Structure-Based Design and Characterization of the Highly Potent and Selective Covalent Inhibitors Targeting the Lysine Methyltransferases G9a/GLP

Abstract

Protein lysine methyltransferases G9a and GLP, which catalyze mono- and di-methylation of histone H3K9 and nonhistone proteins, play important roles in diverse cellular processes. Overexpression or dysregulation of G9a and GLP has been identified in various types of cancer. Here, we report the discovery of a highly potent and selective covalent inhibitor 27 of G9a/GLP via the structure-based drug design approach following structure–activity relationship exploration and cellular potency optimization. Mass spectrometry assays and washout experiments confirmed its covalent inhibition mechanism. Compound 27 displayed improved potency in inhibiting the proliferation and colony formation of PANC-1 and MDA-MB-231 cell lines and exhibited enhanced potency in reducing the levels of H3K9me2 in cells compared to noncovalent inhibitor 26. In vivo, 27 showed significant antitumor efficacy in the PANC-1 xenograft model with good safety. These results clearly indicate that 27 is a highly potent and selective covalent inhibitor of G9a/GLP.

Discovery of a Highly Potent Lysine Methyltransferases G9a/NSD2 Dual Inhibitor to Treat Solid Tumors

“…Recovery of the G9a inhibitory activity for compound 15 was expected to show high cellular potency against the solid tumor cell lines. On the other hand, C7-pyrrolidinylpropoxy side chain of the quinazoline scaffold is beneficial for retaining high G9a potency as previous reported, presented by compounds 3–7 . − Accordingly, we replaced the C7-methoxy group of compound 15 by a 3-(pyrrolidin-1-yl)propoxy side chain and designed compound 16 with the aim of developing a highly potent G9a/NSD2 dual inhibitor to show high antiproliferative activity against solid tumors. Intriguingly, compound 16 not only displayed good NSD2 inhibitory activity (IC 50 = 0.017 μM) but also showed high G9a inhibitory potency (IC 50 = 0.241 μM).…”

“…However, these compounds suffered from weak antiproliferative activity against solid tumors, such as prostate cancer cell line PANC-1 and triple negative breast cancer cell line MDA-MB-231. In order to improve the cellular potencies of these noncovalent G9a inhibitors, our team and independently the Jin group, recently reported the identification and characterization of potent and selective G9a covalent inhibitors, as presented by compounds 6 and 7 . Compared to the noncovalent G9a inhibitors, compound 7 showed much enhanced cellular potency, but still exhibited moderate antiproliferative activity against PANC-1 and MDA-MB-231 .…”

“…In order to improve the cellular potencies of these noncovalent G9a inhibitors, our team and independently the Jin group, recently reported the identification and characterization of potent and selective G9a covalent inhibitors, as presented by compounds 6 and 7 . Compared to the noncovalent G9a inhibitors, compound 7 showed much enhanced cellular potency, but still exhibited moderate antiproliferative activity against PANC-1 and MDA-MB-231 . More G9a inhibitors with novel mechanism of action are still needed to achieve high cellular potency.…”

Discovery of the First-in-Class RORγ Covalent Inhibitors for Treatment of Castration-Resistant Prostate Cancer

“…Compared to reversible noncovalent inhibitors, covalent inhibitors provide various potential advantages, including improved pharmacological potency and selectivity, prolonged duration of action, and reduced propensity for acquiring resistance. − Interestingly, Cys320 is identified in the binding site of reported RORγ reversible inhibitors as evidenced by the co-crystal structures of RORγ in complex with compounds 3 , 5 , and 6 (Figure ), which is suitable for the design of RORγ covalent inhibitors. In this regard, we performed a structure-based drug design approach to facilitate the development of the first-in-class RORγ covalent inhibitors.…”

C2H2 Zinc Finger Transcription Factors Associated with Hemoglobinopathies

“…This hypothesis is based on the following: (1) G9a and DNMT1 directly interact, 145 (2) DNA methylation is intricately connected to the presence of H3K9 methylation, 146 and (3) DNA methylation is required for the MBD2-NuRD complex to occupy the γ-globin promoters. 65 Although G9a/GLP enzymatic inhibitors are being tested in preclinical models as potential anticancer agents, 147 – 153 UNC0638, the most commonly-used small molecule inhibitor of G9a, induces HbF expression in erythroid cells. 154 – 156 …”