Abstract 5221: Target engagement approaches to validate small-molecule binders of the pioneering transcription factor FOXA1

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Abstract The discovery of small-molecules that disrupt or promote the function of transcription factors, often considered ‘undruggable’ targets, is considered a significant challenge.[1] The development of general and systematic approaches to discover small-molecule probes of transcription factors is therefore of substantial value. In this study, a high-throughput small-molecule microarray (SMM) screen was used to identify initial binders of FOXA1, which were studied using a variety of biochemical approaches to identify and characterize target engagement of these small-molecule probes in a cellular context. Tools for validating FOXA1 as a therapeutic target do not currently exist, although FOXA1 is a known mediator of androgen receptor (AR) action in Prostate Cancer (PCa), and has been demonstrated to both assist and antagonize AR activity, dependent upon the cellular context.[2,3] Using the SMM technology, potential small-molecule binders of FOXA1 were identified, and an enrichment for an interesting class of endogenous metabolites was observed. While an interaction between these compounds and FOXA1 has not previously been reported, they were found to exhibit meaningful inhibition of FOXA1-controlled transcription in prostate cancer cell lines. Compound-anchored beads were used to pull-down FOXA1 in whole cell lysates, and we demonstrated via western blot that the endogenous metabolite engages the transcription factor in this setting. Target engagement in live cells was demonstrated using bi-functional chemical probes, which contain a photo-reactive functional group that transforms transient protein-ligand interactions into stable covalent bonds upon UV irradiation. The probes were also modified with a bioorthogonal, or click chemistry handle, which enabled for assay read-outs using fluorescent labeling and mass spectrometry.[4] Using these approaches, we successfully validated the bimolecular interaction between the endogenous metabolites and FOXA1 in cell lysates and live cells, respectively. This report details the first known small-molecule to bind FOXA1 in a cellular setting and suggests a new perspective on the potential role of this class of endogenous compound in prostate cancer.