G2019S selective LRRK2 kinase inhibitor abrogates mitochondrial DNA damage

Abstract

Pathogenic mutations in LRRK2 cause Parkinson's disease (PD). The G2019S variant is the most common, which results in abnormally high kinase activity. Compounds that target LRRK2 kinase activity are currently being developed and tested in clinical trials. We recently found that G2019S LRRK2 causes mitochondrial DNA (mtDNA) damage and treatment with multiple classes of LRRK2 kinase inhibitors at concentrations associated with dephosphorylation of LRRK2 reversed mtDNA damage to healthy control levels. Because maintaining the normal function of LRRK2 in heterozygous G2019S LRRK2 carriers while specifically targeting the G2019S LRRK2 activity could have an advantageous safety profile, we explored the efficacy of a G2019S mutant selective LRRK2 inhibitor to reverse mtDNA damage in G2019S LRRK2 models and patient cells relative to non-selective LRRK2 inhibitors. Potency of LRRK2 kinase inhibition by EB-42168, a G2019S mutant LRRK2 kinase inhibitor, and MLi-2, a nonselective inhibitor, was determined by measuring phosphorylation of LRRK2 at Ser935 and/or Ser1292 using quantitative western immunoblot analysis. The Mito DNADX assay, a novel system that allows for the accurate real-time quantification of mtDNA damage in a 96-well platform, was performed in parallel. We confirmed that EB-42168 selectively inhibits LRRK2 phosphorylation on G2019S LRRK2 relative to wild-type LRRK2. On the other hand, MLi-2 was equipotent for wild-type and G2019S LRRK2. Acute treatment with EB-42168 inhibited LRRK2 phosphorylation and also restored mtDNA damage to healthy control levels. Precision medicine is a common approach in modern day cancer research that is not yet routinely applied to neurodegenerative diseases. Abrogation of mtDNA damage with mutant selective tool inhibitor EB-42168 demonstrates the promise of a precision medicine approach for LRRK2 G2019S PD. Levels of mtDNA damage may serve as a potential pharmacodynamic biomarker of altered kinase activity that could be useful for small molecule development and clinical trials.

A blood-based marker of mitochondrial DNA damage in Parkinson’s disease

“…LRRK2 G2019S PD patient-derived LCLs also demonstrated increased mtDNA damage relative to age-matched controls (Fig. 2A), similar to previously published findings (15,20,37). mtDNA copy number was not different between idiopathic PD patientderived LCLs compared with age-matched healthy controls (Fig.…”

“…Many promising compounds or treatments targeting mitochondrial impairment in PD are in the pipeline (66,67). Yet, to date, a functional mitochondrial blood marker that could be used to establish a PD mitochondrial subtype that reflects this underlying pathophysiology has not been developed, thereby allowing a precision medicine-based approach to PD clinical trials, which may yield a higher chance of success (37,68). Our data provide evidence to support inclusion of mtDNA damage as a blood-based candidate marker for PD in future clinical trials.…”

“…We previously demonstrated that LRRK2 G2019S in vitro neuronal models and cells derived from patients with PD display increased mtDNA damage (15,16,20,37). To investigate whether LRRK2 G2019S causes mtDNA damage in vivo, the ventral midbrain was dissected from wild-type and Lrrk2 G2019S knock-in (GKI) heterozygous or homozygous mice (44), the DNA was purified, and the Mito DNA DX assay was performed.…”

“…Moreover, LRRK2 G2019S increased mtDNA damage in induced pluripotent stem cell (iPSC)derived neurons from patients with PD, which was reversed by gene correction (16). In addition, LRRK2 kinase inhibition (with three structurally distinct inhibitors and an LRRK2 G2019S mutation selective compound) achieved full reversal of mtDNA damage in LRRK2 G2019S PD patient-derived cells; hence, mtDNA damage is a sensitive surrogate measure of altered LRRK2 kinase activity (15,20,37). However, even in patients with PD-linked LRRK2 mutations, there are heterogeneous clinical and neuropathological manifestations that may reflect multiple disease pathways (5,38,39).…”

Inactive S. aureus Cas9 downregulates alpha-synuclein and reduces mtDNA damage and oxidative stress levels in human stem cell model of Parkinson’s disease

“…Previously, we reported that lymphoblastoid cell lines and iPSC-derived neuronal cultures from LRRK2 p.G2019S mutation carriers have increased mtDNA damage 64 , 65 . When we genetically corrected the LRRK2 mutation or treated the cultures with a LRRK2 kinase inhibitor mtDNA damage was no longer detectable.…”