Abstract
Autophagy is a critical cellular homeostatic mechanism, the dysfunction of which has been linked to a wide variety of disease states. It is regulated through the activity of specific kinases, in particular Unc-51 like autophagy activating kinase 1 (ULK1) and Phosphatidylinositol 3-kinase vacuolar protein sorting 34 (VPS34), which have both been suggested as potential targets for drug development. To identify new chemical compounds that might provide useful chemical tools or act as starting points for drug development, we screened each protein against the Published Kinase Inhibitor Set (PKIS), a library of known kinase inhibitors. In vitro screening and analysis of the published selectivity profiles of the hits informed the selection of three relatively potent ATP-competitive inhibitors against each target that presented the least number of off-target kinases in common. Cellular assays confirmed potent inhibition of autophagy in response to two of the ULK1 inhibitors and all three of the VPS34 inhibitors. These compounds represent not only a new resource for the study of autophagy but also potential chemical starting points for the validation or invalidation of these two centrally important autophagy kinases in disease models.
Highlights
Macroautophagy is a lysosome-dependent degradative pathway that recycles intracellular components such as proteins and/or whole organelles
vacuolar protein sorting 34 (VPS34) catalyses the formation of the lipid phosphatidylinositol 3-phosphate (PI3P) at the site of autophagosome formation, which regulates the recruitment of PI3P-binding proteins such as WD repeat domain (WIPI2) and double FYVE-Containing Protein 1 (DFCP1) to aid in the expansion of the growing autophagosome [10]
The Published Kinase Inhibitor Set (PKIS) library of 856 compounds was screened at 100 nM and 1 mM against Unc-51 like autophagy activating kinase 1 (ULK1) in the adenosine diphosphate (ADP) Hunter Plus assay
Summary
Macroautophagy (referred to as autophagy here for simplicity) is a lysosome-dependent degradative pathway that recycles intracellular components such as proteins and/or whole organelles. In an effort to promote research on functionally uncharacterised kinases and to encourage the sharing of data, GlaxoSmithKline (GSK) have made available two Published Kinase Inhibitor Sets (PKIS) that together constitute a library of 856 compounds, which were formerly part of GSK’s kinase inhibitor research programmes [17,18,19]. This library provides a diversity of chemical scaffolds, comprising mainly of kinase hinge binding motifs, which have been screened for inhibition against a very wide range of kinases. We describe the output from two screens of the PKIS against recombinant ULK1 and VPS34, which, following assessment of the published selectivity profiles, resulted in the identification of two new ULK1 inhibitors and three new VPS34 inhibitors that exhibit good efficacy in cellular assays of autophagy
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