m1A去甲基化酶ALKBH3的小分子调控研究

Chemical probe is a selective small-molecule modulator of an undruggable protein’s function that allows chemical biologist to answer these mechanistic and phenotypic questions about its molecular target in biochemical, cell-based or animal studies. The proper usage of the high-value chemical probes has showed both academic and industry perspectives on its values throughout the course of fundamental biology and preclinical research. ALKBH3 has been identified as a new biomarker of the prostate cancer. As a key member in the family of AlkB-repair enzymes, ALKBH3 has primarily been known to selectively remove the methyl group in the m1A lesion in the single-stranded nucleic acids. It is very interesting that this enzyme has recently been involved in the regulation of the cellular abundance of m1A modification within mRNA. How the function of ALKBH3 impacts on the cellular abundance of m1A, and what’s the functional connection between ALKBH3 demethylation and gene expression? All these fundamental questions are unclear due to lacking of high-value chemical probes of ALKBH3. In this project, we aim to develop small-molecule inhibitors of ALKBH3 demethylation in vitro and test its ability to modulate the cellular m1A in mRNA. The major chemical biology based techniques will be further developed and fully applied in order to understand the inhibitor mechanism of action, the target engagement, and the in vivo selectivity. Hopefully, with the support of these studies we would be able to put forth the hypothesis that ALKBH3 demethylase might be an effective pharmacological target for cancer treatment.

作为选择性调节蛋白质功能的小分子工具，化学探针（chemical probe）的正确使用可以从分子、细胞、以及整体等多层面描述“非药靶”（undruggable，尚无药物可及）蛋白质的生物学功能和分子机制。高质量化学探针的匮乏阻碍蛋白质功能与结构的研究，制约了新靶点发现与确证进度。ALKBH3是前列腺癌标记物，主导了m1A碱基损伤修复，还通过调控基因表达影响细胞表型。那么ALKBH3的去甲基化功能以及m1A丰度如何影响肿瘤的发生？ALKBH3在肿瘤中如何影响下游基因表达，是通过调控信使RNA上m1A的水平和分布，还是存在未知的蛋白-蛋白相互作用？针对这些科学问题，本课题致力于发现高质量化学探针，运用化学干扰重现选择性调节ALKBH3相关的生理功能与表型，揭示ALKBH3新功能以及m1A分布和调控的作用机理。重点关注小分子的作用机制、靶标相关性、选择性以及调节表型的化学生物学表征。

ALKBH3去甲基化酶是前列腺癌标记物，作为AlkB修复酶家族的一个重要成员，ALKBH3介导了单链DNA和RNA上m1A碱基损伤修复和mRNA上m1A修饰的动态调控。然而ALKBH3如何影响细胞内mRNA上m1A丰度，以及ALKBH3去甲基化与基因表达调控有何关联尚不清楚。因此，本课题希望通过发展靶向ALKBH3的高质量小分子抑制剂用于相关的干预表型和分子机制研究。本课题首先利用已发展的AlkB家族泛抑制剂大黄酸开展胞内作用靶向性及抑制细胞活性的研究。此外设计了基于蛋白质的高通量筛选方法，筛选了不同来源的5万个小分子化合物库，发现新的骨架分子作为潜在的抑制剂。幸运的是，筛选发现一个正在开展临床前实验研究的化合物C5-H10，其对ALKBH3及其同源家族去甲基化酶具有较好的选择抑制活性。基于结构的设计与合成优化获得衍生物Y6在细胞内能够调控m1A丰度，并具有抗肿瘤活性。本课题的结果初步证明靶向干预ALKBH3去甲基化活性，调控m1A修饰可能是个有效的抗肿瘤策略，深入的化学生物学表型和机制研究仍在进行中。