title: Unique functional and structural properties of the LRRK2 protein ATP-binding pocket.
abstract: Pathogenic mutations in the LRRK2 gene can cause late-onset Parkinson disease. The most common mutation, G2019S, resides in the kinase domain and enhances activity. LRRK2 possesses the unique property of cis-autophosphorylation of its own GTPase domain. Because high-resolution structures of the human LRRK2 kinase domain are not available, we used novel high-throughput assays that measured both cis-autophosphorylation and trans-peptide phosphorylation to probe the ATP-binding pocket. We disclose hundreds of commercially available activity-selective LRRK2 kinase inhibitors. Some compounds inhibit cis-autophosphorylation more strongly than trans-peptide phosphorylation, and other compounds inhibit G2019S-LRRK2 more strongly than WT-LRRK2. Through exploitation of structure-activity relationships revealed through high-throughput analyses, we identified a useful probe inhibitor, SRI-29132 (11). SRI-29132 is exquisitely selective for LRRK2 kinase activity and is effective in attenuating proinflammatory responses in macrophages and rescuing neurite retraction phenotypes in neurons. Furthermore, the compound demonstrates excellent potency, is highly blood-brain barrier-permeant, but suffers from rapid first-pass metabolism. Despite the observed selectivity of SRI-29132, docking models highlighted critical interactions with residues conserved in many protein kinases, implying a unique structural configuration for the LRRK2 ATP-binding pocket. Although the human LRRK2 kinase domain is unstable and insoluble, we demonstrate that the LRRK2 homolog from ameba can be mutated to approximate some aspects of the human LRRK2 ATP-binding pocket. Our results provide a rich resource for LRRK2 small molecule inhibitor development. More broadly, our results provide a precedent for the functional interrogation of ATP-binding pockets when traditional approaches to ascertain structure prove difficult.
authors.affiliation: From the Center for Neurodegeneration and Experimental Therapeutics, Department of Neurology and Center for Biophysical Sciences and Engineering, Department of Optometry, The University of Alabama at Birmingham, Birmingham, Alabama 35294 and.
authors.forename: Zhiyong
authors.initials: Z
authors.lastname: Liu
authors.affiliation: the Drug Discovery Division, Southern Research Institute, Birmingham, Alabama 35294.
authors.forename: Robert A
authors.initials: RA
authors.lastname: Galemmo
authors.affiliation: From the Center for Neurodegeneration and Experimental Therapeutics, Department of Neurology and.
authors.forename: Kyle B
authors.initials: KB
authors.lastname: Fraser
authors.affiliation: From the Center for Neurodegeneration and Experimental Therapeutics, Department of Neurology and.
authors.forename: Mark S
authors.initials: MS
authors.lastname: Moehle
authors.affiliation: From the Center for Neurodegeneration and Experimental Therapeutics, Department of Neurology and.
authors.forename: Saurabh
authors.initials: S
authors.lastname: Sen
authors.affiliation: From the Center for Neurodegeneration and Experimental Therapeutics, Department of Neurology and.
authors.forename: Laura A
authors.initials: LA
authors.lastname: Volpicelli-Daley
authors.affiliation: the Drug Discovery Division, Southern Research Institute, Birmingham, Alabama 35294.
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: DeLucas
authors.affiliation: the Drug Discovery Division, Southern Research Institute, Birmingham, Alabama 35294.
authors.forename: Larry J
authors.initials: LJ
authors.lastname: Ross
authors.affiliation: the Drug Discovery Division, Southern Research Institute, Birmingham, Alabama 35294.
authors.forename: Jacob
authors.initials: J
authors.lastname: Valiyaveettil
authors.affiliation: the Drug Discovery Division, Southern Research Institute, Birmingham, Alabama 35294.
authors.forename: Omar
authors.initials: O
authors.lastname: Moukha-Chafiq
authors.affiliation: the Drug Discovery Division, Southern Research Institute, Birmingham, Alabama 35294.
authors.forename: Ashish K
authors.initials: AK
authors.lastname: Pathak
authors.affiliation: the Drug Discovery Division, Southern Research Institute, Birmingham, Alabama 35294.
authors.forename: Subramaniam
authors.initials: S
authors.lastname: Ananthan
authors.affiliation: the Drug Discovery Division, Southern Research Institute, Birmingham, Alabama 35294.
authors.forename: Hollis
authors.initials: H
authors.lastname: Kezar
authors.affiliation: the Drug Discovery Division, Southern Research Institute, Birmingham, Alabama 35294.
authors.forename: E Lucile
authors.initials: EL
authors.lastname: White
authors.affiliation: the Drug Discovery Division, Southern Research Institute, Birmingham, Alabama 35294.
authors.forename: Vandana
authors.initials: V
authors.lastname: Gupta
authors.affiliation: the Drug Discovery Division, Southern Research Institute, Birmingham, Alabama 35294.
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Maddry
authors.affiliation: the Drug Discovery Division, Southern Research Institute, Birmingham, Alabama 35294.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Suto
authors.affiliation: From the Center for Neurodegeneration and Experimental Therapeutics, Department of Neurology and abwest@uab.edu.
authors.forename: Andrew B
authors.initials: AB
authors.lastname: West
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D055162:Biocatalysis / Q000187:drug effects; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D056945:Hep G2 Cells; D006801:Humans; D007700:Kinetics; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009154:Mutation; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011724:Pyridazines / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D054852:Small Molecule Libraries / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D013329:Structure-Activity Relationship; D014230:Triazoles / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology
chemical_list: C000595193:1-(piperidin-1-yl)-2-((3-(thiophen-2-yl)-(1,2,4)triazolo(4,3-b)pyridazin-6-yl)thio)butan-1-one; D047428:Protein Kinase Inhibitors; D011724:Pyridazines; D054852:Small Molecule Libraries; D014230:Triazoles; D000255:Adenosine Triphosphate; C495280:LRRK2 protein, human; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D017346:Protein Serine-Threonine Kinases
keywords: Alphascreen; High-throughput Screening (HTS); Kinase Inhibitor; Leucine-rich Repeat Kinase 2 (LRRK2); Neurodegeneration; Park8; Protein Kinase; Recombinant Protein Expression