High-throughput screening (HTS) is a proven method for discovering new lead matter for drug discovery and chemical biology. To maximize the likelihood of identifying genuine binders to a molecular target, and avoid wasting resources following up compounds with unproductive/nonspecific mechanisms of action, it is important to employ a range of assays during an HTS campaign that build confidence of target engagement for hit compounds. Biophysical methods that measure direct target/compound engagement have established themselves as key techniques in generating this confidence, and they are now integral to the latter stages of HTS triage at the European Lead Factory (ELF). One relatively new technique that the ELF is using is microscale thermophoresis (MST), which measures the differences in rate of movement through a temperature gradient that are caused when single molecular species form complexes. Here we provide an overview of the MST assay development workflow that the ELF employs and a perspective of our experience to date of using MST to triage the output of HTS campaigns and how it compares and contrasts with the use of other biophysical techniques.
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.
The molecular complex between Keap 1 (Kelch-like ECH-associated protein) and Nrf2 (nuclear factor erythroid 2-related factor 2) plays a major role in the regulation of cyto-protective responses to oxidative stress and electrophilic agents. The Keap1-Nrf2 pathway has been established as a therapeutic target for oxidative stress-related conditions including inflammatory, cardiovascular, neurodegenerative diseases and cancer. The European Lead Factory (ELF) is a major European project generating new lead structures for drug discovery programs in the public and private sectors. This is achieved by screening molecular targets against the Joint European Compound Library (JECL) combining compounds contributed by participating pharmaceutical companies or synthesised by chemistry SMEs. Target proposals are submitted to the ELF by European academics and SMEs and selected programs are screened against the library within the European Screening Centre. Hit compounds are provided to the program owner who gains exclusive rights to exploit them for drug discovery or as novel pharmacological tools. The aim of this project was to identify non-electrophilic inhibitors of the Keap1-Nrf2 interaction. Protein-protein interaction (PPI) targets are typically difficult to drug due to large, often quite shallow interaction surfaces between proteins, which along with other factors is associated with PPI screens often returning a high proportion of false positives. Therefore, designing a high-throughput screening (HTS) triage process that confirms target engagement of hits is essential, ideally involving the use of orthogonal biophysical assays. Here we present the successful development of a label-free MicroScale Thermophoresis (MST) assay to identify inhibitors of the Keap1-Nrf2 interaction. 318,132 compounds from the JECL were tested in a fluorescence polarization assay using a fluorescein-labelled Nrf2 peptide mimic. The HTS campaign yielded a hit rate of 0.4% and many false positives compounds were deselected due to fluorescence interference or redox reactivity. Following analytical assessment, visual inspection and legal clearance, 8 compounds were selected for further characterization. Assay development indicated that label-free MST was a suitable platform to confirm target engagement prior to investing in a Chemistry program. Two structurally related hits showed a concentration-dependent binding response to the full-length Keap1 protein in MST, which was abolished after denaturation of the Keap1 protein. These compounds were competitive with one another and with an unlabeled Nrf2 peptide mimic confirming that they were able to disrupt the interaction between Keap1 and Nrf2. Both compounds contain a chiral center and synthesis of the individual enantiomers confirmed that binding was specific to the S-stereoisomer. This data package provided the confidence to initiate a full analogue program with over 110 compounds synthesized and eventually led to ligand-bound crystal structures which helped rationalize the structure-activity relationships. Citation Format: Julie M. Rainard, Angus J. Morrison, Andrew D. Pannifer, Philip S. Jones, Richard J. Mead, Stuart P. McElroy. Characterization of small molecule inhibitors of the Nrf2-Keap1 interaction using MicroScale Thermophoresis [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-B17.
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