Abstract 3091: Development and optimization of a fluorescence polarization-based assay for the discovery of inhibitors of the RBBP4/7-Histone H3 interaction

Abstract

Abstract Polycomb repressive complex 2 (PRC2) is a multi-subunit epigenetic complex critical for the maintenance of stem cells. Its dysregulation has been implicated in various diseases, including cancer. There are four core subunits in PRC2: the catalytic SET domain-containing histone methyltransferase EZH2 and three core accessory proteins (SUZ12, EED, and RBBP4 or 7), which together mediate gene repression via trimethylation of histone H3 on lysine 27 (H3K27me3). Importantly, overexpression of PRC2 subunits is associated with poor clinical outcome in several cancers. While numerous inhibitors of EZH2 have been developed and showed pre-clinical efficacies in hematological malignancies they have yet to show as much efficacy in solid tumors. One perhaps better alternative for inhibiting PRC2 is to disrupt its scaffolding function, thereby preventing the complex formation. Therefore, the discovery of novel inhibitors of PRC2, outside of the active site, has been a growing interest. Our preliminary observations have suggested that the PRC2 subunits retinoblastoma binding protein 4 and 7 (RBBP4/7) could be a specifically attractive alternative for PRC2 inhibition. Knockdown of RBBP4/7 significantly decreased cell growth, reduced H3K27me3 levels and inhibited mammosphere formation of triple negative breast cancer cell lines. In PRC2, RBBP4 and RBBP7 are responsible for nucleosomal association of the complex by binding to the N-terminal tail of histone H3. Here we describe the development and optimization of a fluorescence polarization (FP) based assay to determine the binding affinities (Kd) of the RBBP4/7-histone H3 interaction and the Ki of potential inhibitors. Using a 384-well format, the assay measures the competitive binding of a fluorescein-labeled H3 peptide (aa 1-21) to RBBP4 (Kd = 400 nM). It exhibits a Z’ of > 0.7 and a dynamic range of 65. It reaches equilibrium after 5 minutes and is stable for over 24 hours. Furthermore, using this assay, we completed a systematic analysis of the RBBP4-H3 interaction by truncation and modification studies and uncovered the smallest H3 peptide required for the interaction (aa 1-9). We have successfully developed an FP assay for the interaction of human RBBP4 protein and an optimized H3 probe. High throughput screening (HTS) is being carried out with this assay using a diverse set of small molecule and natural products obtained from commercial, government and academic collections. This study, along with our biological data, will provide a concrete basis for the development of novel PRC2 inhibitors. Citation Format: Rebecca Reed, Miao-Chia Lo, Chang-Ching Lin, Duxin Sun. Development and optimization of a fluorescence polarization-based assay for the discovery of inhibitors of the RBBP4/7-Histone H3 interaction. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3091.