title: In silico, in vitro and cellular analysis with a kinome-wide inhibitor panel correlates cellular LRRK2 dephosphorylation to inhibitor activity on LRRK2.
abstract: Leucine-rich repeat kinase 2 (LRRK2) is a complex, multidomain protein which is considered a valuable target for potential disease-modifying therapeutic strategies for Parkinson's disease (PD). In mammalian cells and brain, LRRK2 is phosphorylated and treatment of cells with inhibitors of LRRK2 kinase activity can induce LRRK2 dephosphorylation at a cluster of serines including Ser910/935/955/973. It has been suggested that phosphorylation levels at these sites reflect LRRK2 kinase activity, however kinase-dead variants of LRRK2 or kinase activating variants do not display altered Ser935 phosphorylation levels compared to wild type. Furthermore, Ser910/935/955/973 are not autophosphorylation sites, therefore, it is unclear if inhibitor induced dephosphorylation depends on the activity of compounds on LRRK2 or on yet to be identified upstream kinases. Here we used a panel of 160 ATP competitive and cell permeable kinase inhibitors directed against all branches of the kinome and tested their activity on LRRK2 in vitro using a peptide-substrate-based kinase assay. In neuronal SH-SY5Y cells overexpressing LRRK2 we used compound-induced dephosphorylation of Ser935 as readout. In silico docking of selected compounds was performed using a modeled LRRK2 kinase structure. Receiver operating characteristic plots demonstrated that the obtained docking scores to the LRRK2 ATP binding site correlated with in vitro and cellular compound activity. We also found that in vitro potency showed a high degree of correlation to cellular compound induced LRRK2 dephosphorylation activity across multiple compound classes. Therefore, acute LRRK2 dephosphorylation at Ser935 in inhibitor treated cells involves a strong component of inhibitor activity on LRRK2 itself, without excluding a role for upstream kinases. Understanding the regulation of LRRK2 phosphorylation by kinase inhibitors aids our understanding of LRRK2 signaling and may lead to development of new classes of LRRK2 kinase inhibitors.
authors.affiliation: Laboratory for Biomolecular Modelling, Division of Biochemistry, Molecular and Structural Biology, Department of Chemistry, KU Leuven Leuven, Belgium.
authors.forename: Renée
authors.initials: R
authors.lastname: Vancraenenbroeck
authors.affiliation: Laboratory for Biomolecular Modelling, Division of Biochemistry, Molecular and Structural Biology, Department of Chemistry, KU Leuven Leuven, Belgium.
authors.forename: Joren
authors.initials: J
authors.lastname: De Raeymaecker
authors.affiliation: Laboratory for Neurobiology and Gene Therapy, Department of Neurosciences, KU Leuven Leuven, Belgium.
authors.forename: Evy
authors.initials: E
authors.lastname: Lobbestael
authors.affiliation: Laboratory for Neurobiology and Gene Therapy, Department of Neurosciences, KU Leuven Leuven, Belgium.
authors.forename: Fangye
authors.initials: F
authors.lastname: Gao
authors.affiliation: Laboratory for Biomolecular Modelling, Division of Biochemistry, Molecular and Structural Biology, Department of Chemistry, KU Leuven Leuven, Belgium.
authors.forename: Marc
authors.initials: M
authors.lastname: De Maeyer
authors.affiliation: Laboratory for Biomolecular Modelling, Division of Biochemistry, Molecular and Structural Biology, Department of Chemistry, KU Leuven Leuven, Belgium ; Zhang Initiative Research Unit, Riken Saitama, Japan.
authors.forename: Arnout
authors.initials: A
authors.lastname: Voet
authors.affiliation: Laboratory for Neurobiology and Gene Therapy, Department of Neurosciences, KU Leuven Leuven, Belgium.
authors.forename: Veerle
authors.initials: V
authors.lastname: Baekelandt
authors.affiliation: Laboratory for Neurobiology and Gene Therapy, Department of Neurosciences, KU Leuven Leuven, Belgium.
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Taymans
keywords: LRRKtide; MOE; Parkinson's disease; docking; inhibitor; kinase; phosphorylation; receiver operator characteristic