Development of covalent inhibitors of Jumonji-C histone lysine demethylases

在真核生物中，染色体DNA包裹在组蛋白周围；翻译后组蛋白修饰对于调节基因激活和沉默（“表观遗传学”）很重要，例如，组蛋白N(Epsilon)-赖氨酸氨基和精氨酸胍基的可逆甲基化。组蛋白赖氨酸甲基转移酶（KMTs）和组蛋白赖氨酸去甲基化酶（kdm）在调节健康细胞组蛋白甲基化-去甲基化稳态中具有互补的功能。这些酶的失调与癌症等疾病有关，使kdm成为开发抗癌疗法的重要治疗靶点。kdm根据其序列同源性和机制可分为两个亚族。第一个家族，即KDM1，使用黄素腺嘌呤二核苷酸（FAD）作为其共底物，而另一类，即Jumonji-C （JmjC）酶KDM2/7、KDM3、KDM4、KDM5和KDM6，使用2-氧葡萄糖酸酯（2-OG）和二氧作为共底物，并使用非血红素铁（II）作为辅因子（图1）。几种KDM1A抑制剂，如丙氨酰环丙胺、ladademstat等，正在进行癌症治疗的临床试验然而，只有少数强效和选择性的JmjC kdm抑制剂被报道在细胞中显示出作用，其中大多数通过非共价模式抑制。该项目的目标是开发KDM4A-C、KDM5B和KDM6B的有效和选择性抑制剂，这些抑制剂是治疗宫颈癌、胃癌和乳腺癌的潜在靶点；抑制剂将被设计成允许共价抑制。该结果将为其他类型金属酶的抑制剂提供信息，包括那些与抗菌素耐药性有关的金属酶。JmjC KDM抑制剂的开发计划主要集中在通过非共价相互作用抑制的小分子上，而通过共价修饰抑制JmjC KDM的小分子则较少被探索。共价抑制剂可能优于非共价抑制剂，因为蛋白质与配体的相互作用持续时间更长，并且可能是不可逆的，这取决于所选择的官能团。牛津大学最近的工作提供了原理证明，磺酰氟化物以一定程度的选择性抑制JmjC KDM相关的2OG加氧酶FIH和AspH。考虑到KDM6B与FIH的相似性（图2），设计含磺酰氟小分子是开发新型抑制剂的一个有希望的开始，因此被选为目标（图3）。将对磺酰氟化物进行结构活性关系研究，以了解其抑制KDM6B的效力和选择性；例如，侧链将被纳入作为KDM6B抑制剂一部分的小分子中。模型、质谱和晶体学研究将指导结构活性关系研究，并告知共价修饰的位置和化学计量。其他共价反应基团也将被探讨。该项目属于“EPSRC医疗保健技术”研究领域。

In eukaryotes, chromosomal DNA is wrapped around histone proteins; post-translational histone modifications are important to regulate gene activation and silencing ('epigenetics'), for example, reversible methylations at N(Epsilon)-lysine amino groups and at arginine guanidinium groups of histone proteins. Histone lysine methyltransferases (KMTs) and histone lysine demethylases (KDMs) have complementary functions in the regulation of histone methylation-demethylation homeostasis in healthy cells. The dysregulation of these enzymes is linked to diseases such as cancer, rendering KDMs important therapeutic targets to develop anti-cancer therapeutics. KDMs are divided into two subfamilies depending upon their sequence homologies and mechanisms. The first family, that is KDM1, uses flavin adenine dinucleotide (FAD) as its co-substrate, while the other class, that is the Jumonji-C (JmjC) enzymes KDM2/7, KDM3, KDM4, KDM5, and KDM6, use 2-oxoglutarate (2-OG) and dioxygen as co-substrates as well as non-heme Fe(II) as cofactor (Figure 1). Several KDM1A inhibitors like tranylcypromine, ladademstat and others, are undergoing clinical trials for cancer therapy.5 However, only few potent and selective inhibitors of the JmjC KDMs have been reported that show effects in cells, most of them inhibiting via a non-covalent mode. The objective of this project is to develop potent and selective inhibitors for KDM4A-C, KDM5B and KDM6B which are potential targets for the treatment of, for example, cervical, gastric and breast cancer; the inhibitors will be designed to allow for covalent inhibition. The results will inform on the inhibitors of other types of metalloenzymes, including those involved in antimicrobial resistance. JmjC KDM inhibitor development programs have focused on small-molecules which inhibit through non-covalent interactions, while small-molecules which inhibit via covalent modification of JmjC KDMs have been less well explored. Covalent inhibitors can be superior over non-covalent inhibitors as the protein-ligand interactions persist over a longer time period and may be irreversible, depending on the functional group chosen. Recent work in Oxford has afforded proof-of-principle evidence that sulfonylfluorides inhibit the JmjC KDM related 2OG oxygenases FIH and AspH with some level of selectivity. Considering the similarity of KDM6B with FIH (Figure 2), the design of sulfonylfluoride-bearing small-molecules is a promising start for development of novel inhibitors and was therefore chosen as a target (Figure 3). Structure activity relationship studies will be performed on sulfonylfluorides to inform on their potency and selectivity for KDM6B inhibition; for example, side chains will be incorporated into the small-molecules that are part of reported KDM6B inhibitors. Modelling, mass spectrometric, and crystallographic studies will guide the structure activity relationship studies and inform on the position and stoichiometry of the covalent modification. Other covalently reacting groups will also be explored. This project falls within the 'EPSRC healthcare technologies' research area.