Screening for Novel LRRK2 Inhibitors Using a High-Throughput TR-FRET Cellular Assay for LRRK2 Ser935 Phosphorylation

Spencer B Hermanson,Kun Bi,Jing Zhao,Coby B Carlson,Kurt W Vogel,R Jeremy Nichols,Steven M Riddle,Philipp J Kahle

doi:10.1371/journal.pone.0043580

Abstract

BackgroundMutations in the leucine-rich repeat kinase-2 (LRRK2) have been linked to Parkinson’s disease. Recent studies show that inhibition of LRRK2 kinase activity decreased the level of phosphorylation at its own Ser910 and Ser935, indicating that these sites are prime targets for cellular readouts of LRRK2 inhibition.Methodology/Principal FindingsUsing Time-Resolved Förster Resonance Energy Transfer (TR-FRET) technology, we developed a high-throughput cellular assay for monitoring LRRK2 phosphorylation at Ser935. LRRK2-Green Fluorescence Protein (GFP) fusions were expressed in cells via BacMam. Phosphorylation at Ser935 in these cells is detected using a terbium labeled anti-phospho-Ser935 antibody that generates a TR-FRET signal between terbium and GFP. LRRK2 wild-type and G2019S are constitutively phosphorylated at Ser935 in cells as measured by TR-FRET. The phosphorylation level is reduced for the R1441C mutant and little could be detected for the kinase-dead mutant D1994A. The TR-FRET cellular assay was further validated using reported LRRK2 inhibitors including LRRK2-IN-1 and our results confirmed that inhibition of LRRK2 can reduce the phosphorylation level at Ser935. To demonstrate the utility of this assay for screening, we profiled a small library of 1120 compounds. Three known LRRK2 inhibitors were identified and 16 hits were followed up in the TR-FRET and a cytotoxicity assay. Interestingly, out of the top 16 hits, five are known inhibitors of IκB phosphorylation, two CHK1 and two CDC25 inhibitors. Thirteen hits were further tested in a biochemical LRRK2 kinase activity assay and Western blot analysis for their effects on the phosphorylation of Ser910, Ser935, Ser955 and Ser973.Conclusions/SignificanceWe developed a TR-FRET cellular assay for LRRK2 Ser935 phosphorylation that can be applied to the screening for LRRK2 inhibitors. We report for the first time that several compounds such as IKK16, CHK1 inhibitors and GW441756 can inhibit LRRK2 Ser935 phosphorylation in cells and LRRK2 kinase activity in vitro.

Highlights

Parkinson’s disease (PD) is a progressive neurodegenerative disorder that affects 1% of people over age 60 and more than 5 million people worldwide
Consistent with previous reports on the localization of N-terminal tagged Green Fluorescence Protein (GFP)-leucine-rich repeat kinase-2 (LRRK2) [14,21,28], the Cterminal tagged LRRK2 wild-type, G2019S and D1994A mutants are expressed in the cytoplasm mostly in a diffused pattern with occasional aggregates observed in a small number of cells (Figure 1A)
We evaluated the phosphorylation of LRRK2 expressed in U-2 OS via BacMam at Ser910, Ser935, Ser955 and Ser973 and found that LRRK2 was modified at these sites and that they were responsive to LRRK2-IN-1 treatment as reported previously in other systems [22] (Figure S1)

Summary

Introduction

Parkinson’s disease (PD) is a progressive neurodegenerative disorder that affects 1% of people over age 60 and more than 5 million people worldwide. Mutations in leucine-rich repeat kinase 2 (LRRK2) have been linked to autosomal familial and sporadic PD [1,2] and LRRK2 has recently been pursued as a potential therapeutic target for PD [3,4,5]. The most frequent PD associated LRRK2 mutation encodes a glycine-to-serine substitution at residue 2019 (G2019S), within the conserved ‘‘DFG’’ motif of subdomain VII in the activation loop of the kinase domain. This mutation has been shown to increase kinase activity in several reports [5,8]. Recent studies show that inhibition of LRRK2 kinase activity decreased the level of phosphorylation at its own Ser910 and Ser935, indicating that these sites are prime targets for cellular readouts of LRRK2 inhibition

Methods

Results

Conclusion