DS_DISC766278 – Supplemental material for Discovery of Small-Molecule Antagonists of the H3K9me3 Binding to UHRF1 Tandem Tudor Domain

<p>Supplemental material, DS_DISC766278 for Discovery of Small-Molecule Antagonists of the H3K9me3 Binding to UHRF1 Tandem Tudor Domain by Guillermo Senisterra, Hugh Y. Zhu, Xiao Luo, Hailong Zhang, Guoliang Xun, Chunliang Lu, Wen Xiao, Taraneh Hajian, Peter Loppnau, Irene Chau, Fengling Li, Abdellah Allali-Hassani, Peter Atadja, Counde Oyang, En Li, Peter J. Brown, Cheryl H. Arrowsmith, Kehao Zhao, Zhengtian Yu, and Masoud Vedadi in SLAS Discovery</p

Discovery and Molecular Basis of a Diverse Set of Polycomb Repressive Complex 2 Inhibitors Recognition by EED

<div><p>Polycomb repressive complex 2 (PRC2), a histone H3 lysine 27 methyltransferase, plays a key role in gene regulation and is a known epigenetics drug target for cancer therapy. The WD40 domain-containing protein EED is the regulatory subunit of PRC2. It binds to the tri-methylated lysine 27 of the histone H3 (H3K27me3), and through which stimulates the activity of PRC2 allosterically. Recently, we disclosed a novel PRC2 inhibitor EED226 which binds to the K27me3-pocket on EED and showed strong antitumor activity in xenograft mice model. Here, we further report the identification and validation of four other EED binders along with EED162, the parental compound of EED226. The crystal structures for all these five compounds in complex with EED revealed a common deep pocket induced by the binding of this diverse set of compounds. This pocket was created after significant conformational rearrangement of the aromatic cage residues (Y365, Y148 and F97) in the H3K27me3 binding pocket of EED, the width of which was delineated by the side chains of these rearranged residues. In addition, all five compounds interact with the Arg367 at the bottom of the pocket. Each compound also displays unique features in its interaction with EED, suggesting the dynamics of the H3K27me3 pocket in accommodating the binding of different compounds. Our results provide structural insights for rational design of novel EED binder for the inhibition of PRC2 complex activity.</p></div

EED inhibitors are non-competitive with SAM or H3K27me0 peptide.

<p><b>a.</b> EED inhibitors are non-competitive with SAM. Enzymatic assays were carried out at 1 x and 10 x SAM with H3K27me0 in excess. There is no IC50 shift when increasing SAM concentration. <b>b</b>. EED inhibitors are non-competitive with H3K27me0. Enzymatic assays were carried out at 1 x and 10 x H3K27me0 with SAM in excess. There is no IC50 shift when increasing H3K27me0 concentration. <b>c</b>. Binding affinity determination of EED210 to EED by ITC. The stoichiometry of binding between EED210 and EED was approximately 1:1 molar ratio with N = 0.78. The enthalpy change is -20.14 ±5.29 Kcal/mol and the entropy change is -46.5cal/mol/deg. <b>d</b>. Concentration dependent SPR analysis of EED210 binding to EED (residues 40–441).</p

The molecular basis of H3K27me3 competitive inhibitors recognition by EED.

<p><b>a.</b> Electrostatic surface potentials of EED666 binding pocket (left); Blue—positive charge, red—negative charge; The blue, black and pink dashed circles indicate the deep pocket, aromatic packing region and the edge of pocket, respectively; Five compounds are aligned to underline the common binding features (right). Chemical groups embedded in the deep pocket are colored blue; chemical groups packed against Tyr365, Tyr148 and Phe97 are colored black; chemical groups located in the edge of the pockets are colored pink. Chemical groups involved in three binding regions are divided by two dashed lines. <b>b.</b> Binding mode of each co-structure. Interacting residues in EED are labeled and shown as sticks. Water molecules are shown as red sphere. Yellow dashed lines are hydrogen bonds.</p

Identification of allosteric PRC2 inhibitors through EED binding.

