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

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

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

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

Discovery and Characterization of Allosteric WNK Kinase Inhibitors

Protein kinases are known for their highly conserved adenosine triphosphate (ATP)-binding site, rendering the discovery of selective inhibitors a major challenge. In theory, allosteric inhibitors can achieve high selectivity by targeting less conserved regions of the kinases, often with an added benefit of retaining efficacy under high physiological ATP concentration. Although often overlooked in favor of ATP-site directed approaches, performing a screen at high ATP concentration or stringent hit triaging with high ATP concentration offers conceptually simple methods of identifying inhibitors that bind outside the ATP pocket. Here, we applied the latter approach to the With-No-Lysine (K) (WNK) kinases to discover lead molecules for a next-generation antihypertensive that requires a stringent safety profile. This strategy yielded several ATP noncompetitive WNK1–4 kinase inhibitors, the optimization of which enabled cocrystallization with WNK1, revealing an allosteric binding mode consistent with the observed exquisite specificity for WNK1–4 kinases. The optimized compound inhibited rubidium uptake by sodium chloride cotransporter 1 (NKCC1) in HT29 cells, consistent with the reported physiology of WNK kinases in renal electrolyte handling

Allosteric Inhibition of SHP2: Identification of a Potent, Selective, and Orally Efficacious Phosphatase Inhibitor

SHP2 is a nonreceptor protein tyrosine phosphatase (PTP) encoded by the <i>PTPN11</i> gene involved in cell growth and differentiation via the MAPK signaling pathway. SHP2 also purportedly plays an important role in the programmed cell death pathway (PD-1/PD-L1). Because it is an oncoprotein associated with multiple cancer-related diseases, as well as a potential immunomodulator, controlling SHP2 activity is of significant therapeutic interest. Recently in our laboratories, a small molecule inhibitor of SHP2 was identified as an allosteric modulator that stabilizes the autoinhibited conformation of SHP2. A high throughput screen was performed to identify progressable chemical matter, and X-ray crystallography revealed the location of binding in a previously undisclosed allosteric binding pocket. Structure-based drug design was employed to optimize for SHP2 inhibition, and several new protein–ligand interactions were characterized. These studies culminated in the discovery of 6-(4-amino-4-methylpiperidin-1-yl)-3-(2,3-dichlorophenyl)­pyrazin-2-amine (SHP099, <b>1</b>), a potent, selective, orally bioavailable, and efficacious SHP2 inhibitor