Evaluation of 5-phenylindazoles on LRRK2 inhibition using cellular-based assay and molecular docking

Abstract

LRRK2 has been widely recognised as a critical drug target as it is relevant for both familial and idiopathic cases of Parkinson’s disease (PD). Increased LRRK2 kinase activity has been observed in PD patients regardless of their genetic status. This study investigated seventeen 5-phenylindazole derivatives as LRRK2 kinase inhibitors by monitoring Rab10 phosphorylation at Thr73 (a direct LRRK2 substrate) and LRRK2 phosphorylation at Ser935 in HEK293 cells expressing either wild- type or G2019S LRRK2. Molecular docking was used to analyse protein-ligand interactions at the atomic level. In wild-type LRRK2 cells, four small molecules (4h, 4i, 4n and 4o) induced a modest, non-significant inhibition of Rab10 Thr73 phosphorylation (ranging from 27% to 35%), but LRRK2 Ser935 phosphorylation remained unaffected. Whereas no significant inhibition of either LRRK2 Ser935 and Rab10 Thr73 phosphorylation was observed in the LRRK2-G2019S cells treated with any of the 5-phenylindazole derivatives. Computational analysis revealed that these compounds preferentially bind to the open-inactive conformation of LRRK2, a characteristic of type II kinase inhibitors. However, due to their compact molecular scaffolds, these small molecules only partially occupied the extended type II binding pocket, limiting the allosteric engagement necessary for potent inhibition. In conclusion, this structure-activity relationship study demonstrates that while type II binding mode selectivity was modestly achieved, scaffold extension is required to increase molecular size and achieve fuller occupancy of the extended type II pocket.