blunted IL-22 induction by FICZ. These data reveal the AhR-dependent transcriptome in human CD4 T cells and suggest the mechanism through which the AhR regulates T cell function may be partially dependent on Gq-coupled receptors including GPR68

A ternary complex model of Sirtuin4-NAD+-Glutamate dehydrogenase

Sirtuin4 (Sirt4) is one of the mammalian homologues of Silent information regulator 2 (Sir2), which promotes the longevity of yeast, C. elegans, fruit flies and mice. Sirt4 is localized in the mitochondria, where it contributes to preventing the development of cancers and ischemic heart disease through regulating energy metabolism. The ADP-ribosylation of glutamate dehydrogenase (GDH), which is catalyzed by Sirt4, downregulates the TCA cycle. However, this reaction mechanism is obscure, because the structure of Sirt4 is unknown. We here constructed structural models of Sirt4 by homology modeling and threading, and docked nicotinamide adenine dinucleotide+ (NAD+) to Sirt4. In addition, a partial GDH structure was docked to the Sirt4-NAD+ complex model. In the ternary complex model of Sirt4-NAD+-GDH, the acetylated lysine 171 of GDH is located close to NAD+. This suggests a possible mechanism underlying the ADP-ribosylation at cysteine 172, which may occur through a transient intermediate with ADP-ribosylation at the acetylated lysine 171. These results may be useful in designing drugs for the treatment of cancers and ischemic heart disease

Comprehensive Assessment of GPR68 Expression in Normal and Neoplastic Human Tissues Using a Novel Rabbit Monoclonal Antibody

GPR68 (OGR1) belongs to the proton-sensing G protein-coupled receptors that are involved in cellular adaptations to pH changes during tumour development. Although expression of GPR68 has been described in many tumour cell lines, little is known about its presence in human tumour entities. We characterised the novel rabbit monoclonal anti-human GPR68 antibody 16H23L16 using various cell lines and tissue specimens. The antibody was then applied to a large series of formalin-fixed, paraffin-embedded normal and neoplastic human tissue samples. Antibody specificity was demonstrated in a Western blot analysis of GPR68-expressing cells using specific siRNAs. Immunocytochemical experiments revealed pH-dependent changes in subcellular localisation of the receptor and internalisation after stimulation with lorazepam. In normal tissue, GPR68 was present in glucagon-producing islet cells, neuroendocrine cells of the intestinal tract, gastric glands, granulocytes, macrophages, muscle layers of arteries and arterioles, and capillaries. GPR68 was also expressed in neuroendocrine tumours, where it may be a positive prognostic factor, in pheochromocytomas, cervical adenocarcinomas, and endometrial cancer, as well as in paragangliomas, medullary thyroid carcinomas, gastrointestinal stromal tumours, and pancreatic adenocarcinomas. Often, tumour capillaries were also strongly GPR68-positive. The novel antibody 16H23L16 will be a valuable tool for basic research and for identifying GPR68-expressing tumours during histopathological examinations