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Updated: Apr 23, 2026
Assaying the Kinase Activity of LRRK2 in vitro
Published on: January 18, 2012
Unique functional and structural properties of the LRRK2 protein ATP-binding pocket
Zhiyong Liu1, Robert A Galemmo2, Kyle B Fraser3
1From 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.
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.
Insights
Researchers developed novel assays to identify selective LRRK2 kinase inhibitors for Parkinson disease. A probe inhibitor, SRI-29132, shows promise in cellular models despite metabolic challenges.
Area of Science:
- Biochemistry
- Neuroscience
- Drug Discovery
Background:
- Pathogenic mutations in the Leucine-rich repeat kinase 2 (LRRK2) gene are a common cause of late-onset Parkinson disease.
- The G2019S mutation enhances LRRK2 kinase activity and is frequently observed in Parkinson disease patients.
- LRRK2 exhibits unique cis-autophosphorylation of its GTPase domain, a process not fully understood due to structural data limitations.
Purpose of the Study:
- To develop novel high-throughput assays to probe the LRRK2 ATP-binding pocket and identify selective kinase inhibitors.
- To characterize structure-activity relationships of LRRK2 inhibitors, differentiating between cis-autophosphorylation and trans-peptide phosphorylation.
- To identify and validate a potent and selective LRRK2 inhibitor for potential therapeutic applications in Parkinson disease.
Main Methods:
- Utilized novel high-throughput assays to measure both cis-autophosphorylation and trans-peptide phosphorylation of LRRK2.
- Screened hundreds of commercially available kinase inhibitors for LRRK2 activity selectivity.
- Employed docking models and structure-activity relationship analyses to guide inhibitor design and selection.
- Engineered an ameba LRRK2 homolog to mimic human LRRK2 ATP-binding pocket characteristics for structural studies.
Main Results:
- Identified numerous LRRK2 kinase inhibitors with varying selectivity profiles for cis-autophosphorylation versus trans-peptide phosphorylation, and for wild-type versus G2019S mutant LRRK2.
- Discovered and validated SRI-29132 as a highly selective LRRK2 kinase inhibitor.
- SRI-29132 demonstrated efficacy in attenuating proinflammatory responses in macrophages and rescuing neurite retraction in neurons, with good potency and blood-brain barrier penetration.
- Observed rapid first-pass metabolism for SRI-29132, indicating a limitation for in vivo efficacy.
Conclusions:
- The developed high-throughput assays provide a valuable resource for discovering LRRK2 small molecule inhibitors.
- SRI-29132 represents a promising chemical probe for LRRK2 research, despite its metabolic liabilities.
- The study demonstrates a viable approach for functional interrogation of ATP-binding pockets when traditional structural methods are challenging.
- Findings offer insights into the unique structural configuration of the LRRK2 ATP-binding pocket and guide future inhibitor development for Parkinson disease.
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