<i>De Novo</i> Peptide Design and Experimental Validation of Histone Methyltransferase Inhibitors

<div><p>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 <i>de novo</i> 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 <i>de novo</i> peptide design method. The computationally discovered peptides were experimentally validated <i>in vitro</i> using dose titrations and mechanism of action enzymatic assays. The peptide with the highest <i>in vitro</i> response, SQ037, was validated <i>in nucleo</i> 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.</p></div

<i>In nucleo</i> Experiments to Test Effects of the Inhibitor Peptide on HMT Activity.

<p>Quantitative proteomics distinguish newly generated “heavy” histone methylation at H3K27 from old, “light” methylation. MS monitoring of new H3K27me3 from the in nucleo reaction with (A) SAM only, (B) SAM plus the scrambled control peptide added, and (C) SAM plus SQ037 inhibitor peptide added. Relative abundance levels across all conditions for (D) H3K27me2, (E) H3K27me3 and (F) H3K9me3 are shown. Statistical significance between abundance levels is indicated, *P<0.002 and **P<0.0001. Three biological replicates were used. Note that the specified 12 Da shift is nominal, exact value is 12.062 Da.</p

Potency of Top Designed Peptide vs. Native and Mutated Peptides.

<p>A high throughput radiometric assay was used to determine the inhibitory potential of the top candidate peptide, SQ037, versus the native unmethylated peptide, the native methylated peptide, and a peptide with a point mutation K27A. Shown is the absolute EZH2 HMT activity (counts per minute, cpm).</p

Selected SET Domain Template Structure.

<p>(A) Composition of PRC2 showing the complex of SUZ12, EZH2 (containing the SET domain), EED and the association of PHF1. (B) Design template of a histone fragment bound to vSET (grey), PDB code: 2G46. The first nine residues of the histone fragment, which make no contacts with vSET, are colored yellow, while the 21 residues that do make contacts are colored red. The SAH cofactor is colored blue.</p

Three-Stage <i>De Novo</i> Peptide Design Workflow Diagram.

<p>Stage I is an optimization-based sequence selection stage. Stage II is a fold specificity calculation to determine how well designed sequences fold into the desired template structure compared to the native sequence. Stage III is an approximate binding affinity calculation to determine how well the designed sequences binds to the target protein.</p

Designed Peptides Competitively Inhibit EZH2 Catalytic Activity.

<p>(A) A radiometric assay was used to determine the EZH2 catalytic activity in the absence (lane 1) or presence of 125 µM of candidate EZH2 inhibitor peptides (lanes 2–11). The inhibitory potential of native H3 peptide (lane 12) and an unrelated peptide (random; lane 13) was assessed. A reaction without peptide, but heat inactivated at 95°C for 5 min prior to incubation, was used to determine the background (lane 14). Shown is a fluorographic image of [³H]-labeled methyl groups incorporated on histone H3 (upper panel). Histones were visualized by Coomassie Blue staining (lower panel). (B) A high throughput radiometric assay was used to determine the inhibitory potential of candidate peptides. Shown is the absolute EZH2 HMT activity (counts per minute, cpm). (C,D) The catalytic activity of EZH2(C) and EZH1(D) was assessed in the absence (lane 2) or presence (lane 3) of SQ037 [125 µM]. Shown is a fluorographic image of [³H]-labeled methyl groups incorporated on histone H3 (upper panel). Histones and PRC2 constituents were visualized by Coomassie Blue staining (lower panel).</p

Mechanism of Action Enzymatic Assay Results.

<p>(A) Peptide dose response curve in the EZH2 HMT assay using 100 nM (10× enzyme) and 10 nM (1×enzyme) of reconstituted, recombinant PRC2. (B) Peptide dose response curve in the EZH2 HMT assay using 2.4 µM (10× SAM) and 0.24 µM (1× SAM) of ³H-labeled SAM. (C) Peptide dose response curve in the EZH2 HMT assay using 60 µM (10× substrate) and 6 µM (1× substrate) of H3K27me1.</p