Discovery and Development of Potent and Selective Inhibitors of Histone Methyltransferase G9a

G9a is a histone lysine methyltransferase responsible for the methylation of histone H3 lysine 9. The discovery of A-366 arose from a unique diversity screening hit, which was optimized by incorporation of a propyl-pyrrolidine subunit to occupy the enzyme lysine channel. A-366 is a potent inhibitor of G9a (IC₅₀: 3.3 nM) with greater than 1000-fold selectivity over 21 other methyltransferases

A-366 inhibits H3K9me2 similar to UNC0638, but does not inhibit cell growth of MOLT-16 and HT-1080 cells.

<p>(A) The T-cell ALL cell line MOLT-16 was treated for 5 days with A-366 or UNC0638 before assessing their proliferation. (B) MOLT-16 cells were treated for 3 days with compounds and lysed. Lysates were normalized and western blot analysis was performed for H3K9me2, total H3 and G9a. (C) HT-1080 fibrosarcoma cells were treated for 2 or 5 days with A-366 or UNC0638 before assessing their proliferation. (D) H3K9me2 AlphaLISA was performed on HT-1080 cells treated for 2 days with compounds and % inhibition of H3K9me2 was calculated compared to untreated control cells.</p

A-366 does not impact the proliferation of MCF-7 cells despite inhibition of H3K9me2.

<p>(A) MCF-7 and MDA-MB-231 breast cancer cell lines were treated with the indicated concentrations of A-366 or UNC0638 for 14 days before assessing colony formation. (B) MCF-7 and MDA-MB-231 cells were treated with the indicated concentrations of A-366 or UNC0638 for 3 days and subjected to western blot analysis of global H3K9me2 and total histone H3.</p

<i>In vivo</i> efficacy study of A-366 in MV4;11 flank xenografts.

<p>(A) MV4;11 cells were implanted subcutaneously in SCID/bg mice and allowed to establish tumors of ~200 mm³. A-366 was administered to tumor-bearing mice at 30 mg/kg/day by osmotic mini-pump for 14 days. Tumors were measured at the indicated time points and tumor growth was plotted as a function of time. (B) In a parallel PK/PD arm carried out in MV4;11 tumor-bearing animals, plasma and tissues were collected at the indicated time points and analyzed for A-366 levels. (C) A-366-treated tumors from the indicated time points were harvested and analyzed by AlphaLISA for reductions in H3K9me2 relative to vehicle.</p

A-366 has cellular activity comparable to known G9a/GLP inhibitors.

<p>(A) The chemical structure of A-366. (B) PC-3 prostate adenocarcinoma cells were incubated in triplicate with DMSO or the indicated concentrations of A-366 or UNC0638 for 72 hours. H3K9me2 levels were assessed by In-Cell Western assay. (C) In-Cell Western assay for total histone H3 levels from the same plate as shown in (B).</p