Abstract
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.
Highlights
Parkinson’s disease (PD) is a progressive neurodegenerative disorder, affecting 2% of the population over the age of 60 [1,2]
Given that 68 potently inhibits Leucine-rich repeat kinase-2 (LRRK2) GTP binding and kinase activity in vitro [13], we kept its scaffold structure to retain the inhibition of GTP binding and kinase activity
FX2149 at 100 nM concentration significantly reduced G2019S-LRRK2 phosphorylation at residues S935 and S2032 by ~90% (Fig. 3A and 3B). We further validated these results by in vitro kinase assays showing the similar inhibition of G2019S-LRRK2 kinase activity by FX2149 (Fig. 3C). These findings indicated that FX2149 (100 nM) reduced LRRK2 kinase activity similar to that of 68 at 10 nM concentration [13]
Summary
Parkinson’s disease (PD) is a progressive neurodegenerative disorder, affecting 2% of the population over the age of 60 [1,2]. PD patients display a loss of dopaminergic neurons in the substantia nigra and the presence of Lewy bodies in their brains [1,2]. LRRK2 GTP-Binding Inhibitor and Parkinson's Disease pharmacotherapy for PD patients is limited to symptomatic treatment, which only temporarily reduces motor symptoms but does not prevent neurodegeneration. There are no disease modifying drugs to prevent dopaminergic neuron loss and abnormal protein deposition in the brains. There is a strong demand for neuroprotective therapies to prevent or attenuate dopaminergic neuron degeneration
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