Mimicking H3 Substrate Arginine in the Design of G9a Lysine Methyltransferase Inhibitors for Cancer Therapy: A Computational Study for Structure-Based Drug Design

G9a protein methyltransferase is a potential epigenetic drug target in different cancers and other disease conditions overexpressing the enzyme. G9a is responsible for the H3K9 dimethylation mark, which epigenetically regulates gene expression. Arg8 and Lys9 of the H3 substrate peptide are the two crucial residues for substrate-specific recognition and methylation. Several substrate competitive inhibitors are reported for the potent inhibition of G9a by incorporating lysine mimic groups in the inhibitor design. In this study, we explored the concept of arginine mimic strategy. The hydrophobic segment of the reported inhibitors BIX-01294 and UNC0638 was replaced by a guanidine moiety (side-chain moiety of arginine). The newly substituted guanidine moieties of the inhibitors were positioned similar to the Arg8 of the substrate peptide in molecular docking. Additionally, improved reactivity of the guanidine-substituted inhibitors was observed in density functional theory studies. Molecular dynamics, molecular mechanics Poisson–Boltzmann surface area binding free energy, linear interaction energy, and potential mean force calculated from steered molecular dynamics simulations of the newly designed analogues show enhanced conformational stability and improved H-bond potential and binding affinity toward the target G9a. Moreover, the presence of both lysine and arginine mimics together shows a drastic increase in the binding affinity of the inhibitor towards G9a. Hence, we propose incorporating a guanidine group to imitate the substrate arginine’s side chain in the inhibitor design to improve the potency of G9a inhibitors.G9a蛋白甲基转移酶（G9a protein methyltransferase）是多种过表达该酶的癌症及其他疾病中极具潜力的表观遗传药物靶点。该酶负责催化H3K9二甲基化（H3K9 dimethylation）标记，以此表观遗传调控基因表达。H3底物肽（H3 substrate peptide）的精氨酸8位（Arg8）与赖氨酸9位（Lys9）是决定底物特异性识别与甲基化过程的两个关键残基。目前已有多项研究报道了多种底物竞争性抑制剂，通过在抑制剂设计中引入赖氨酸模拟基团，实现对G9a的强效抑制。本研究探索了精氨酸模拟策略的应用思路：将已报道的抑制剂BIX-01294与UNC0638的疏水片段替换为胍基（guanidine moiety，即精氨酸的侧链基团）。分子对接（molecular docking）结果显示，抑制剂中新引入的胍基位点定位与底物肽的Arg8高度相似。此外，密度泛函理论（density functional theory）研究表明，胍基取代的抑制剂反应活性有所提升。针对新设计的类似物开展的分子动力学（molecular dynamics）、分子力学-泊松-玻尔兹曼表面积结合自由能（molecular mechanics Poisson–Boltzmann surface area binding free energy）、线性相互作用能（linear interaction energy），以及由拉伸分子动力学（steered molecular dynamics）模拟计算得到的平均力势（potential mean force）分析结果显示，此类类似物具有更优异的构象稳定性，且与靶标G9a之间的氢键作用潜力与结合亲和力均得到显著改善。值得注意的是，同时包含赖氨酸与精氨酸模拟基团的抑制剂，其与G9a的结合亲和力出现了大幅提升。因此，我们提出在G9a抑制剂的设计中引入胍基以模拟底物精氨酸的侧链，从而有效提升抑制剂的活性效力。