Hancock, RM; (2013) The discovery and evaluation of inhibitors of the KEAP1-NRF2 protein-protein interaction. Doctoral thesis (PhD), UCL (University College London).

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Abstract

Disruption of the interaction between the ubiquitination facilitator protein Keap1 and the transcription factor Nrf2 is a potential strategy for enhancing the expression of antioxidant and detoxification gene products that are regulated by this cap ‘n’ collar basic-region leucine zipper transcription factor. Agents that disrupt this protein-protein interaction may be useful pharmacological probes and future cancer chemopreventive agents. The activity of Nrf2 is thought to be regulated by at least two other proteins; the nuclear protein prothymosin α and the proteasome associated sequestosome-1 that compete for the Keap1 binding site. The aim of this project is to develop compounds that directly inhibit the Keap1-Nrf2 protein-protein interaction and thereby enhance Nrf2 activity by a mechanism different to that of existing chemopreventive agents. Linear and cyclic peptides based on the binding motifs of Nrf2, prothymosin α and sequestosome-1 were designed and synthesised. An optimised series of peptides was then developed with further changes to the sequence to improve the binding profile. The peptides were shown to inhibit the Keap1-Nrf2 protein-protein interaction, determined using a fluorescence polarisation assay. A small series of stearoyl capped peptides were also developed for use in cell based assays. The peptides can be ranked in order of affinity and used to determine a structure activity relationship for interaction with the Keap1 protein. Cyclic inhibitors were generally more potent than the linear peptides, however, the most potent peptide was a linear hybrid sequence based upon the Nrf2 and sequestosome-1 binding motifs, and with an IC50 of 115 nmol/L was more active than either native sequence alone and equivalent in activity to the Nrf2 Neh2 domain protein. Finally, a small library of peptidomimetics designed using the peptide template and molecular modelling studies were also synthesised, resulting in five small molecules with IC50 values in the micromolar range.

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