Functionally active modulators targeting the LRRK2 WD40 repeat domain identified by FRASE-bot in CACHE Challenge #1

Abstract

Critical Assessment of Computational Hit-Finding Experiments (CACHE) Challenges emerged as real-life stress tests for computational hit-finding strategies. In CACHE Challenge #1, 23 participants contributed their original workflows to identify small-molecule ligands for the WD40 repeat (WDR) of LRRK2, a promising Parkinson's target. We applied the FRASE-based hit-finding robot (FRASE-bot), a platform for interaction-based screening allowing a drastic reduction of the explorable chemical space and a concurrent detection of putative ligand-binding sites. In two screening rounds, 84 compounds were procured for experimental testing and 8 were confirmed to bind LRRK2-WDR with dissociation constants (K _d) ranging from 3 to 41 μM. To investigate the functional effect of WDR ligands, they were tested for their ability to modify the LRRK2 activity markers in HEK293T cells. Two compounds showed statistically significant increases in the kinase activity of WT LRRK2, and two compounds affected the conformation and kinase activity of major LRRK2 mutants.

Fig. 1. FRASE database screening. (A) 2544 ligand fragments (magenta sticks) seeded in the LRRK2 WDR structure (orange ribbons); (B) 85 ligand fragments with scores >0.4 are grouped into five distinct clusters (1–5); (C) potential ligand-binding pockets corresponding to clusters 1–5.

Fig. 2. The experimentally confirmed Round-1 hits. Chemical structures, LRRK2 K_d values, solubility, and SPR dose–response data for the Round-1 hits 1 (A) and 2 (B); docking poses for 1 (C) and 2 (D).

Fig. 3. The experimentally confirmed Round-2 hits. Chemical structures and LRRK2 K_d values, and solubility for the Round-3 hits 3–8.

Fig. 4. Cellular effects of compounds on LRRK2 kinase activity. (A) Example of HEK293T cells transfected with a 3×Flag-tagged LRRK2 WT or Y1699C pathogenic mutant and treated with DMSO, 30 nM MLi-2, a potent kinase domain inhibitor (ref. 35), or 30 μM of the different hits for 2 h, cell lysates analyzed by western blotting for Flag, pS1292 LRRK2, pS935 LRRK2, RAB10, pT73 RAB10 and α-tubulin as the loading control. (B) Quantification of the pS935 LRRK2/LRRK2 ratio of blots depicted in (A). *p < 0.05. (C) Quantification of the pS1292 LRRK2/LRRK2 ratio of blots depicted in (A). *p < 0.05, **p < 0.01. (D) Quantification of the pT73 RAB10/RAB10 ratio of blots depicted in (A). *p < 0.05, **p < 0.01, and ****p < 0.001.