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Updated: May 30, 2025
Rab10 Phosphorylation Detection by LRRK2 Activity Using SDS-PAGE with a Phosphate-binding Tag
Published on: December 14, 2017
Functionally active modulators targeting the LRRK2 WD40 repeat domain identified by FRASE-bot in CACHE Challenge #1
Akhila Mettu1, Marta Glavatskikh2, Xiaowen Wang1
1University of Missouri - Columbia, Department of Chemistry USA dmitri.kireev@missouri.edu.
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.
Insights
Computational hit-finding strategies were tested in the CACHE Challenge. The FRASE-bot identified 8 LRRK2-WDR binders, with 2 compounds modulating Parkinson
Area of Science:
- Computational chemistry and drug discovery
- Neuroscience and Parkinson's disease research
Background:
- The WD40 repeat (WDR) of Leucine-Rich Repeat Kinase 2 (LRRK2) is a key target for Parkinson's disease therapeutics.
- Computational hit-finding strategies require rigorous validation through real-world challenges.
Purpose of the Study:
- To assess the efficacy of computational hit-finding workflows in identifying LRRK2-WDR ligands.
- To evaluate the performance of the FRASE-based hit-finding robot (FRASE-bot) in a competitive challenge setting.
- To investigate the functional impact of identified ligands on LRRK2 activity.
Main Methods:
- Application of the FRASE-bot, an interaction-based screening platform, for hit identification.
- Procurement and experimental testing of 84 selected compounds.
- Biochemical assays to determine binding affinity (Kd) and cellular assays to assess LRRK2 activity modulation.
Main Results:
- Eight compounds were confirmed to bind the LRRK2-WDR domain, with dissociation constants ranging from 3 to 41 μM.
- Two identified compounds demonstrated a statistically significant increase in wild-type LRRK2 kinase activity.
- Two compounds modulated the conformation and kinase activity of key LRRK2 mutants.
Conclusions:
- The FRASE-bot successfully identified validated binders for the LRRK2-WDR target.
- Identified ligands show potential for modulating LRRK2 activity, offering therapeutic avenues for Parkinson's disease.
- The Critical Assessment of Computational Hit-Finding Experiments (CACHE) challenge provides a valuable platform for validating computational methods.
