Identifying small molecule inhibitors of chromatin-binding proteins

Within the nucleus of the eukaryotic cell, DNA wraps around histone proteins to form the nucleosome, the most basic unit of chromatin structure. Specialized protein domains, called histone "readers," bind chromatin based on the post-translational modification state of histones to effect critical functions in gene regulation. Many histone reader proteins are implicated in cancer and are thus attractive targets for small molecule inhibitors. To this end, several biochemical assays utilizing AlphaScreen, HaloTag, and fluorescence polarization technologies were developed for high throughput small molecule screening against histone binding by reader proteins. These assays were harnessed to perform small molecule screening against two histone reader domains, the third plant homeodomain (PHD) finger of JARID1A and the linked tandem tudor domain-PHD finger (TTD-PHD) of UHRF1. Small molecule inhibitors of JARID1A PHD finger 3 included disulfiram, amiodarone, and tegaserod. Disulfiram ejects zinc from the structural fold of the PHD finger, while amiodarone and tegaserod are competitive inhibitors for the histone-binding groove. Medicinal chemistry was performed to generate amiodarone derivatives that were at least 10-fold more potent than amiodarone against JARID1A PHD finger 3. Small molecule inhibitors of UHRF1 TTD-PHD included SCH-202,676, sanguinarine, and α-NETA. These compounds inhibit UHRF1 TTD-PHD through the PHD finger region in a cysteine-dependent manner, resulting in zinc release. UHRF1 TTD-PHD inhibitors also inhibit a number of other histone reader domains, including non-zinc finger reader domains, in a cysteine-dependent manner. Collectively, these studies resulted in an integrated high throughput screening strategy targeting histone reader proteins, and highlighted several distinct mechanisms by which histone readers can be inhibited by small molecules.