Development of class IIa selective histone deacetylase inhibitors with chemosensitizing properties as anticancer agents

组蛋白脱乙酰酶(HDAC)是细胞表观遗传调控中的重要酶。它们在癌症发展和治疗中的重要性被公认为4种HDAC抑制剂(HDACi)的批准。批准的HDACi大多是广谱HDACi，这可能是观察到的严重副作用的原因。因此，表观遗传药物的发现在一定程度上转向了类和异构体选择性HDACi的发展。库尔茨和卡萨克实验室的研究活动导致了具有体外抗癌活性和IIa类偏好的HDACi的开发。LMK235是一种IIa类优先的HDACi，由于与顺铂的协同作用，能够逆转顺铂耐药癌细胞的耐药性。此外，LMK235可防止卵巢癌细胞对顺铂产生耐药性。因此，我们的工作假设是，IIa类HDAC是治疗表达IIa类HDAC的特定类型癌症的有希望的靶点，例如结肠癌和头颈癌。这项赠款提案的目的是开发有效和选择性的IIa HDACi类药物，并评估其抗癌潜力。问题是，抑制IIa类HDAC是否与先前研究中观察到的LMK235的抗癌作用有关，以及这种抑制是否可以作为提高DNA靶向药物(如铂化合物)的敏感性和防止耐药性发展的适当策略。已建立的药物化学策略将用于逐步改善LMK235s IIa类HDAC对IIb类HDAC异构体的偏好，并改善已经很好的对I类HDAC的选择性。为了实现这两个目标，我们打算瞄准IIa类HDAC的高度保守的催化位点。IIa类选择性HDACi的开发将以分子对接为指导。合成和结构阐明将在T.Kurz教授的小组中进行。库尔茨实验室的新化合物将在M.Kassack教授的团队中进行HDAC抑制和抗癌效果的生物学评估。参考HDACi用于比较。将评估化合物在头颈和结肠癌细胞系中的细胞毒性、对细胞HDAC的影响、诱导细胞凋亡、细胞周期停滞、基因表达的变化以及HDAC目标蛋白的乙酰化状态。非癌细胞充当选择性控制。此外，将应用酶促HDAC分析来探索合成化合物的HDAC选择性分布。然后，对化合物进行逆转和防止对DNA损伤剂的化学耐药性的测试。潜在的分子机制将被探索。总而言之，这项联合研究项目将深入了解IIa类HDAC对癌症发展迄今鲜为人知的贡献，以及它们作为抗癌靶点的用途。此外，该项目将提供选择性的IIa类HDAC抑制剂作为药理工具。

Histone deacetylases (HDACs) are important enzymes in epigenetic modulation of cells. Their importance in cancer development and treatment is accepted as seen by the approval of 4 HDAC inhibitors (HDACi). Approved HDACi are mostly broad spectrum HDACi which may account for observed severe side effects. Epigenetic drug discovery is thus in part shifting towards the development of class- and isoform-selective HDACi. Research activities of the Kurz and the Kassack laboratories have resulted in the development of HDACi with in vitro anticancer activity and class IIa preference. LMK235 is a class IIa preferential HDACi able to revert chemoresistance in cisplatin-resistant cancer cell lines due to synergistic activity with cisplatin. Furthermore, LMK235 prevents the development of cisplatin resistance in an ovarian cancer cell line. Thus, our working hypothesis is that class IIa HDACs are promising targets for the treatment of specific types of cancer expressing class IIa HDACs such as colon and head neck cancers. The objective of this grant proposal is to develop potent and selective class IIa HDACi and to evaluate their anticancer potential. The question will be addressed whether inhibition of class IIa HDACs is responsible for the anticancer effects of LMK235 observed in previous studies and if such inhibition can serve as a suitable strategy to increase sensitivity of DNA-targeting drugs such as platinum compounds and to prevent the development of resistance. Established strategies of medicinal chemistry will be used for the stepwise improvement of LMK235s class IIa preference over class IIb HDAC isoforms and to improve the already good selectivity over class I HDACs. In order to accomplish both goals, we intend to target the highly conserved catalytic site of class IIa HDACs. The development of class IIa selective HDACi will be guided by molecular docking. Synthesis and structural elucidation will be performed in the group of Prof. T. Kurz. New compounds from the Kurz lab will undergo biological evaluation for HDAC inhibition and anticancer effects in the group of Prof. M. Kassack. Reference HDACi are used for comparison. Compounds will be evaluated in head-neck and colon cancer cell lines for cytotoxicity, effects on cellular HDACs, induction of apoptosis, cell cycle arrest, changes in gene expression and in the acetylation status of HDAC target proteins. Non-cancer cells serve as selectivity controls. Further, enzymatic HDAC assays will be applied to explore the HDAC selectivity profile of the synthesized compounds. Then, compounds are tested for reversal and prevention of chemoresistance against DNA-damaging agents. Underlying molecular mechanism will be explored. In conclusion, this joint research project will yield insight into the so far little known contribution of class IIa HDACs to cancer development and their use as anticancer targets. Furthermore, this project will provide selective class IIa HDAC inhibitors as pharmacological tools.