Identification of Novel Series of Potent and Selective Relaxin Family Peptide Receptor 1 (RXFP1) Agonists.

Relaxin H2 is a clinically relevant peptide agonist for relaxin family peptide receptor 1 (RXFP1), but a combination of this hormone's short plasma half-life and the need for injectable delivery limits its therapeutic potential. We sought to overcome these limitations through the development of a potent small molecule (SM) RXFP1 agonist. Although two large SM HTS campaigns failed in identifying suitable hit series, we uncovered novel chemical space starting from the only known SM RXFP1 agonist series, represented by ML290. Following a design-make-test-analyze strategy based on improving early dose to man ranking, we discovered compound 42 (AZ7976), a highly selective RXFP1 agonist with sub-nanomolar potency. We used AZ7976, its 10 000-fold less potent enantiomer 43 and recombinant relaxin H2 to evaluate in vivo pharmacology and demonstrate that AZ7976-mediated heart rate increase in rats was a result of RXFP1 agonism. As a result, AZ7976 was selected as lead for continued optimization.

TG1019/OXE, a Galpha(i/o)-protein-coupled receptor, mediates 5-oxo-eicosatetraenoic acid-induced chemotaxis.

We have previously identified a Galpha(i/o)-protein-coupled receptor (TG1019/OXE) using 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-oxo-ETE) as its ligand. We investigated signal transduction from TG1019 following stimulation with 5-oxo-ETE and role of TG1019 in 5-oxo-ETE-induced chemotaxis, using Chinese hamster ovary cells expressing TG1019 (CHO/TG1019 cells). 5-Oxo-ETE induced intracellular calcium mobilization and rapid activation of MEK/ERK and PI3K/Akt pathways in CHO/TG1019 cells. CHO/TG1019 cells stimulated with 5-oxo-ETE and other eicosanoids exhibited chemotaxis with efficacies related to agonistic activity of each eicosanoid for TG1019. Pretreatment of the cells with pertussis toxin, a phospholipase C (PLC) inhibitor (U73122) or a PI3K inhibitor (LY294002), markedly suppressed 5-oxo-ETE-induced chemotaxis, whereas pretreatment with a MEK inhibitor (PD98059) had no significant effect on the chemotaxis. Our results show that TG1019 mediates 5-oxo-ETE-induced chemotaxis and that signals from TG1019 are transduced via Galpha(i/o) protein to PLC/calcium mobilization, MEK/ERK, and PI3K/Akt, among which PLC and PI3K would play important roles in the chemotaxis.

Identification of a novel human eicosanoid receptor coupled to G(i/o).

We have conducted an in silico data base search for and cloned a novel G-protein-coupled receptor (GPCR) named TG1019. Dot and Northern blotting analyses showed that transcripts of the novel GPCR were expressed in various tissues except brain, and the expression was more intense in liver, kidney, peripheral leukocyte, lung, and spleen than in other tissues. By GTP gamma S binding assay using the TG1019-G alpha(i1)-protein fusion expressed in insect cells, eicosanoids, and polyunsaturated fatty acids such as 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-oxo-ETE), 5(S)-hydroperoxy-6E,8Z, 11Z,14Z-eicosatetraenoic acid, and arachidonic acid were identified to exhibit agonistic activities against TG1019. 5-oxo-ETE was the most potent to enhance the specific binding by 6-fold at a maximum effect dose of submicromolar to micromolar order with an ED(50) value of 5.7 nM. Conversely, polyunsaturated fatty acids such as docosahexaenoic acid and eicosapentaenoic acid showed antagonistic activities against TG1019. In Chinese hamster ovary cells transiently expressing TG1019, the forskolin-stimulated production of cAMP was inhibited up to approximately 70% by 5-oxo-ETE, with an IC(50) value of 33 nM. This inhibition was sensitive to pretreatment of the cells with pertussis toxin.