Unique Functional and Structural Properties of the LRRK2 ATP-Binding Pocket [Molecular Bases of Disease]

September 16th, 2014 by Liu, Z., Galemmo, R. A., Fraser, K. B., Moehle, M. S., Sen, S., Volpicelli-Daley, L. A., DeLucas, L. J., Ross, L. J., Valiyaveettil, J., Moukha-Chafiq, O., Pathak, A. K., Ananthan, S., Kezar, H., White, E. L., Gupta, V., Maddry, J. A., Suto, M. J., W

Pathogenic mutations in the LRRK2 gene can cause late-onset Parkinson's 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. As high-resolution structures of the human LRRK2 kinase domain are not available, we used novel high-throughput assays that measured both cis-auto-phosphorylation 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-auto-phosphorylation 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 pro-inflammatory responses in macrophages and rescuing neurite retraction phenotypes in neurons. Further, 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. While the human LRRK2 kinase domain is unstable and insoluble, we demonstrate that the LRRK2-homolog from amoeba 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.