Differential Inhibition of LRRK2 in Parkinson's Disease Patient Blood by a G2019S Selective LRRK2 Inhibitor

Abstract

Background: A common genetic mutation that causes Parkinson's disease (PD) is the G2019S LRRK2 mutation. A precision medicine approach that selectively blocks only excess kinase activity of the mutant allele could yield a safe and effective treatment for G2019S LRRK2 PD.

Objective: To determine the activity of a G2019S mutant selective leucine-rich repeat kinase 2 (LRRK2) kinase inhibitor as compared to a nonselective inhibitor in blood of subjects with genetic and idiopathic PD on two LRRK2 biomarkers, pSer935 LRRK2 and pThr73 Rab10.

Methods: Blood was collected from 13 subjects with or without a G2019S LRRK2 mutation with PD and one healthy control. Peripheral blood mononuclear cells were treated ex vivo with a novel G2019S LRRK2 inhibitor (EB-42168) or the nonselective inhibitor MLi-2. Quantitative western immunoblot analyses were performed.

Results: EB-42168 was 100 times more selective for G2019S LRRK2 when compared to wild-type (WT) LRRK2. Concentrations that inhibited phosphorylation of pSer935 LRRK2 by 90% in homozygous G2019S LRRK2 patients, inhibited pSer935 LRRK2 by 36% in heterozygous patients, and by only 5% in patients carrying only the WT allele. Similar selectivity was seen for pThr73 Rab10. MLi-2 showed an equivalent level of inhibition across all genotypes.

Conclusions: These findings demonstrate that EB-42168, a G2019S LRRK2 selective inhibitor, lowers mutant G2019S LRRK2 phosphorylated biomarkers while simultaneously sparing WT LRRK2. Selective targeting of G2019S LRRK2 with a small molecule lays the foundation for a precision medicine treatment of G2019S LRRK2 PD. © 2021 ESCAPE Bio, Inc. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Keywords: G2019S LRRK2; Parkinson's disease; biomarker; peripheral blood mononuclear cells; pharmacodynamic.

© 2021 ESCAPE Bio, Inc. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Figures

FIG. 1

(A) The concentration of EB‐42168 that inhibited pSer935 LRRK2 by 90% in homozygous (HOM) G2019S LRRK2 PBMCs (172 nM) resulted in significantly less inhibition of pSer935 leucine‐rich repeat kinase 2 (LRRK2) in heterozygous (HET) G2019S LRRK2 PBMCs (36 ± 7.5%, P < 0.0001) and noncarriers (NC) peripheral blood mononuclear cells (PBMCs) (5 ± 3%, P < 0.0001). (B) The concentration of EB‐42168 that inhibited pThr73 Rab10 by 90% in HOM (582 nM) resulted in significantly less inhibition of pThr73 Rab10 in HET G2019S LRRK2 (44 ± 15%, P < 0.0001) and NC PBMCs (9 ± 12%, P < 0.0001). (C) The IC90 concentration of MLi‐2 for pSer935 LRRK2 inhibition in HOM G2019S LRRK2 PBMCs (10 nM) also inhibited the other genotypes by >90% (HET G2019S LRRK2 = 95 ± 2.7%, P = 0.047; NC = 92 ± 2.2%). (D) The MLi‐2 IC90 concentration for pT73 Rab10 (33 nM) resulted in a similarly high level of inhibition in HET G2019S LRRK2 (90 ± 3.6%) and NC (96 ± 2.9%). Bar height represents the average signal quantitated from immunoblots for pSer935 LRRK2 normalized to total LRRK2 relative to DMSO control (A and C) or pThr73 Rab10 normalized to total Rab10 relative to DMSO control (B and D). Error bars represent standard deviation (SD) of n = 3 HOM, 5 HET, and 5 NC of the G2019S LRRK2 variant. **P < 0.005, ***P < 0.0001.

FIG. 2

(A) EB‐42168 concentration response curves (range, 0.38 to 100 μM) for inhibition of pSer935 leucine‐rich repeat kinase 2 (LRRK2) are shown as the geometric mean for each genotype as indicated; correspondingly colored droplines show the aggregate IC50 values by genotype; error bars show geometric standard deviation (SD). (B) Similar representation for pThr73 Rab10. (C) Individual patient IC50 values for pSer935 LRRK2 ranged from 1579 to 2530 nM in G2019S LRRK2 NC (open green circles, orange fill indicates presence of L119P LRRK2 variant), 205 to 743 nM in heterozygous (HET) (open blue circles, turquoise and magenta fills indicate presence of additional HET and homozygous (HOM) GBA1 variants, respectively), and 15 to 27 nM in HOM G2019S LRRK2 (open red circles). (D) Individual patient IC50 values for pThr73 Rab10 ranged from 2022 to 9444 nM in G2019S LRRK2 NC, 174 to 2480 nM in HET, and 30 to 64 nM in HOM G2019S LRRK2 (colors of circles represent similar genotypes as in C).

FIG. 3

(A) MLi‐2 concentration response curves (range, 0.038 to 10 μM) for inhibition of pSer935 leucine‐rich repeat kinase 2 (LRRK2) are shown as the geometric mean for each genotype as indicated; correspondingly colored droplines show aggregate IC50 values by genotype; error bars show geometric standard deviation (SD). (B) Similar representation for pThr73 Rab10. (C) Individual patient IC50 values for pSer935 LRRK2 ranged from 0.8 to 1.7 nM in G2019S LRRK2 noncarriers (NC) (open green circles, orange fill indicates presence of L119P LRRK2 variant), 0.7 to 2.1 nM in heterozygous (HET) (open blue circles, turquoise and magenta fills indicate presence of additional HET and homozygous (HOM) GBA1 variants, respectively), and 1.4 to 1.8 nM in HOM G2019S LRRK2 (open red circles). (D) Individual patient IC50 values for pThr73 Rab10 ranged from 2.7 to 8.5 nM in G2019S LRRK2 NC, 3.1 to 4.9 nM in HET, and 2.5 to 4.0 nM in HOM G2019S LRRK2 (colors of circles represent similar genotypes as in C).