ABSTRACT: Targeting LRRK2 for Drug Discovery

Mutations in Leucine Rich repeat Kinase 2 (LRRK2) lead to a form of autosomal dominant Parkinson’s Disease (PD) with symptoms and pathological hallmarks that are similar to sporadic forms of the disorder.  A number of these mutations result in increased kinase activity of the protein, and it is hypothesized that inhibition of LRRK2’s kinase activity may slow or halt progression of this disease.  Hence, our objective is to identify LRRK2 kinase inhibitors for development as disease modifying therapies for PD patients. Furthermore, these studies will further elucidate the role played by LRRK2 in cells, and highlight intracellular pathways altered during the progression of PD.

To characterize the kinase activity of LRRK2, understand its enzymological properties, and develop assays suitable for compound screening, LRRK2 protein was purified using baculovirus expression systems.  LRRK2 was found to robustly phosphorylate a surrogate peptide substrate, LRRKtide, and also leads to a slow autophosphorylation of LRRK2.  One of the most prevalent human LRRK2 mutations, G2019S, was found to significantly increase the rate of both autophosphorylation and LRRKtide phosphorylation compared to wild-type. Furthermore, G2019S LRRK2 demonstrates a 2-fold lower binding affinity for ATP compared to wild-type LRRK2.  

In preparation for HTS we developed a Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET)-based assay (LanthaScreen^TM).  An analysis of five clinically observed LRRK2 mutations using either radiometric or Lanthascreen^TM assays revealed that the G2019S mutation results in the greatest enhancement of kinase activity using LRRKtide as substrate.  To develop secondary assays allowing the prioritization of hits obtained using in vitro assays, we have generated inducible stable cell lines expressing wild-type and mutant LRRK2, and can demonstrate that LRRK2 immunoprecipitated from these cells retains kinase activity. 

These cell-free and cell-based assays and reagents are also being used to identify relevant LRRK2 substrates and pathways in combination with peptide positional motif scanning and Protoarray^TM assays.  Initial results are highlighting preferred phosphorylation motifs and hence identifying putative cellular substrates.  Together, these studies facilitate the identification of substrates phosphorylated by human mutations of LRRK2, the identification of pathways modulated by this kinase, and the identification of selective LRRK2 kinase inhibitors for development as disease modifying therapies for the treatment of PD.

Steven P. Braithwaite¹, Michael Monaghan¹, Robert Martone¹, Vasanti S. Anand¹, Andres Hurtado-Lorenzo¹, Pooja P. Pungaliya², Brian Bates², Warren D. Hirst¹, Peter H. Reinhart¹

¹Wyeth Research, Princeton, NJ 08543  ²Wyeth Research, Cambridge, MA 02140