Design, synthesis, and characterization of species-independent h/mGPR35 agonists for MAFLD/MASH

G protein-coupled receptor 35 (GPR35) has emerged as a potential therapeutic target for metabolic dysfunction-associated fatty liver disease (MAFLD) and metabolic dysfunction-associated steatohepatitis (MASH). However, the sequence identity between human and mouse GPR35 is only 72%, which severely limits the development of species-independent active ligands for GPR35 and consequently hinders pharmacological studies targeting GPR35 in MAFLD/MASH. In this work, a series of novel GPR35 agonists based on the coumarin scaffold were developed, exhibiting low species differences between hGPR35 and mGPR35. Some of these agonists demonstrated low nanomolar potency, such as compound 6l (hEC₅₀ = 30 nM, mEC₅₀ = 21 nM) and compound 9f (hEC₅₀ = 12 nM, mEC₅₀ = 2 nM). The key binding sites of 6l and 9f with the h/mGPR35 receptor were further identified, respectively. Both compounds exhibited excellent lipid-lowering effects in in vitro and in vivo models. In a high-fat diet-induced mouse model of MASH, 6l and 9f significantly alleviated key pathological drivers of disease progression, including hepatic steatosis, lipid metabolism disorders, liver inflammation and fibrosis. Collectively, this study provides a class of novel agonists with high potency and low species differences for h/mGPR35, laying a foundation for further exploration of the pharmacological relationship between GPR35 and MAFLD/MASH.