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  • The physiological and pharmacological roles of GPR remain la


    The physiological and pharmacological roles of GPR81 remain largely unclear because of several reasons. The low potency of lactate agonizing GPR81 in the millimolar range, together with its fast metabolic turnover, renders as a challenge in in vivo studies using lactate. Furthermore, there are no potent and selective surrogate agonists of GPR81 that enable in vivo pharmacological studies. In the present study, we demonstrated that GPR81 is required for the lactate-induced inhibition of lipolysis. In addition, we described the identification of a potent and highly selective small-molecule GPR81 agonist by high-throughput screening (HTS), followed by chemical optimization. Finally, intraperitoneal (i.p.) administration of the compound suppressed lipolysis in mice at doses that did not cause cutaneous flushing. Taken together, these results suggest that GPR81 is an attractive drug target for treating dyslipidemia and does not have potential to cause cutaneous flushing.
    Materials and methods
    Discussion The present study aimed to identify potent and selective small-molecule agonists for GPR81 for use as tool compounds to understand the pharmacological role and therapeutic potential of GPR81 and as lead compounds that can be further studied to develop a drug for treating dyslipidemia. These aims were successfully accomplished by performing HTS, followed by a series of profiling tests and chemical optimizations. HTS and follow-up studies yielded four chemical series of small-molecule GPR81 agonists. Among these DTP3 of compounds, we chose the aminothiazole derivatives as potential leads for further evaluation. The initial hit compound had modest potency against GPR81, and its use was limited by its poor solubility. This chemical series was optimized through the synthesis and profiling of 70 close analogs, which generated SAR and improved physical and chemical parameters, ultimately yielding Compound 2. Compound 2 showed good solubility, was approximately 10-fold more potent against GPR81 than the original hit compound, and was selective over GPR109a. In addition, Compound 2 showed good exposure in the mouse in vivo when administered intraperitoneally, which is acceptable for use in later in vivo experiments. Our present findings indicate that Compound 2 is an attractive and promising lead compound for the following reasons: First, Compound 2 has high potency for both human and mouse GPR81, with EC50 values of 58±5.4nM and 50±15nM, respectively, as well as remarkable selectivity against GPR109a. Although activation of GPR109a exerts a therapeutic benefit by inhibiting lipolysis, mainly in adipocytes, it is thought to cause cutaneous flushing by activating Langerhans cells and keratinocytes in the skin (Benyo et al., 2005, Hanson et al., 2010, Jacobson, 2010, Pike, 2005, Pike and Wise, 2004). Therefore, a high potency to GPR81 and great selectivity against GPR109a indicates that this compound is expected to exert antidyslipdemic effects without the risk of GPR109a-mediated flushing, which has been a major issue for this class of drug. Second, Compound 2 inhibits lipolysis in differentiated 3T3-L1 adipocytes. Because recombinant overexpressing systems that enable signal amplification were used to identify and evaluate compounds, it was important to confirm the activity of the compounds in a more physiologically relevant cell system. Our data demonstrated that Compound 2 inhibited lipolysis in differentiated 3T3-L1 adipocytes, thereby strongly indicating that Compound 2 would have the potential to demonstrate efficacy in vivo where the expression level is considered to be lower than that in recombinant overexpressing systems. Compound 2 shows desirable pharmacokinetic properties with good exposure in mice and reduces the FFA content at doses that do not show any signs of cutaneous flushing. In contrast, nicotinic acid causes cutaneous flushing and reduction in the FFA content at similar doses in these models. Whereas hydroxybenzoic acid derivatives were previously identified to have antilipolytic effects in vivo by agonizing GPR81 (Dvorak et al., 2012), it is noteworthy that in vitro mouse GPR81 EC50 of our compound is over 400 times more potent, achieving efficacy at lower plasma concentration and that we experimentally confirmed our compound is effective in vivo without causing flushing, as theoretically expected.