The G2019S mutation in LRRK2 imparts resiliency to kinase inhibition

Abstract

The G2019S mutation in LRRK2 is one of the most common known genetic causes of neurodegeneration and Parkinson disease (PD). LRRK2 mutations are thought to enhance LRRK2 kinase activity. Efficacious small molecule LRRK2 kinase inhibitors with favorable drug properties have recently been developed for pre-clinical studies in rodent models, and inhibitors have advanced to safety trials in humans. Rats that express human G2019S-LRRK2 protein and G2019S-LRRK2 knock-in mice provide newly characterized models to better understand the ostensible target for inhibitors. Herein, we explore the relationships between LRRK2 kinase inhibition in the brain and the periphery to establish the link between LRRK2 kinase activity and protein stability, induction of lysosomal defects in kidney and lung, and how G2019S-LRRK2 expression impacts these phenotypes. Using a novel ultra-sensitive scalable assay based on protein capillary electrophoresis with LRRK2 kinase inhibitors included in-diet, G2019S-LRRK2 protein was resilient to inhibition compared to wild-type (WT)-LRRK2 protein, particularly in the brain. Whereas WT-LRRK2 kinase activity could be completed blocked without lowering LRRK2 protein levels, higher inhibitor concentrations were necessary to fully reduce G2019S-LRRK2 activity. G2019S-LRRK2 expression afforded robust protection from inhibitor-induced kidney lysosomal defects, suggesting a gain-of-function for the mutation in this phenotype. In rodents treated with inhibitors, parallel measurements of phospho-Rab10 revealed a poor correlation to phospho-LRRK2, likely due to cells that express Rab10 but poorly express LRRK2 in heterogenous tissues and cell isolates. In summary, our results highlight several challenges associated with the inhibition of the G2019S-LRRK2 kinase that might be considered in initial clinical efforts.

Neurodegeneration caused by LRRK2-G2019S requires Rab10 in select dopaminergic neurons

“…The existence of Rab10 in the tyrosine hydroxylase positive neurons controlling vision and proboscis movement argues that LRRK2 might indeed phosphorylate Rab10 directly. Thus our in vivo results both support the in vitro (biochemical and cell culture) data in which LRRK2 directly phosphorylates Rab10 (3)(4)(5)(6)(7)(8), but also suggest a separate pathway for LRRK2-G2019S action. Further, while Rab3 is also phosphorylated by LRRK2 in vitro (3), it did not synergise with LRRK2-G2019S in the visual assay, and was not detected in the MC neurons .…”

“…A number of studies highlighted a diverse range of >30 proteins that might be phosphorylated by LRRK2, suggesting it is a generalised kinase (2). However, several research teams have recently reported that LRRK2 is a more specific kinase, phosphorylating Rab GTPases (3)(4)(5)(6)(7)(8). However, it is not clear which of the more than 60 Rabs are actually phosphorylated in vivo.…”

“…Biochemically, about 8 seem to be directly phosphorylated by LRRK2 [ Rabs 3,5,8,10,12,29,35 and 43 (3)]. In mammals, analysis of the role of the Rabs is complex because individual Rabs may have similar, or even compensatory functions, which may differ by tissue (8,13). The situation is simpler in the fly, because there are fewer Rabs -only 23 mammalian orthologs.…”

Dopaminergic Neurodegeneration Induced by Parkinson’s Disease-Linked G2019S LRRK2 is Dependent on Kinase and GTPase Activity

“…S2A). These data suggest that pT73-Rab10 does not serve as a sensitive readout of LRRK2 kinase activity in the brain of this rat model, similar to prior reports in rodent brain (41). Instead, LRRK2 kinase activity was monitored by autophosphorylation at Ser1292 in brain tissue.…”

“…Third, the genetic analyses of LRRK2 kinase activity were complemented by pharmacological studies with PF-360, a potent and selective generation "3" LRRK2 inhibitor. PF-360 is highly potent with an in vivo IC50 of ~3 nM for LRRK2, and exhibits an improved selectivity profile, oral bioavailability and brain permeability compared to earlier generations of LRRK2 inhibitors (41,47). We demonstrate full inhibition of endogenous LRRK2 kinase activity in peripheral tissues and brain of our rat model with chronic in-diet dosing of PF-360 at 175 mg/kg chow.…”