Promising Outcomes of Original Grant:
Our original goal was to (1) establish electron microscopy (EM)-based dimeric structural assays for the LRRK2 protein and (2) develop high-throughput kinase assays and screen for small chemical inhibitors of LRRK2 kinase activity (including the FDA-approved library). Our study has achieved the main goals in establishing an EM map of LRRK2 protein that serves as a structural framework to understand LRRK2 activity regulation. In addition, we have performed screening of two chemical libraries (>25,000) and identified 4 compounds (in the sub-micromolar range) as potential LRRK2 kinase inhibitors (FDA-approved compounds). These inhibitors are now being evaluated in cell-based assays and various LRRK2 pre-clinical models.
Objectives for Supplemental Investigation:
The structure-function relationship of LRRK2 kinase is poorly understood. Uncertainty of what constitutes brain-specific LRRK2 complexes (i.e., what type of post-translational modifications and adaptor proteins modulate the activity of pathogenic LRRK2 complexes) and a lack of structural information on these complexes impede progress in the understanding of LRRK2; this in turn impedes development of effective therapeutic strategies for Parkinson's disease. The goal of our current application is to expand on the original project by performing three-dimensional reconstruction of brain LRRK2 protein complexes by electron cryo-microscopy (cryo-EM, as well as determining the effect of brain-specific post-translational modifications (including phosphorylations and other types of potential modifications) and adaptor protein binding in modulating dimeric LRRK2 structure and kinase/GTPase activity.
Importance of This Research for the Development of a New PD Therapy:
We anticipate these studies will lead to (I) an improved resolution of our current structure of LRRK2, and (II) insight into the composition and chemical modifications in brain-specific LRRK2 that modulate structure and enzymatic activity of LRRK2. These results are expected to provide enhanced structural information to evaluate small chemicals of LRRK2 inhibitors in regulating LRRK2 kinase/GTPase activity and better understand their potential in the development of drugs to treat Parkinson’s disease.