Cache: Utilizing ultra-large library screening in Rosetta to identify novel binders of the WD-repeat domain of Leucine-Rich Repeat Kinase 2

Abstract In this study, we present a pipeline for identifying novel ligands targeting the Tryptophan-Aspartate-Repeat domain 40 (WDR40) of Leucine-Rich Repeat Kinase 2 (LRRK2), a protein associated with Parkinson’s disease, as part of the first Critical Assessment of Computational Hit-finding Experiments (CACHE) challenge, a blind benchmark experiment for drug discovery. Mutations in this protein are the most common genetic cause of familial Parkinson’s disease, yet this target remains understudied. We conducted an ultra-large library screening (ULLS) of the Enamine REAL space using a newly developed evolutionary algorithm, RosettaEvolutionaryLigand (REvoLd), which allows for efficient screening of combinatorial compound libraries. The protocol involved refining the target structure with molecular dynamic simulations, identifying a binding site via blind-docking, and optimizing compounds through REvoLd, culminating in a manual selection amongst the top-scoring REvoLd hits. A single binder molecule was identified that derived from the combination of two Enamine building blocks. In the second round, derivatives of the hit compound were used as input for REvoLd to further sample within the Enamine REAL space. Ultimately, a total of five molecules were identified, from which three show a measurable dissociation constant K $$_D$$ D value better than 150 $$\upmu$$ μ μm, showcasing the effectiveness of this approach. However, it also highlighted shortcomings, such as the preference for nitrogen-rich rings in the RosettaLigand scoring function. Scientific contribution We introduce the first real-world application for REvoLd, an evolutionary docking algorithm enabling efficient ultra-large library screening for flexible protein targets. Our approach identified novel binders for the WDR40 domain of LRRK2 within the CACHE challenge #1, representing the first prospective validation of REvoLd. Here, we present a preparation pipeline to allow exploration of a large protein pocket with unspecific binding areas, and unlike prior brute-force docking efforts, our method integrates receptor flexibility and combinatorial chemistry optimization. Graphical Abstract

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