Li Tianxia, He Xinhua, Thomas Joseph M, Yang Dejun, Zhong Shijun, Xue Fengtian, Smith Wanli W
Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD, United States of America.
Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore, MD, United States of America; Beijing Institute of Pharmacology and Toxicology, Beijing, China.
PLoS One. 2015 Mar 27;10(3):e0122461. doi: 10.1371/journal.pone.0122461. eCollection 2015.
Leucine-rich repeat kinase-2 (LRRK2), a cytoplasmic protein containing both GTP binding and kinase activities, has emerged as a highly promising drug target for Parkinson's disease (PD). The majority of PD-linked mutations in LRRK2 dysregulate its GTP binding and kinase activities, which may contribute to neurodegeneration. While most known LRRK2 inhibitors are developed to target the kinase domain, we have recently identified the first LRRK2 GTP binding inhibitor, 68, which not only inhibits LRRK2 GTP binding and kinase activities with high potency in vitro, but also reduces neurodegeneration. However, the in vivo effects of 68 are low due to its limited brain penetration. To address this problem, we reported herein the design and synthesis of a novel analog of 68, FX2149, aimed at increasing the in vivo efficacy. Pharmacological characterization of FX2149 exhibited inhibition of LRRK2 GTP binding activity by ~90% at a concentration of 10 nM using in vitro assays. Furthermore, FX2149 protected against mutant LRRK2-induced neurodegeneration in SH-SY5Y cells at 50-200 nM concentrations. Importantly, FX2149 at 10 mg/kg (i.p.) showed significant brain inhibition efficacy equivalent to that of 68 at 20 mg/kg (i.p.), determined by mouse brain LRRK2 GTP binding and phosphorylation assays. Furthermore, FX2149 at 10 mg/kg (i.p.) attenuated lipopolysaccharide (LPS)-induced microglia activation and LRRK2 upregulation in a mouse neuroinflammation model comparable to 68 at 20 mg/kg (i.p.). Our results highlight a novel GTP binding inhibitor with better brain efficacy, which represents a new lead compound for further understanding PD pathogenesis and therapeutic studies.
富含亮氨酸重复激酶2（LRRK2）是一种兼具GTP结合和激酶活性的胞质蛋白，已成为帕金森病（PD）极具前景的药物靶点。LRRK2中大多数与PD相关的突变会使其GTP结合和激酶活性失调，这可能导致神经退行性变。虽然大多数已知的LRRK2抑制剂是针对激酶结构域开发的，但我们最近鉴定出了首个LRRK2 GTP结合抑制剂68，它不仅在体外能高效抑制LRRK2的GTP结合和激酶活性，还能减轻神经退行性变。然而，由于68的脑渗透性有限，其体内效果较差。为了解决这个问题，我们在此报告了一种新型的68类似物FX2149的设计与合成，旨在提高体内疗效。FX2149的药理学特性显示，在体外实验中，浓度为10 nM时对LRRK2 GTP结合活性的抑制率约为90%。此外，FX2149在50 - 200 nM浓度下可保护SH - SY5Y细胞免受突变型LRRK2诱导的神经退行性变。重要的是，通过小鼠脑LRRK2 GTP结合和磷酸化实验确定，10 mg/kg（腹腔注射）的FX2149显示出与20 mg/kg（腹腔注射）的68相当的显著脑抑制效果。此外，在小鼠神经炎症模型中，10 mg/kg（腹腔注射）的FX2149可减轻脂多糖（LPS）诱导的小胶质细胞活化和LRRK2上调，效果与20 mg/kg（腹腔注射）的68相当。我们的结果突出了一种具有更好脑内疗效的新型GTP结合抑制剂，它代表了一种新的先导化合物，有助于进一步理解PD的发病机制并开展治疗研究。
