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 non-selective inhibitor, was determined by measuring phosphorylation of LRRK2 at Ser935 and/or Ser1292 using quantitative western immunoblot analysis. The Mito DNA_DX assay, which 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. We further investigated the relationship between LRRK2 kinase activity, mtDNA damage and mitophagy. Levels of mtDNA damage caused by G2019S LRRK2 were fully re-established within 2 h of a LRRK2 inhibitor wash out and recovery experiment, indicating the mtDNA damage phenotype is highly dynamic. G2019S LRRK2 mitophagy defects were not alleviated with LRRK2 kinase inhibition, suggesting that mitophagy is not mechanistically regulating LRRK2 kinase-mediated reversal of mtDNA damage in this acute timeframe. Abrogation of mtDNA damage with the mutant selective tool inhibitor EB-42168 demonstrates the potential 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.

LRRK2的致病性突变会导致帕金森病 (PD)。G2019S 变体是最常见的，它会导致异常高的激酶活性。目前正在开发针对 LRRK2 激酶活性的化合物并进行临床试验。我们最近发现 G2019S LRRK2 会导致线粒体 DNA (mtDNA) 损伤，并且使用与 LRRK2 去磷酸化相关浓度的多种类 LRRK2 激酶抑制剂进行治疗，可将 mtDNA 损伤逆转至健康对照水平。由于在杂合子 G2019S LRRK2携带者中维持 LRRK2 的正常功能，同时专门针对 G2019S LRRK2 活性可能具有有利的安全性，因此我们探索了 G2019S 突变型选择性 LRRK2 抑制剂逆转 G2019S LRRK2 模型和患者细胞中 mtDNA 损伤的功效。非选择性 LRRK2 抑制剂。通过使用定量蛋白质免疫印迹分析测量 LRRK2 Ser935 和/或 Ser1292 的磷酸化，确定 EB-42168（一种 G2019S 突变 LRRK2 激酶抑制剂）和 MLi-2（一种非选择性抑制剂）抑制 LRRK2 激酶的效力。并行进行Mito DNA _DX测定，可在 96 孔平台中准确实时定量 mtDNA 损伤。我们证实，相对于野生型 LRRK2，EB-42168 选择性抑制 G2019S LRRK2 上的 LRRK2 磷酸化。另一方面，MLi-2 对于野生型和 G2019S LRRK2 是等效的。EB-42168 的急性治疗可抑制 LRRK2 磷酸化，并将 mtDNA 损伤恢复至健康对照水平。我们进一步研究了 LRRK2 激酶活性、mtDNA 损伤和线粒体自噬之间的关系。G2019S LRRK2 引起的 mtDNA 损伤水平在 LRRK2 抑制剂清洗和恢复实验后 2 小时内完全重建，表明 mtDNA 损伤表型是高度动态的。G2019S LRRK2 线粒体自噬缺陷并未因 LRRK2 激酶抑制而得到缓解，这表明在这一急性时间范围内，线粒体自噬并不能从机制上调节 LRRK2 激酶介导的 mtDNA 损伤逆转。使用突变选择性工具抑制剂 EB-42168 消除 mtDNA 损伤，证明了针对 LRRK2 G2019S PD 的精准医学方法的潜力。mtDNA 损伤水平可能作为激酶活性改变的潜在药效生物标志物，可用于小分子开发和临床试验。