An Evolutionarily Conserved Structural Platform for PRC2 Inhibition by a Class of Ezh2 Inhibitors

Matthew Bratkowski,Xin Liu,Xin Yang

doi:10.1038/s41598-018-27175-w

Abstract

Polycomb repressive complex 2 (PRC2) mediates trimethylation of histone H3K27 (H3K27me3), an epigenetic hallmark for repressed chromatin. Overactive mutants of the histone lysine methyltransferase subunit of PRC2, Ezh2, are found in various types of cancers. Pyridone-containing inhibitors such as GSK126 compete with S-adenosylmethionine (SAM) for Ezh2 binding and effectively inhibit PRC2 activity. PRC2 from the thermophilic fungus Chaetomium thermophilum (ct) is functionally similar to the human version in several regards and has the added advantage of producing high-resolution crystal structures, although inhibitor-bound structures of human or human/chameleon PRC2 are also available at up to 2.6 Å resolution. We solved crystal structures of both human and ctPRC2 bound to GSK126 and the structurally similar inhibitor GSK343. While the two organisms feature a disparate degree of inhibitor potency, surprisingly, GSK126 binds in a similar manner in both structures. Structure-guided protein engineering of the drug binding pocket allowed us to introduce humanizing mutations into ctEzh2 to produce a ctPRC2 variant that is more susceptible to GSK126 inhibition. Additional analysis indicated that an evolutionarily conserved structural platform dictates a unique mode of GSK126 binding, suggesting a mechanism of drug selectivity. The existing drug scaffold may thus be used to probe the function and cellular regulation of PRC2 in a wide spectrum of organisms, ranging from fungi to humans.

Highlights

Crystal structures of a human/American chameleon (h/Ac) Polycomb repressive complex 2 (PRC2) bound to inhibitor 1 and a human PRC2 bound to a CPI-1205 derivative (CPI-1205d) show that the body and variable elongated arm regions of the drugs are caged by Ezh[2] residues Y661 and Y111, which helps to explain why mutation at these sites results in loss of drug potency[21,27,28,29]
In order to gain a more detailed understanding of the interaction of PRC2 with pyridone inhibitors and extend the existing drug scaffold to the study of fungal PRC2, we solved the crystal structure of human and ctPRC2 bound to GSK126
Pyridone inhibitors are promising therapeutics for lymphoma and gaining a clearer molecular understanding of their mechanism of PRC2 inhibition will allow for the design of better future drugs

Summary

Introduction

The SAM binding pocket of ctEzh[2] is structurally similar to the human version, but the drug gating residues in the SAL and SET of human Ezh[2] that are necessary for pyridone inhibitor potency[27,28] do not appear to have homologues in fungal Ezh[2] based on sequence analysis (Fig. S1). Humanizing mutations increased GSK126 inhibition and allowed us to solve additional structures of humanized ctPRC2 bound to GSK126 and GSK343 that revealed drug interactions for a model more similar to human PRC2 at high resolution Overall, these studies demonstrated increased diversity among the binding modes of pyridone inhibitors to PRC2 and provided feasibility for the study of PRC2 function in multiple organisms by chemical genetics

Methods

Results

Conclusion