The Rational Design of LRRK2 Inhibitors for Parkinson's Disease

Abstract

Parkinson’s disease is a chronic neurodegenerative disorder that affects 1-2% of the world’s population over the age of 65. Current treatments that reduce the severity of symptoms cause numerous side-effects and lose efficacy over the course of disease progression. Leucine-rich repeat kinase 2 (LRRK2) is a novel drug target for the development of disease modifying therapeutics for Parkinson’s disease. LRRK2 mutants have elevated kinase activity and, as such, chemical inhibitors have therapeutic potential. The physiological benefits that arise from chemically inhibiting LRRK2 have been proven through the use of generic kinase inhibitors and more recently the selective benzodiazepinone compound LRRK2IN1. LRRK2IN1 is a highly potent inhibitor, exhibiting a half-maximal inhibitory concentration (IC50) of 9 nM in cellular assays. However, LRRK2IN1 is not biologically available in the brain because it has poor physicochemical and pharmacokinetic properties. In previous research we rationally designed a LRRK2IN1 analogue (IN1_G) that was predicted to have improved metabolic stability and blood-brain barrier permeability. Preliminary biological analysis indicated that both LRRK2IN1 and IN1_G potently inhibited LRRK2-associated neuro-inflammation in vitro. However, the high molecular weight, topological polar surface area and lipophilicity of LRRK2IN1 and IN1_G were predicted to be incompatible with functional activity in vivo. Structural modifications were thus required to optimise compounds as neuro-protective treatments for Parkinson’s disease. Biological evaluation of the structural components of LRRK2IN1 and IN1_G indicated that the aniline-bipiperidine 1 motif was a moderately potent inhibitor of neuro-inflammation, whilst the tricyclic diazepinone motif IN1_H had no anti-inflammatory efficacy. In the current research a series of truncated LRRK2IN1/IN1_G analogues were rationally designed to determine if the diazepinone motif could be replaced with low molecular weight bioisosteres without affecting functional potency. In silico property predictions and scoring functions were used to guide the design of truncated analogues. The Schrödinger suite programs LigPrep, QikProp and Marvin were used to predict the physicochemical and pharmacokinetic properties of analogues. The recently described central nervous system multi-parameter optimisation score was used to select analogues that were likely to possess favourable pharmacokinetic and safety profiles. Analogues were docked in a homology model of the LRRK2 kinase domain that was developed in our previous research. Analogues that conformed to the binding mode of known kinase inhibitors and were predicted by GLIDE to bind to the LRRK2 homology model with high affinity were prioritised for synthesis. Twenty analogues were synthesised using methods known in the literature. The substrate scope of Buchwald-Hartwig chemistry was explored. Novel “all-water” chemistry was employed to synthesise N-benzyl aniline analogues. Methodology recently developed in our group was used to synthesise diazepine and oxazepine analogues of IN1_H. Analogues were assessed for anti-inflammatory efficacy in two cell-based assays. Four truncated analogues — 25, 30, 31 and 39 — had equivalent functional efficacy to LRRK2IN1/IN1_G, inhibiting the secretion of pro-inflammatory cytokines from stimulated primary human microglia by more than 43% at concentrations of 1 µM. These analogues were all predicted to have improved pharmacokinetic properties compared to LRRK2IN1/IN1_G and are excellent candidates for further development. The synthetic intermediate 63 was found to be highly potent (57% inhibition of cytokine secretion at 1 µM), which has suggested options for the development of future analogues. The potency of analogues 25, 30, 31 and 39 indicated that the tricyclic diazepinone motif was not essential for anti-inflammatory efficacy. Analogues from this research have been used to identify a role for LRRK2 in the pathology of severe brain cancer glioblastoma. Although their mechanisms of action have not yet been determined, it is clear that analogues developed in this research have potential applications in the treatment of numerous disorders driven by an inflammatory microenvironment. Further optimisation of the analogues developed in this research will provide the first disease-modifying therapeutics for Parkinson’s disease.