Abstract 6313: A transcription block survival assay for the discovery of functional inhibitors of transcription factors

Abstract

Abstract Transcription factor dysregulation is often key to oncogenesis and cancer progression. Direct inhibition of transcription factors provides a logical therapeutic strategy but the broad and shallow interaction interfaces of their protein-protein and protein-DNA interactions are challenging to target using traditional small-molecule approaches. The larger size of peptides provides an opportunity to span such interfaces with the exquisite affinity and selectivity required, and a number of peptide-based inhibitors are starting to emerge. In this study we focused on inhibiting basic leucine zipper transcription factors (bZIPs) as proof of principle. A key therapeutically relevant protein-protein interaction of bZIPs is the dimerization interface. Developing efficient peptide inhibitors of bZIP transcription factors has generally focused on either rational design or screen-based assays to identify peptides best able to inhibit dimerization. However, a major step in which bZIPs induce cancer initiation and progression is through DNA binding and the most effective inhibitors of bZIP dimerization do not necessarily translate into those best able to block the bZIP-DNA interaction. An assay that is able to screen for peptides that inhibit dimerization of the target transcription factor and – importantly – also selects according to the ability to inhibit protein-DNA interactions, holds significant promise in the development of more potent inhibitors. To achieve this aim we have developed a Transcription Block Survival assay. Here key binding sites are placed into a gene essential for E.coli survival. The target transcription factor then blocks transcription of this essential gene by binding to these sites, leading to cell death. When a peptide inhibitor from a peptide library is introduced into the system that is able to effectively inhibit DNA binding of the transcription factor, the gene is transcribed, restoring growth. Libraries can have up to two million peptides and allow the assay to select for peptides that not only bind to the target transcription factor, but that are also able to differentiate peptides on their ability to inhibit the transcription factor from binding DNA. The assay has been fully optimized for one bZIP target, and is currently undergoing the final stages of optimization for two other bZIP targets. Future work will focus on screening peptide libraries against these systems. This assay provides a route towards more efficient selection of potent inhibitors of transcription factors for the treatment of cancer. Citation Format: Sarah K. Madden, Jody M. Mason. A transcription block survival assay for the discovery of functional inhibitors of transcription factors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6313.