No abstract

ObjectivesGPR40 (FFAR1), a clinically proven anti-diabetes target, is a Gq-coupled receptor for long chain fatty acids (LCFA) stimulating insulin secretion directly and mediating a major part of the dietary triglyceride-induced secretion of the incretins GLP-1 and GIP. In phase-II studies the GPR40 agonist TAK-875 decreased blood glucose but surprisingly without stimulating incretins.Methods and resultsHere we find that GPR40 can signal through not only Gq and IP3 but also Gs and cAMP when stimulated with certain agonists such as AM-1638 and AM-5262 in contrast to the endogenous LCFA ligands and agonists such as TAK-875 and AM-837, which only signal through Gq. In competition binding against [3H]AM-1638 and [3H]L358 the Gq + Gs and the Gq-only agonists either competed for or showed positive cooperativity by increasing the binding of the two different radio-ligands, in opposite ways. Nevertheless, both the Gq-only and the Gq + Gs agonists all docked surprisingly well into the binding site for TAK-875 in the X-ray structure of GPR40. In murine intestinal primary cell-cultures the endogenous LCFAs and the Gq-only agonists stimulated GLP-1 secretion with rather poor efficacy as compared with the high efficacy Gq + Gs GPR40 agonists and a prototype GPR119 agonist. Similarly, in fasting both male and female mice the Gq + Gs agonists showed significantly higher efficacy than the Gq-only agonists in respect of increasing plasma GLP-1 and plasma GIP in a GPR40-dependent manner.ConclusionsIt is concluded that stimulation of GPR40 by endogenous LCFAs or by Gq-only synthetic agonists result in a rather limited incretin response, whereas Gq + Gs GPR40 agonists stimulate incretin secretion robustly.

Clinical studies indicate that partial agonists of the G-protein-coupled, free fatty acid receptor 1 GPR40 enhance glucose-dependent insulin secretion and represent a potential mechanism for the treatment of type 2 diabetes mellitus. Full allosteric agonists (AgoPAMs) of GPR40 bind to a site distinct from partial agonists and can provide additional efficacy. We report the 3.2-Å crystal structure of human GPR40 (hGPR40) in complex with both the partial agonist MK-8666 and an AgoPAM, which exposes a novel lipid-facing AgoPAM-binding pocket outside the transmembrane helical bundle. Comparison with an additional 2.2-Å structure of the hGPR40-MK-8666 binary complex reveals an induced-fit conformational coupling between the partial agonist and AgoPAM binding sites, involving rearrangements of the transmembrane helices 4 and 5 (TM4 and TM5) and transition of the intracellular loop 2 (ICL2) into a short helix. These conformational changes likely prime GPR40 to a more active-like state and explain the binding cooperativity between these ligands.

Naïve T cells, when activated by specific antigen and cytokines, up-regulate adhesion molecules as well as chemokine receptors on their surface, which allows them to migrate to inflamed tissues. Human studies have shown that CXCR3 is one of the chemokine receptors that is induced during T cell activation. Moreover, CXCR3-positive T cells are enriched at inflammatory sites in patients with autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. In this study, we use a mouse model of inflammation to demonstrate that CXCR3 is required for activated T cell transmigration to inflamed tissue. Using an anti- mCXCR3 antibody, we have shown that in vitro-differentiated T helper (Th) 1 and Th2 cells up-regulated CXCR3 upon stimulation with specific antigen/major histocompatibility complex. However, only Th1 cells, when adoptively transferred to syngeneic recipients, are efficiently recruited to the peritoneum in an adjuvant-induced peritonitis model. Furthermore, the neutralizing anti-mCXCR3 antibody profoundly inhibits the recruitment of Th1 cells to the inflamed peritoneum. Real-time, quantitative reverse transcriptase-polymerase chain reaction analysis demonstrates that the CXCR3 ligands, interferon (IFN)-inducible protein 10 (CXCL10) and IFN-inducible T cell alpha chemoattractant (CXCL11), are among the many chemokines induced in the adjuvant-treated peritoneum. The anti-mCXCR3 antibody is also effective in inhibiting a delayed-type hypersensitivity response, which is largely mediated by enhanced trafficking of activated T cells to peripheral inflammatory sites. Collectively, our results suggest that CXCR3 has a critical role in T cell transmigration to sites of inflammation and thus, may serve as a molecular target for anti-inflammatory therapies.

The discovery of vibegron, a potent and selective human β3-AR agonist for the treatment of overactive bladder (OAB), is described. An early-generation clinical β3-AR agonist MK-0634 (3) exhibited efficacy in humans for the treatment of OAB, but development was discontinued due to unacceptable structure-based toxicity in preclinical species. Optimization of a series of second-generation pyrrolidine-derived β3-AR agonists included reducing the risk for phospholipidosis, the risk of formation of disproportionate human metabolites, and the risk of formation of high levels of circulating metabolites in preclinical species. These efforts resulted in the discovery of vibegron, which possesses improved druglike properties and an overall superior preclinical profile compared to MK-0634. Structure-activity relationships leading to the discovery of vibegron and a summary of its preclinical profile are described.