LRRK2-specific DARPins as tools to investigate kinase activation and regulation in Parkinson's Disease

About This Grant

PROJECT SUMMARY Parkinson’s disease (PD) is the second fastest growing aging-related neurodegenerative disease in the world. One of the most commonly mutated genes in PD is Leucine Rich Repeat Kinase 2 (LRRK2). LRRK2 mutations that are linked to PD result in hyperactive kinase activity, but unmutated LRRK2 can also be hyperactive in idiopathic forms of PD, making its kinase the most actionable target for PD therapeutics. Thus, it is critical to understand how LRRK2’s kinase activity is regulated, something that remains poorly understood. LRRK2 encodes a multi-domain protein, whose N-terminal half is comprised of three protein-protein interaction repeat domains that form an arm-like structure that drapes across its kinase and GTPase domains, to dock at the C- terminal WD40 domain. When these arm-like repeats are docked on the WD40 domain, LRRK2’s kinase is autoinhibited. Here, I will use LRRK2-specific Designed Ankyrin Repeat Proteins (DARPins), which I was involved in developing, to determine how LRRK2’s kinase activity is regulated. We identified three DARPins, E11, C12 and G10, which bind tightly to LRRK2 at partially overlapping sites on its WD40 domain, yet have distinct effects on LRRK2’s kinase activity. Our data suggest that DARPin C12 activates LRRK2’s kinase activity, while DARPin E11 and G10 inhibit it. Our preliminary cryo-EM data support the hypothesis that DARPin C12 relieves autoinhibition by blocking binding of LRRK2’s repeat domains to the WD40 domain. My preliminary data also supports the hypothesis that some PD alleles outside of the kinase active site effectively act like C12: they activate the kinase by relieving autoinhibition. Based on the binding sites of E11 and C12, we hypothesize that E11 may be blocking binding of an activating factor, while C12 maybe stabilizing the autoinhibited conformation of LRRK2. Thus, DARPin E11 is an excellent tool to identify novel activators of LRRK2, as well as to screen existing small molecule libraires to identify allosteric inhibitors of LRRK2’s kinase. I expect to determine how mutations in LRRK2 that are distal to the kinase domain increase kinase activity. In addition, my goal to identify novel activators of LRRK2’s kinase and allosteric inhibitors of its kinase will lead to new therapeutic strategies for targeting LRRK2 for the treatment of PD.