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.