Misregulation of chromatin-modifying proteins is a common alteration in human cancers, and aberrant activities of these proteins are implicated in various aspects of tumorigenesis and cancer progression, as well as in treatment resistance. Histone demethylases that belong to KDM5 subfamily are epigenetic ?eraser? proteins that antagonizes methylation of lysine 4 of histone H3. Two members of KDM5 family, KDM5A and KDM5B, are frequently amplified and overexpressed in cancer. Elevated expression of these demethylases is critical for tumorigenesis, proliferation, migration and metastasis in cancers such as breast, prostate, lung, gastric and colon cancer, as well as hepatocellular carcinoma and neuroblastoma. Furthermore, elevated expression of these proteins promotes resistance to radiation therapy and targeted therapy. Several orthosteric inhibitors have been developed to target the binding pocket of the obligatory co-substrate of this family of enzyme, ?-ketoglutarate (?-KG), however the high cellular concentrations of ?-KG impede cellular effectiveness of these orthosteric chemical probes. We hypothesize that chemical probes that do not compete with this abundant cellular metabolite can have significant advantages in the context of a cell. Our recent work has identified PHD1 domain, one of the three chromatin reader domains within KDM5A, as an allosteric regulatory site in this demethylase. Here we propose to develop allosteric small molecule modulators of KDM5A by targeting its PHD1 domain. We will address development of PHD1-directed chemical probes using high-throughput screening. Specifically, hits will be identified in a fluorescence polarization (FP)-based high-throughput screen of a structurally diverse 250,000 compounds library available at UCSF's Small Molecule Discovery Center. Hits will be prioritized based on their potency and through cheminformatics filters, and validated by an orthogonal FP-based assay as well as by surface plasmon resonance-based binding assay. Selectivity of prioritized hits and their available derivatives will be assessed in a comprehensive counter-screen against related chromatin reader domains. Using protein NMR, we will determine binding poses of the most potent and selective hits. A series of activity assays will be used to assess activity and mode of action of identified ligands, both in vitro and in cells. The proposed research has a potential to yield chemical probes for PHD1, expanding repertoire of small molecules that target epigenetic reader domains, and to enable allosteric modulation of KDM5A and KDM5B.
Chromatin-interacting proteins regulate gene expression and their misregulaton can drive cancer. Validation of these proteins as potential therapeutic targets necessitates development of effective chemical probes, and may lead to novel therapeutic interventions.