Abstract C57: The biological evaluation of small molecule inducers of Nrf2 transcriptional activity that inhibit the Keap1-Nrf2 protein-protein interaction.

Abstract

Abstract The transcription factor Nrf2 up-regulates a range of cytoprotective enzymes with antioxidant response elements in their promoter regions and thus can protect cells against oxidative damage. Nrf2 is negatively regulated by the ubiquitination facilitator protein Keap1 that facilitates degradation of Nrf2. A number of natural products, including the isothiocyanate sulforaphane up-regulate Nrf2 by interacting with Keap1 in a covalent manner to stall its activity. Inhibition of the protein-protein interaction (PPI) between Keap1 and Nrf2 with small molecules constitutes an alternative strategy for developing pharmacological tool compounds and potential anti-inflammatory and chemopreventive therapeutic agents. We have applied structure-based drug design methods, including in silico screening, to develop a series of new inhibitors of the Keap1-Nrf2 interaction that mimic Nrf2 peptide motifs that bind to the Keap1 Kelch domain. Initial screening demonstrated that a series of substituted diaryl heterocycles were capable of inhibiting the PPI in vitro and inducing the expression of Nrf2 dependent gene products. The early hit compound HB229 is not cytotoxic (IC50>250 µM), induced the expression of NAD(P)H oxidoreductase-1 (NQO1) and hemeoxygenase-1 (HO1) at low micromolar concentrations and increased the nuclear accumulation of Nrf2 in cell based assays using mouse Hepa1c1c7 hepatoma and human Hela and HEK293 cell lines. HB229 inhibited the PPI with an apparent IC50 of ∼7.1 µM in a fluorescence polarisation assay. Further biological evaluation of HB229, and a derivative of a recently described bis-sulphonamide Keap1-Nrf2 PPI inhibitor, revealed a consistent time and dose dependent Nrf2 induction. Additionally, both compounds appear to be capable of interacting with the aryl hydrocarbon receptor (AhR) pathway to some extent. For instance, both compounds induce nuclear accumulation of the AhR in cells, however this behaviour was not observed with the prototype irreversible Nrf2 inducer sulforaphane. The significance of these findings will be discussed. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C57. Citation Format: Adrian Fowkes, Marjolein Schaap, Helene Bertrand, Clarisse Thiollier, Hiroko Kaatchi, Geoff Wells. The biological evaluation of small molecule inducers of Nrf2 transcriptional activity that inhibit the Keap1-Nrf2 protein-protein interaction. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C57.