Development of Next-Generation Macrocyclic Histone Deacetylase Inhibitors

表观遗传机制在基因表达的调控中起着至关重要的作用，并决定了哪些基因在哪些细胞和什么时间表达。这既发生在健康细胞中，也发生在调节异常的患病细胞中。这些机制既表现在对DNA本身的化学修饰，也表现在对DNA包裹的组蛋白蛋白质的化学修饰上。清除这些标记的“橡皮擦”酶，如依赖锌的组蛋白脱乙酰酶(HDAC)，已被公认为新兴的药物靶点，特别是在肿瘤学中。一些有效但非选择性的抑制剂已经进入临床，本项目旨在开发基于azumamide(一种天然存在的环四肽家族)支架的这类酶的新型抑制剂。该项目中将产生的抑制剂将研究一种新的作用模式，该模式不依赖于强大的活性部位金属结合，而是依赖于破坏HDAC与其蛋白质伙伴的蛋白质-蛋白质相互作用。将I类HDAC从其多蛋白复合体中分离出来也会导致酶活性的急剧下降。关于HDAC与其蛋白质伙伴的复合体的结构知识是可以获得的，这将氮胺抑制剂的一个氨基酸残基放置在靠近蛋白质-蛋白质相互作用界面的位置。通过对该残基和金属结合残基进行化学修饰，将生成一个新的氮胺衍生物文库，并测试其对HDAC的抑制作用。将建立一种概念上的新的体外实验，通过荧光偏振来研究HDAC蛋白质-蛋白质之间的相互作用。这可以用来证明这些抑制剂对复合体形成的破坏。这种迄今未被探索的抑制机制允许引入关于HDAC亚家族甚至单个同种类型的选择性，因为它们各自具有不同的蛋白质复合体伙伴。在与生化测试数据的反馈的系统的SAR研究中，结构指导迭代优化新的氮胺类药物将产生有效的双机制抑制剂。通过平衡这两种机制，可以为一种HDAC亚型生成比其他亚型更具可调效力和选择性的化合物。细胞培养实验将验证癌细胞株的抗增殖特性和底物特异性乙酰化的变化。使用表观遗传HDAC酶的四肽抑制剂代表了一种独特的替代时间基因调控的方法。如所概述的，所设计的双机制化合物将允许同时抑制酶活性以及它们在蛋白质-蛋白质复合体中的支架功能。由此可以预期的增强的生物效应有可能使这些下一代HDAC抑制剂成为表征单个酶的生物学作用以及潜在的候选药物的宝贵的工具化合物。

Epigenetic mechanisms play a vital role in the regulation of gene expression and determine, which genes are expressed by which cells and at what times. This occurs both in healthy and, when aberrantly regulated, diseased cells. These mechanisms are manifested in chemical modifications both to DNA itself as well as to histone proteins, around which DNA is wrapped. “Eraser” enzymes that remove such marks like e.g. zinc-dependent histone deacetylases (HDACs) have been recognized as emerging drug targets, in particular in oncology. Some potent, yet unselective, inhibitors have already reached the clinic.This project aims at the development of novel inhibitors of this enzyme class based on the scaffold of azumamides, a family of naturally occurring cyclotetrapeptides. The inhibitors that will be generated in this project will investigate a novel mode of action, which does not rely on strong active site metal binding, but on disruption of protein-protein interactions of HDACs with their protein partners. Isolation of class I HDACs from their multiprotein complexes also leads to drastically reduced enzymatic activity.Structural knowledge is available about HDACs in complex with their protein partners, which positions one amino acid residue of the azumamide inhibitors in close proximity to the protein-protein interaction interface. Through chemical modifications to this residue as well as to the metal-binding residue, a library of novel azumamide derivatives will be generated and tested for their HDAC inhibition. A conceptually new in vitro assay will be established, which investigates HDAC protein-protein interactions by fluorescence polarization. This can be used to demonstrate disruption of complex formation by these inhibitors. This heretofore unexplored mechanism of inhibition allows for the introduction of selectivity with regard to HDAC subfamilies or even individual isotypes as they each have different protein complex partners. Structure-guided iterative optimization of the novel azumamides in a systematic SAR study in feedback with data from biochemical testing will yield potent dual-mechanism inhibitors. By balancing of these two mechanisms, compounds can be generated with tunable potency and selectivity for one HDAC subtype over the others. Cell culture experiments will validate the antiproliferative properties on cancer cell lines and substrate-specific changes in acetylation.The use of tetrapeptidic inhibitors of epigenetic HDAC enzymes represents a unique alternative method to temporal gene regulation. As outlined, the designed dual-mechanism compounds will allow for simultaneous inhibition of enzyme activity as well as their scaffolding function in protein-protein complexes. The enhanced biological effects that can be expected from this have the potential to make these next-generation HDAC inhibitors valuable tool compounds for the characterization of the biological role of individual enzymes as well as potential drug candidates.