CSIR- Indian Institute of Integrative Medicine, Sanatnagar, Srinagar, Kashmir, India; Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, India.
Department of Biotechnology, Babasaheb Bhimrao Ambedkar University, Vidya Vihar, Raebareli Road, Lucknow 226025, Uttar Pradesh, India.
Biochim Biophys Acta Gen Subj. 2023 Sep;1867(9):130399. doi: 10.1016/j.bbagen.2023.130399. Epub 2023 Jun 8.
G9a is a histone H3K9 methyltransferase enzyme found highly upregulated in many cancers. H3 binds to the rigid I-SET domain and the cofactor, S-adenosyl methionine, binds to the flexible post-SET domain of G9a. Inhibition of G9a is known to inhibit the growth of cancer cell-lines.
Recombinant G9a and H3 were used to develop radioisotope-based inhibitor screening assay. The identified inhibitor was evaluated for isoform selectivity. The mode of enzymatic inhibition was studied by enzymatic assays and bioinformatics. Anti-proliferative activity of the inhibitor was studied in cancer cell lines by utilizing MTT assay. The mechanism of cell death was studied by western blotting and microscopy.
We developed a robust G9a inhibitor screening assay that led to the discovery of SDS-347 as a potent G9a inhibitor with IC of 3.06 μM. It was shown to reduce the levels of H3K9me2 in cell-based assay. The inhibitor was found to be peptide competitive and highly specific as it did not show any significant inhibition of other histone methyltransferases and DNA methyltransferase. Docking studies showed that SDS-347 could form direct bonding interaction with Asp1088 of the peptide-binding site. SDS-347 showed anti-proliferative effect against various cancer cell lines especially the K562 cells. Our data suggested that SDS-347 mediated antiproliferative action via ROS generation, induction of autophagy and apoptosis.
Overall, the findings of the current study include development of a new G9a inhibitor screening assay and identification of SDS-347, as a novel, peptide competitive and highly specific G9a inhibitor with promising anticancer potential.
G9a 是一种组蛋白 H3K9 甲基转移酶，在许多癌症中高度上调。H3 与刚性 I-SET 结构域结合，辅因子 S-腺苷甲硫氨酸与 G9a 的柔性后 SET 结构域结合。已知抑制 G9a 可抑制癌细胞系的生长。
使用重组 G9a 和 H3 开发基于放射性同位素的抑制剂筛选测定法。鉴定的抑制剂用于评估同工型选择性。通过酶促测定和生物信息学研究研究了酶抑制的模式。通过 MTT 测定法在癌细胞系中研究抑制剂的抗增殖活性。通过蛋白质印迹和显微镜研究研究了细胞死亡的机制。
我们开发了一种强大的 G9a 抑制剂筛选测定法，该测定法导致 SDS-347 被发现为一种有效的 G9a 抑制剂，IC 为 3.06 μM。它显示可降低细胞测定中的 H3K9me2 水平。该抑制剂被发现是肽竞争抑制剂，并且高度特异性，因为它对其他组蛋白甲基转移酶和 DNA 甲基转移酶没有显示出任何明显的抑制作用。对接研究表明，SDS-347 可以与肽结合位点的 Asp1088 形成直接键合相互作用。SDS-347 对各种癌细胞系（特别是 K562 细胞）表现出抗增殖作用。我们的数据表明，SDS-347 通过 ROS 生成、自噬和细胞凋亡诱导来介导抗增殖作用。
总体而言，本研究的结果包括开发一种新的 G9a 抑制剂筛选测定法和鉴定 SDS-347，作为一种新型的、肽竞争和高度特异性的 G9a 抑制剂，具有有前途的抗癌潜力。
