Objective/Rationale:
Mutations have been found in two places in the LRRK2 protein in patients with familial Parkinson’s disease. These LRRK2 mutations cause increased LRRK2 protein activity, leading to neurotoxicity in these patients, so reducing such activity would be beneficial. One of these two mutations occurs in an area called the GTPase domain of the LRRK2 protein, and increased LRRK2 activity can be triggered by GTP binding to the GTPase domain. Therefore, we propose that interfering with the ability of GTP to bind with GTPase will inhibit any increase in LRRK2 activity that might be caused by the mutation and aim to develop specific inhibitors of LRRK2 GTPase activation.
Project Description:
We aim to develop a method for identifying compounds that are more likely to bind with LRRK GTPase than GTP, which would block GTP from binding to it. Using this method, we will screen a library of diverse small molecules to identify potential inhibitors of LRRK2 GTPase activation. We will then validate their activity and specificity in vitro using biochemical assays.
Relevance to Diagnosis and Treatment of Parkinson’s Disease:
LRRK2 activity appears to play a key role in the pathogenesis of Parkinson’s disease. We aim to develop compounds that inhibit LRRK2 GTPase activation, which is necessary for all known LRRK2 protein functions. If we are successful, we will test the efficacy and safety of these compounds as a new strategy for the treatment of Parkinson’s disease.
Anticipated Outcome:
We anticipate the discovery and biochemical validation of small molecule(s) that specifically inhibit LRRK2 GTPase activation in vitro.
Final Outcome
After testing multiple sources of LRRK2, we found that only LRRK2 isolated from mammalian cells was associated with significant GTPase activity. We succeeded in purifying recombinant LRRK2 protein from mammalian cells in sufficient quantities for high-throughput screening. Using the isolated LRRK2 protein, we screened about 140,000 compounds using a robust assay for GDP production by LRRK2. We identified multiple hit compounds that reproducibly inhibited GDP production by LRRK2 and not other tested small GTPase. Currently, we are evaluating the effects of the compounds on LRRK2 kinase activity and on cell-based assays of neurodegeneration. Further experiments will be conducted to elucidate the mechanism of action by which the hit compounds LRRK2 GTPase activity.