Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, 100#, Shizi Street, Hongshan Road, Nanjing, 210028, Jiangsu, China.
Laboratory of Cellular and Molecular Biology, Jiangsu Province Academy of Traditional Chinese Medicine, Nanjing, 210028, China.
Cell Commun Signal. 2020 Apr 3;18(1):55. doi: 10.1186/s12964-020-00536-7.
IDH2/R140Q mutation is frequently detected in acute myeloid leukemia (AML). It contributes to leukemia via accumulation of oncometabolite D-2-HG. Therefore, mutant IDH2 is a promising target for AML. Discovery of IDH2 mutant inhibitors is in urgent need for AML therapy.
Structure-based in silico screening and enzymatic assays were used to identify IDH2/R140Q inhibitors. Molecular docking, mutant structure building and molecular dynamics simulations were applied to investigate the inhibitory mechanism and selectivity of CP-17 on IDH2/R140Q. TF-1 cells overexpressed IDH2/R140Q mutant were used to study the effects of CP-17 on cellular proliferation and differentiation, the wild-type TF-1 cells were used as control. The intracellular D-2-HG production was measured by LC-MS. The histone methylation was evaluated with specific antibodies by western blot.
CP-17, a heterocyclic urea amide compound, was identified as a potent inhibitor of IDH2/R140Q mutant by in silico screening and enzymatic assay. It exhibits excellent inhibitory activity with IC of 40.75 nM against IDH2/R140Q. More importantly, it shows poor activity against the wild-type IDH1/2, resulting in a high selectivity of over 55 folds, a dramatic improvement over previously developed inhibitors such as AGI-6780 and Enasidenib. Molecular simulations suggested that CP-17 binds to IDH2/R140Q at the allosteric site within the dimer interface through extensive polar and hydrophobic interactions, locking the enzyme active sites in open conformations with abolished activity to produce D-2-HG. Cellular assay results demonstrated that CP-17 inhibits intracellular D-2-HG production and suppresses the proliferation of TF-1 erythroleukemia cells carrying IDH2/R140Q mutant. Further, CP-17 also restores the EPO-induced differentiation that is blocked by the mutation and decreases hypermethylation of histone in the TF-1(IDH2/R140Q) cells.
These results indicate that CP-17 can serve as a lead compound for the development of inhibitory drugs against AML with IDH2/R140Q mutant. Video abstract.
IDH2/R140Q 突变在急性髓系白血病（AML）中经常被检测到。它通过积累致癌代谢物 D-2-HG 导致白血病。因此，突变型 IDH2 是 AML 的一个有前途的治疗靶点。迫切需要发现 IDH2 突变抑制剂来治疗 AML。
基于结构的计算机虚拟筛选和酶活性测定用于鉴定 IDH2/R140Q 抑制剂。分子对接、突变体结构构建和分子动力学模拟用于研究 CP-17 对 IDH2/R140Q 的抑制机制和选择性。过表达 IDH2/R140Q 突变的 TF-1 细胞用于研究 CP-17 对细胞增殖和分化的影响，野生型 TF-1 细胞作为对照。通过 LC-MS 测量细胞内 D-2-HG 的产生。通过 Western blot 用特异性抗体评估组蛋白甲基化。
CP-17 是一种杂环脲酰胺化合物，通过计算机虚拟筛选和酶活性测定被鉴定为 IDH2/R140Q 突变体的有效抑制剂。它对 IDH2/R140Q 的抑制活性 IC 为 40.75nM，具有优异的抑制活性。更重要的是，它对野生型 IDH1/2 的活性较差，选择性超过 55 倍，与先前开发的抑制剂如 AGI-6780 和 Enasidenib 相比有显著提高。分子模拟表明，CP-17 通过广泛的极性和疏水相互作用与二聚体界面内的 IDH2/R140Q 结合在别构部位，将酶活性部位锁定在开放构象中，从而阻止 D-2-HG 的产生。细胞试验结果表明，CP-17 抑制细胞内 D-2-HG 的产生，并抑制携带 IDH2/R140Q 突变的 TF-1 红白血病细胞的增殖。此外，CP-17 还能恢复突变阻断的 EPO 诱导的分化，并降低 TF-1(IDH2/R140Q)细胞中组蛋白的过度甲基化。
这些结果表明，CP-17 可作为开发针对 IDH2/R140Q 突变 AML 抑制药物的先导化合物。
