In Vitro Characterization of VPS34 Lipid Kinase Inhibition by Small Molecules

Abstract

VPS34 is a class III phosphoinositide 3-kinase that acts on vesicle trafficking. This kinase has recently attracted significant attention because of the function it plays in the machinery involved in the early steps of autophagy. Moreover, because significant progress had been made in the optimization of specific kinase inhibitors, its potential to be targeted by catalytic inhibitors has been investigated by different groups. The aim of this review is to present the key in vitro assays necessary for characterizing inhibitors of the catalytic activity of VPS34. The review covers catalytic (IC₅₀ on purified recombinant protein) and binding assays (K_D, k_a, k_d on purified recombinant protein), and a cell-based assay (IC₅₀ in GFP-FYVE expressing cell line). The methodology for crystallization of VPS34 protein is also presented as it can provide guidance for the design by medicinal chemistry of small molecular mass kinase inhibitor.

Keywords: Autophagy; Endosomes; Lipid kinase; PI3K; PIK3C3/VPS34; Vesicle trafficking.