Targeting GTPases in Parkinson's disease: comparison to the historic path of kinase drug discovery and perspectives

Abstract

Neurological diseases have placed heavy social and financial burdens on modern society. As the life expectancy of humans is extended, neurological diseases, such as Parkinson's disease, have become increasingly common among senior populations. Although the enigmas of Parkinson's diseases await resolution, more vivid pictures on the cause, progression, and control of the illness are emerging after years of research. On the molecular level, GTPases are implicated in the etiology of Parkinson's disease and are rational pharmaceutical targets for their control. However, targeting individual GTPases, which belong to a superfamily of proteins containing multiple members with a conserved guanine nucleotide binding domain, has proven to be challenging. In contrast, pharmaceutical pursuit of inhibition of kinases, which constitute another superfamily of proteins with more than 500 members, has been fairly successful. We reviewed the breakthroughs in the history of kinase drug discovery to provide guidance for the GTPase field. We summarize recent progress made in the regulation of GTPase activity. We also present an efficient and cost effective approach to drug screening, which uses multiplex flow cytometry and mixture-based positional scanning libraries. These methods allow simultaneous measurements of both the activity and the selectivity of the screened library. Several GTPase activator clusters were identified which showed selectivity against different GTPase subfamilies. While the clusters need to be further deconvoluted to identify individual active compounds, the method described here and the structure information gathered create a foundation for further developments to build upon.

Keywords: GTPase; Parkinson’s; drug; kinase; multiplex.

FIGURE 1

GTPase domain core structure. (A) Human Ras small GTPase with GTP bound. Six stranded β-sheets are surrounded by five α-helices (PDB: 121p). (B) Dictyostelium dynamin large GTPase with GDP bound. Eight stranded β-sheets are surrounded by nine α-helices (PDB: 1jwy). Helices are shown in red, β-sheets in blue, switch region in white, and the guanine nucleotide in yellow. Though the nucleotide identities are different for the two structures, the compact and globular arrangement is conserved. Both Tiff files were taken from www.endocytosis.org/Dynamin/GTPbinding-motifs.htm. (C) Overlaid structures of three inhibitors with GDP bound K-Ras G12C. These allosteric inhibitors bind to switch-II region and induce an inactive GDP binding conformation (Ostrem et al., 2013).

FIGURE 2

Physiological processes related to the Parkinson’s disease and the GTPases and the effectors involved. GTPases LRRK2, Rab7L1, Rab5, and GTPase effector ArfGAP1 regulate Golgi and α-synuclein aggregate clearance; large GTPases MFN1, MFN2, OPA1, and DRP1 regulate mitochondria fission and fusion; MIRO and Rheb have a role in organelle transport and axon maintenance; GTPase Rho, Rac1, and kinase effector ROCK are involved in oxidative stress management and neuroinflammation; Rho, Rit, and Rin are involved in neuronal differentiation and survival. LRRK2, leucine-rich repeat kinase 2; ArfGAP1, ADP-ribosylation factor GTPase-activating protein 1; MFN1 and MFN2, mitofusin-1 and mitofusin-2; OPA1, optic atrophy 1; DRP1, dynamin-related protein 1; MIRO, mitochondrial Rho-GTPase; Rheb, Ras homolog enriched in brain; ROCK, Rho-associated protein kinase.

FIGURE 3

A flow chart showing the progress in the development of GTPase activity modulators. The initial GTPase inhibitors were natural products that inhibit GTPase prenylation and signal transduction. In the last several years, through in silico screening and rational design, inhibitors that blocked protein interactions and GEF enzymatic activities were identified. On the other hand, single compound library screening revealed molecules that interfere with either protein interactions or nucleotide binding. The most recent progress lies in the combinatorial library screening in a multiplex format.