RXFP4 is a G protein-coupled receptor (GPCR) in the relaxin family. It has recently been recognised that this receptor and its cognate ligand INSL5 may have a role in the regulation of food intake, gut motility, and other functions relevant to metabolic health and disease. Recent data from reporter-mice showed co-location of Rxfp4 and serotonin (5-HT) in the lower gut. We used human single-cell RNA sequence data (scRNASeq) to show that RXFP4 is in a subset of gut enterochromaffin cells that produce 5-HT in humans. We also used RNAScope to show co-location of Rxfp4 mRNA and 5-HT in mouse colon, confirming prior findings. To understand whether RXFP4 might regulate serotonin production, we developed a cell model using Colo320, a human gut-derived immortalised cell line that produces and releases serotonin. Overexpression of RXFP4 in these cells resulted in a constitutive decrease in cAMP levels in both the basal state and in cells treated with forskolin. Treatment of cells with two RXFP4 agonists, INSL5 derived peptide INSL5-A13 and small molecule compound-4, further reduced cAMP levels. This was paralleled by a reduction in expression of mRNA for TPH1, the enzyme controlling the rate limiting step in the production of serotonin. Overexpression of RXFP4 also attenuated the cAMP-induced release of serotonin from Colo320 cells. Together this demonstrates that serotonin producing enterochromaffin cells are the major site of RXFP4 expression in the gut and that RXFP4 can have inhibitory functional impacts on cAMP production as well as TPH1 expression and serotonin release.
© 2023 The Author(s).

GPR88 is an orphan G protein-coupled receptor mainly expressed in the brain, whose endogenous ligand has not yet been identified. To elucidate GPR88 functions, our group has developed RTI-13951-33 (1b) as the first in vivo active GPR88 agonist, but its poor metabolic stability and moderate brain permeability remain to be further optimized. Here, we report the design, synthesis, and pharmacological characterization of a new series of RTI-13951-33 analogues with the aim of improving pharmacokinetic properties. As a result, we identified a highly potent GPR88 agonist RTI-122 (30a) (cAMP EC50 = 11 nM) with good metabolic stability (half-life of 5.8 h) and brain permeability (brain/plasma ratio of >1) in mice. Notably, RTI-122 was more effective than RTI-13951-33 in attenuating the binge-like alcohol drinking behavior in the drinking-in-the-dark paradigm. Collectively, our findings suggest that RTI-122 is a promising lead compound for drug discovery research of GPR88 agonists.

Blockade of lysophosphatidic acid receptor 5 (LPA5) by a recently reported antagonist AS2717638 (2) attenuated inflammatory and neuropathic pains, although it showed moderate in vivo efficacy and its structure-activity relationships and the ADME properties are little studied. We therefore designed and synthesized a series of isoquinolone derivatives and evaluated their potency in LPA5 calcium mobilization and cAMP assays. Our results show that substituted phenyl groups or bicyclic aromatic rings such as benzothiophenes or benzofurans are tolerated at the 2-position, 4-substituted piperidines are favored at the 4-position, and methoxy groups at the 6- and 7-positions are essential for activity. Compounds 65 and 66 showed comparable in vitro potency, excellent selectivity against LPA1-LPA4 and >50 other GPCRs, moderate metabolic stability, and high aqueous solubility and brain permeability. Both 65 and 66 significantly attenuated nociceptive hypersensitivity at lower doses than 2 and had longer-lasting effects in an inflammatory pain model, and 66 also dose-dependently reduced mechanical allodynia in the chronic constriction injury model and opioid-induced hyperalgesia at doses that had no effect on the locomotion in rats. These results suggest that these isoquinolone derivatives as LPA5 antagonists are of promise as potential analgesics.
Copyright © 2022 Elsevier Masson SAS. All rights reserved.

Improving morphine tolerance (MT) is an urgent problem in the clinical treatment of bone cancer pain. Considering that β-Elemene is widely used in the treatment of cancer pain, we explored the effects and mechanism of β-Elemene in preventing MT of bone cancer pain.
Bone cancer pain and chronic MT rat model was established by injecting MADB106 cells and morphine (10 mg/kg). SH-SY5Y cells were treated with morphine (10 μg/mL) for 48 h to establish a cell model. The mechanical withdrawal threshold and thermal withdrawal latency of rats were detected by mechanical allodynia and thermal hyperalgesia tests, respectively. The protein expressions of μ-opioid receptor (MOPR), cyclic adenosine monophosphate (cAMP), N-methyl-D-aspartate receptor subunit 2B (NR2B), phosphorylated-calmodulin-dependent protein kinase II (p-CaMKII), and CaMKII were detected by western blot. The viability of SH-SY5Y cells was determined by the cell counting kit-8 assay. cAMP content in SH-SY5Y cells was measured by a LANCE cAMP kit.
Animal experiments showed that MT strengthened over time, while increased β-Elemene dosage alleviated MT. The viability of SH-SY5Y cells was down-regulated by high-dose β-Elemene. In the rat and cell models, long-term morphine treatment decreased the expression of MOPR and increased the cAMP and NR2B expressions and p-CaMKII/CaMKII, while β-Elemene and siNR2B counteracted the effects of morphine treatment. In addition, siNR2B reversed the effects of β-Elemene on related protein expressions and cAMP content in the cell model.
β-Elemene improved MT in bone cancer pain through the regulation of NR2B-mediated MOPR.
Copyright © 2022 Qinfei Zhou et al.

The neuropeptide relaxin-3/RXFP3 system is involved in many important physiological processes such as stress responses, appetite control, and motivation for reward. To date, pharmacological studies of RXFP3 have been limited to peptide ligands. In this study, we report the discovery of the first small-molecule antagonists of RXFP3 through a high-throughput screening campaign. Focused structure-activity relationship studies of the hit compound resulted in RLX-33 (33) that was able to inhibit relaxin-3 activity in a battery of functional assays. RLX-33 is selective for RXFP3 over RXFP1 and RXFP4, two related members in the relaxin/insulin superfamily, and has favorable pharmacokinetic properties for behavioral assessment. When administered to rats intraperitoneally, RLX-33 blocked food intake induced by the RXFP3-selective agonist R3/I5. Collectively, our findings demonstrated that RLX-33 represents a promising antagonist scaffold for the development of drugs targeting the relaxin-3/RXFP3 system.