Identification of Betulinic Acid Derivatives as Potent TGR5 Agonists with Antidiabetic Effects via Humanized TGR5 Mutant Mice.

Takeda G protein-coupled receptor 5 (TGR5) is a promising target for treating metabolic syndrome and inflammatory diseases. Herein, we identified a new series of betulinic acid derivatives as potent TGR5 agonists, which show remarkable activity on human (h) and canine (c) TGR5 but exhibit unpromising activity on murine (m) TGR5. Species difference was also observed with many other reported TGR5 agonists. Therefore, we screened 29 amino acids which were conserved in hTGR5 and cTGR5 but different in mTGR5 and found a key amino acid, H88 in mTGR5 (Y89 in hTGR5), which contributed to the species difference. With the CRISPR/Cas9 system, the mTGR5 mutation was introduced into mice, and the optimized compound displayed a significant glucose-lowering effect and stimulated GLP-1 and insulin secretion in TGR5 mice but not in wild-type animals. Taken together, our study provides a useful tool to bridge the gap of species difference and discovers a potent TGR5 agonist for further investigation.