Epigenetic processes control gene expression and play a major role in cell proliferation, survival and differentiation. Dysregulation of epigenetic mechanisms may lead to alterations in gene expression, resulting in cellular transformation and malignant outgrowth. Epigenetic enzymes are recognized as promising therapeutic targets for the treatment of cancer. Inventiva has several research programs in epigenetics, with specific focus on histone lysine methyltransferases (HKMTs). Emerging evidence suggests that G9a, a HKMT, able to mono and dimethylate H3K9 leading to the inactivation of tumor suppressor genes, plays a predominant role in lung and ovarian cancers. To identify novel G9a inhibitors, we used an AlphaLisa assay, with a histone H3-derived peptide and a specific antibody against H3K9me2. The assay, in 384-well format, displayed a robust Z-factor (0.84) with a signal to noise ratio around 800. We screened Inventiva's proprietary compound library using this assay. Three hundred thirty-six hits inhibited G9a by more than 60% (Hit rate: 0.14%). Despite the advantages offered by AlphaLisa technology, such as high S/N ratios and straightforward automation, this technology has been reported to generate a high number of false positives. In order to confirm the activity of our hits, we used a different technology, the methyltransferase Glo (MT-Glo) assay, which measures the reaction product SAH by luminescence read-out. Out of the 336 identified compounds, 75 were confirmed by this orthogonal screening (Hit confirmation rate: 22%). Additional compound characterization is ongoing to allow the selection of the best hit series for chemical optimization.

In conclusion, the AlphaLisa assay, coupled with MT-Glo assay allowed the identification and confirmation of potent hits against G9a, as chemical starting points for our hit-to-lead optimization program.

Citation Format: Claudia Fromond, Xavier Espanel, Laurent Chene, Philippe Masson, Benaïssa Boubia, Christian Montalbetti, Pierre Broqua. Identification of G9a inhibitors by AlphaLisa™ technology and hit confirmation using MT-Glo™. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 99. doi:10.1158/1538-7445.AM2015-99