A fluorescence based high throughput assay to identify inhibitors of the autophagy associated kinase ULK1


A fluorescence based high throughput assay to identify inhibitors of the autophagy associated kinase ULK1
Puneet Khurana1, Alison Levy1, Ahmad Kamal1, Ryan Mordue1, Michelle Newman1, Simon Osborne1, Barbara Saxty1, Ian Ganley2, Paul D Wright1
1 MRC Technology, Centre for Therapeutics Discovery, 1-3 Burtonhole Lane, Mill Hill, London, NW7 1AD, United Kingdom.
2 MRC Protein Phosphorylation and Ubiquitylation Unit, The Sir James Black Centre , College of Life Sciences, University of Dundee , Dow Street , Dundee , DD1 5EH, United Kingdom.

ULK1 (Unc-51 Like Autophagy Activating Kinase 1) is a serine/threonine-protein kinase, and is crucial for the initiation of autophagy. Autophagy is a homeostatic cell survival mechanism but can also promote growth of established tumors, and act as a cancer cell survival mechanism. By inhibiting the catalytic activity of ULK1, autophagy can be blocked in cancer cells, reducing their capacity to cope with the stresses of tumourigenesis and chemotherapy. Potent ULK1 inhibitors may therefore provide therapeutic benefit in multiple cancer types which rely on autophagy for their survival, as well as a combination approach in cancers with chemotherapeutic-resistant phenotypes.
An HTS (high throughput screening) assay was developed using the TranscreenerTM ADP fluorescence intensity assay (BellBrook Labs, USA). Baculovirus-expressed recombinant full length human GST-ULK1 protein (Stratech, UK) was used, with Myelin Basic Protein (MBP) as the substrate The assay was highly sensitive to low levels of ATP-ADP conversion, and a >6 fold signal window was observed with only 10% ATP turnover. A total of 23434 compounds were screened comprising subsets of MRC Technology proprietary compounds and a kinase focused library. Robust screening statistics were observed, including an average Z’ of 0.79, and an average assay window of 6.37. Following chemistry triage, 324 compounds showing >30 % inhibition of ULK1 activity were selected for full IC50 analysis. A high proportion of these compounds (~72%) showed concentration dependent inhibition of ULK1 activity.
Compounds with potencies <3µM were subsequently screened in an orthogonal radiometric kinase assay, and a good correlation between IC50s was observed across the two assay formats. Subsequently, compounds were confirmed as being ATP-competitive, and also as stoichiometric binders. A subset of compounds were assessed for activity in cell based assays, measuring inhibition of autophagy (reduction in total LC3 accumulation) and inhibition of cell viability.
Chemistry optimisation of lead compounds is on-going. It is hoped that compounds with appropriate ULK1 activity, kinase selectivity and favourable physiochemical properties could be used to further elucidate the role of ULK1 in autophagy and cancer, ultimately leading to novel therapeutics.

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