Strategies Toward Inhibition of the Polycomb Repressive Complex 2 Using PNA Conjugates and Bitopic Peptides

Abstract

H3K27M is a mutation in the H3 histone tail of nucleosomes that is implicated in diffuse intrinsic pontine glioma (DIPG), a fatal tumor in the brainstem that is prevalent among children. The H3K27M mutation inhibits EZH2, the catalytic domain of PRC2, a histone methyltransferase enzyme complex, which results in the reduction of global H3K27me3 levels. Potent peptide-based synthetic inhibitors of PRC2 would serve as useful probes to elucidate the mechanism of PRC2 inhibition by K27M, and would promote the development of potential therapeutics for adult pathologies characterized by hyperactivity of the EZH2 subunit. However, to date, potent inhibitors targeting the PRC2 have not been developed. In this study, two different strategies were used to evaluate PRC2 inhibition. PNA’s (Peptide Nucleic Acids) served to direct PNA-bound H3K27M peptide to the nucleosome, thereby recruiting PRC2 to the nucleosome for potentially multivalent inhibition. Also, bitopic peptides containing binding sites to two domains of PRC2, the EZH2 and RbAp46/48 domains, were synthesized with linkers of varied lengths in order to investigate the effects of bivalent binding in PRC2 inhibition. While the PNA-H3\(_{23-34}\) K27M did not inhibit PRC2 under the specific assay conditions, the bitopic peptides H3\(_{1-10}\)-(PEG)\(_{n}\)-H3\(_{21-37}\) K27Nle (n=1,2,3) showed stronger inhibition than the H3\(_{1-10}\) and H3\(_{21-37}\) K27Nle peptides in trans, demonstrating that the presence of two binding domains in the peptides confers increased potency of inhibition. The shortest bitopic peptide exhibited the strongest inhibition in the series, illuminating geometric preferences of bivalent binding. This study highlights the significance of bitopicity in enhancing inhibition of multivalent complexes such as PRC2, which could lead to the development of the next generation of potent PRC2 inhibitors for cancer therapy.