<p><b>a.</b> Flowchart of EEDi identification and validation from PRC2 high throughput screening. <b>b.</b> Chemical structures of five identified SAM non-competitive inhibitors. The IC50 values were determined using an EED-H3K27me3 AlphaScreen competition binding assay.</p

The crystal structures of H3K27me3 competitive inhibitors binding to EED.

<p><b>a.</b> Structures of EED-EZH2 peptide in complex with EED396, EED666, EED709, EED162 and EED210. The five structures are aligned and shown in the same view. The EZH2 peptide is highlighted as red cylinder. <b>b.</b> A representative highlight of the conformational change of Arg367, Trp364, and Tyr365, in comparison of the EED666 bound (Arg 367 in green, Tyr365 in blue, and Trp364 in red) in and H3K27me3 bound EED structures (top); below, comparison of EED666-bound EED pocket (right) with H3K27me3-bound pocket (left); EED is shown as a surface and colored white. H3K27me3 peptide is shown as ball-and-stick in green color; for clarity, only the surface of residues Arg367 (green), Tyr365 (blue) and Trp364(red) are highlighted. <b>c</b>. The dynamic conformational changes of Arg367, Tyr365, Tyr148 and Phe97 in inhibitor bound EED structures.</p

Data collection and statistics for the structure of EED -inhibitor complexes.

<p>Data collection and statistics for the structure of EED -inhibitor complexes.</p

Discovery of Potent and Selective Allosteric Inhibitors of Protein Arginine Methyltransferase 3 (PRMT3)

PRMT3 catalyzes the asymmetric dimethylation of arginine residues of various proteins. It is crucial for maturation of ribosomes and has been implicated in several diseases. We recently disclosed a highly potent, selective, and cell-active allosteric inhibitor of PRMT3, compound <b>4</b>. Here, we report comprehensive structure–activity relationship studies that target the allosteric binding site of PRMT3. We conducted design, synthesis, and evaluation of novel compounds in biochemical, selectivity, and cellular assays that culminated in the discovery of <b>4</b> and other highly potent (IC₅₀ values: ∼10–36 nM), selective, and cell-active allosteric inhibitors of PRMT3 (compounds <b>29</b>, <b>30</b>, <b>36</b>, and <b>37</b>). In addition, we generated compounds that are very close analogs of these potent inhibitors but displayed drastically reduced potency as negative controls (compounds <b>49</b>–<b>51</b>). These inhibitors and negative controls are valuable chemical tools for the biomedical community to further investigate biological functions and disease associations of PRMT3

Discovery of First-in-Class, Potent, and Orally Bioavailable Embryonic Ectoderm Development (EED) Inhibitor with Robust Anticancer Efficacy

Overexpression and somatic heterozygous mutations of EZH2, the catalytic subunit of polycomb repressive complex 2 (PRC2), are associated with several tumor types. EZH2 inhibitor, EPZ-6438 (tazemetostat), demonstrated clinical efficacy in patients with acceptable safety profile as monotherapy. EED, another subunit of PRC2 complex, is essential for its histone methyltransferase activity through direct binding to trimethylated lysine 27 on histone 3 (H3K27Me3). Herein we disclose the discovery of a first-in-class potent, selective, and orally bioavailable EED inhibitor compound <b>43</b> (EED226). Guided by X-ray crystallography, compound <b>43</b> was discovered by fragmentation and regrowth of compound <b>7</b>, a PRC2 HTS hit that directly binds EED. The ensuing scaffold hopping followed by multiparameter optimization led to the discovery of <b>43</b>. Compound <b>43</b> induces robust and sustained tumor regression in EZH2^MUT preclinical DLBCL model. For the first time we demonstrate that specific and direct inhibition of EED can be effective as an anticancer strategy