针对组蛋白甲基转移酶DOT1L的选择性小分子抑制剂设计和优化研究

Disruptor of telomeric silencing 1-like (DOT1L) is an evolutionarily conserved histone methyltransferase (HMT) that specifically catalyzes the methylation of the histone H3-lysine79 (H3K79) residue in the core domain, which plays an important role in transcriptional regulation, cell cycle regulation, and the DNA damage response, and is proposed to be a catalytic driver of leukemogenesis in mixed lineage leukemia (MLL). Since the discovery of the critical role of DOT1L in MLL leukemia, research has focused on identifying inhibitors of DOT1L. Because almost all methyltransferases use S-adenosyl-L-methionine (SAM) as the methyl donor (enzyme cofactor), a common strategy for designing DOT1L inhibitors is to develop SAM analogs that may compete with SAM. To date, a variety of DOT1L inhibitors have been reported, but most are SAM derivatives with an adenosine group, and their selectivity profile and pharmacokinetic properties are unsatisfactory, e.g., showing low oral bioavailability and high clearance rates. Since the structural diversity of DOT1L inhibitors remains limited, there remains an urgent need to develop new DOT1L inhibitors with novel scaffolds. Meanwhile, there are some peculiarities to the active site of DOT1L, compared to general ligand sites of drug targets, which make traditional structure-based drug design (SBDD) approaches toward DOT1L particularly challenging: extreme flexibility, a large number of polar residues with high charge density, extensive solvent exposure of its binding site. Therefore, widely used docking programs and scoring functions, which account differently for these types of interactions, may not perform as well for DOT1L-ligand complexes. To address these issues, we have developed a customized SBDD approach called SAM score for discovering novel inhibitors of HMTs, and successfully applied this approach in discovering four DOT1L inhibitors with novel scaffolds. In this proposed project, we aim to further employ this approach to guide the lead optimization of our previously identified DOT1L inhibitors, further carry out molecular, cellular, and animal level of antitumor evaluations, investigate their epigenetic regulation mechanisms, and, in the end, obtain new DOT1L inhibitors with high efficiency and specificity, and good drugability properties.

组蛋白赖氨酸甲基转移酶DOT1L特异性催化组蛋白H3第79位赖氨酸（H3K79）发生甲基化，可作为混合细胞系白血病（MLL）的潜在治疗靶标。DOT1L小分子抑制剂及调控机制的研究，为MLL相关性白血病的治疗提供了新的思路。目前，针对DOT1L的抑制剂设计主要围绕辅酶S-腺苷甲硫氨酸的类似物结构改造开展，已报道的活性化合物结构类型单一，普遍存在选择性差、药代性质不好的缺点。此外，DOT1L的活性位点具有开放性高、柔性大和极性强的结构特点，使用传统的合理药物设计方法发现靶向DOT1L的新化学实体的难度较大。针对这些问题，本项目拟通过发展和应用甲基转移酶专用药物设计方法，针对已发现的具有新颖骨架的DOT1L抑制剂进行结构活性关系分析和ADMET性质优化，开展分子、细胞和动物水平的抗肿瘤活性评价及表观调控作用机制研究，以期获得高效、高特异性、高成药性的新型DOT1L抑制剂药物候选化合物。

DOT1L可作为MLL相关白血病的潜在治疗靶标，其小分子抑制剂及调控机制的研究，为MLL相关性白血病的治疗提供了新的思路。本课题中，开展新骨架非核苷类DOT1L选择性抑制剂的药物设计研究。在前期发现的活性化合物的基础上，通过构效关系研究、基于结构的药物设计和ADME/T预测等方法加速活性化合物改造，以获得全新的高特异性、高活性和成药性佳的DOT1L先导化合物，并在分子、细胞、作用网络和动物水平上进一步研究其作用机制。同时，完善DOT1L先导化合物发现的理论设计方法。通过课题组全体成员的努力，各学科紧密合作，我们顺利完成了本课题的各项既定研究任务。目前，本项目已全面地完成了预期的考核指标。基于DOT1L先导化合物2，开展细胞实验和基于结构的优化等工作。合成37个化合物，得到了若干分子、细胞水平活性良好的BRD4抑制剂，最终得到3个成药性较好、对MV4-11等三种肿瘤细胞系有选择性的新型先导化合物。获得化合物29，其在小鼠体内对急性髓系白血病的发展具有显著的治疗效果。发展一系列稳定可靠的甲基转移酶专用药物设计方法，显著提升先导化合物发现和优化效率。在该课题资助下，我们已在Nucleic acids research、Protein & cell、Journal of medicinal chemistry、Bioinformatics、Acta Pharmaceutica Sinica B、Sci China Life Sci.、Chemical Science等杂志共发表致谢本基金号的论文30篇（其中SCI论文28篇，非SCI论文2篇），申请发明专利3项。培养博士研究生4人，硕士研究生3人。本课题的研究内容和结果为发现MLL相关白血病治疗提供新的药物先导结构，为进一步优化和改造非核苷类抑制剂提供了线索。培育的新型药物设计方法，提高了先导化合物发现和优化的效率研究工作。

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