De Novo Peptide Design and Experimental Validation of Histone Methyltransferase Inhibitors

James Smadbeck,Meghan B Peterson,Benjamin A Garcia,Aashna Mago,Christina Lee,Shivani Garapaty,Christodoulos A Floudas,Barry M Zee,Patrick Trojer,Athanassios Giannis,H Sunny Sun

doi:10.1371/journal.pone.0090095

Abstract

Histones are small proteins critical to the efficient packaging of DNA in the nucleus. DNA–protein complexes, known as nucleosomes, are formed when the DNA winds itself around the surface of the histones. The methylation of histone residues by enhancer of zeste homolog 2 (EZH2) maintains gene repression over successive cell generations. Overexpression of EZH2 can silence important tumor suppressor genes leading to increased invasiveness of many types of cancers. This makes the inhibition of EZH2 an important target in the development of cancer therapeutics. We employed a three-stage computational de novo peptide design method to design inhibitory peptides of EZH2. The method consists of a sequence selection stage and two validation stages for fold specificity and approximate binding affinity. The sequence selection stage consists of an integer linear optimization model that was solved to produce a rank-ordered list of amino acid sequences with increased stability in the bound peptide-EZH2 structure. These sequences were validated through the calculation of the fold specificity and approximate binding affinity of the designed peptides. Here we report the discovery of novel EZH2 inhibitory peptides using the de novo peptide design method. The computationally discovered peptides were experimentally validated in vitro using dose titrations and mechanism of action enzymatic assays. The peptide with the highest in vitro response, SQ037, was validated in nucleo using quantitative mass spectrometry-based proteomics. This peptide had an IC50 of 13.5 M, demonstrated greater potency as an inhibitor when compared to the native and K27A mutant control peptides, and demonstrated competitive inhibition versus the peptide substrate. Additionally, this peptide demonstrated high specificity to the EZH2 target in comparison to other histone methyltransferases. The validated peptides are the first computationally designed peptides that directly inhibit EZH2. These inhibitors should prove useful for further chromatin biology investigations.

Highlights

Histones are small proteins critical to the efficient packaging of DNA in the nucleus [1]
De Novo Design of Methyltransferase Inhibitors The full de novo peptide design framework is described in detail in the Methods sections
For the design of inhibitors of the lysine methyltransferase enhancer of zeste homolog 2 (EZH2) target, four iterations of the method were run with different sets of biological constraints on the allowed sequences

Summary

Introduction

Histones are small proteins critical to the efficient packaging of DNA in the nucleus [1]. DNA winds itself around the surface of the histones, forming DNA-protein complexes known as nucleosomes [2]. The N-terminal histone tail protrudes from the nucleosome, allowing for post-translational modification of key histone residues. These post-translational modifications commonly consist of phosphorylation, acetylation, methylation, ubiquitylation, ribosylation, and sumoylation, to name a few [3]. Combinations of such histone modifications take part in the regulation of DNA transcription and constitute an additional level to the genetic code, termed the ‘‘histone code’’. These modifications are dynamically maintained by various histone-modifying enzymes that control their transfer and removal

Objectives

Methods

Results

Conclusion