Understanding and targeting isocitrate-metabolising enzymes involved in tuberculosis and cancer

2016年，130万人死于结核病(1)，结核病是由结核分枝杆菌（Mtb）引起的，而结核分枝杆菌是一种很少用普通抗生素治疗的病原体。多种药物治疗是可行的，但长期（至少6个月）严格的治疗方案对患者构成挑战。耐结核分枝杆菌菌株的出现使治疗越来越无效。2016年，报告了49万例耐多药结核病病例。(1)结核分枝杆菌存活的关键特征包括其代谢灵活性和持续处于高度耐药休眠状态的能力。(2)分枝杆菌的一个重要代谢特征是glyoxylate shunt，这一途径存在于大多数原核生物中，使Mtb能够在脂肪酸等C2碳源上生长。它部分绕过了TCA循环，在这个循环中，异柠檬酸脱氢酶（IDH）催化2-氧戊二酸（2OG）的氧化脱羧，得到异柠檬酸，然后由2OG脱氢酶将异柠檬酸脱羧为琥珀酰辅酶a。然后通过琥珀酰辅酶a合成酶催化的氧化磷酸化生成琥珀酸盐。当TCA循环中的脱羧步骤导致两个羧基作为CO2的损失时，乙醛酸分流绕过这些步骤，从而使简单的碳源化合物能够用于大分子合成（例如葡萄糖）。异柠檬酸裂解酶（ICL）是乙醛酸分流中的第一个酶，它直接将异柠檬酸（而不是2OG）转化为乙醛酸盐和琥珀酸盐。在某些非复制状态下，ICL在Mtb中上调。icl基因敲除降低了结核分枝杆菌的持久性和毒力。(2)因此，乙醛酸盐分流对于结核分枝杆菌在宿主内存活很重要，发现IDH和/或ICL抑制剂可能会带来新的治疗策略。了解结核分枝杆菌的代谢途径需要对TCA循环和乙氧基酸分流之间的分叉点进行详细的生化和生物物理研究，开发针对这些代谢途径的抑制剂对于验证异柠檬酸代谢酶作为药物靶点非常重要。因此，我的工作是针对IDH和ICL及其调控的生化和生物物理研究，包括动力学、质谱（MS）、晶体学和核磁共振谱（NMR）研究，以及早期抑制剂的开发。因此，我将针对这些酶筛选现有的内部文库以及GSK提供的抑制剂文库。命中分子的效力、选择性和结合方式将通过生物化学、生物物理和晶体学研究来确定；并将进一步为化学抑制剂优化提供信息。在我的筛选中发现的有效抑制剂将在弗朗西斯-克里克研究所对分枝杆菌细胞进行测试。该结果将更详细地了解乙醛酸分流的作用及其调控以及分枝杆菌代谢的产量抑制信息。除了开发Mtb IDH/ICL抑制剂外，还将开展类似的人类IDH2及其与癌症相关的变体（R140Q, R172K）的生化和生物物理研究。据报道，这些变异获得了一种新形态功能，产生2-羟基戊二酸酯（2HG），这是一种肿瘤代谢物，也是由Mtb IDH产生的(3)。这些研究将支持我们对结核病和人类异柠檬酸代谢的理解；因此，可以为新的癌症治疗铺平道路。该项目属于英国生物科学委员会的研究领域“对抗抗菌素耐药性”，并符合“了解已知耐药性流行的生物体的基本微生物学，以便了解耐药性是如何产生和维持的，并制定缓解战略”和“支持开发新型抗菌素和抗菌素替代品”的优先事项。该项目涉及牛津大学（University of Oxford）、弗朗西斯•克里克研究所（Francis Crick Institute）和GSK。

In 2016, 1.3 million people died from tuberculosis (TB),(1) which is caused by Mycobacterium tuberculosis (Mtb), a pathogen that is rarely treated with common antibiotics. Multi-drug treatments are available but the long-term (at least six months) strict treatment regimens pose challenges for patients. The emergence of Mtb resistant strains is making treatment increasingly ineffective. In 2016, 490,000 cases of multidrug-resistant TB were reported.(1) Key characteristics for the survival of Mtb involve its metabolic flexibility and its ability to persist in a highly antibiotic-resistant dormant state.(2) An important metabolic feature of mycobacteria is the glyoxylate shunt, a pathway that exists in most prokaryotes and enables Mtb to grow on C2 carbon sources such as fatty acids. It partly bypasses the TCA cycle, in which isocitrate dehydrogenase (IDH) catalyses the oxidative decarboxylation of 2-oxoglutarate (2OG) giving isocitrate, which is then decarboxylated to succinyl-CoA by 2OG dehydrogenase. Succinate is then generated via an oxidative phosphorylation as catalysed by succinyl-CoA synthetase. While the decarboxylation steps in the TCA cycle lead to the loss of two carboxyl groups as CO2, the glyoxylate shunt bypasses these steps, hence enabling simple carbon source compounds to be used for macromolecule synthesis (e.g. glucose).Isocitrate lyase (ICL), the first enzyme in the glyoxylate shunt, converts isocitrate (instead of 2OG) into glyoxylate and succinate directly. ICL is upregulated in Mtb in certain non-replicating states. An icl knockout attenuated the persistence and virulence of Mtb.(2) Thus, the glyoxylate shunt is important for Mtb to survive inside the host and finding inhibitors of IDH and/or ICL may enable new treatment strategies. Detailed biochemical and biophysical studies of the bifurcating point between the TCA cycle and the glyoxylate shunt are required for understanding the metabolic pathways in Mtb and developing inhibitors for them are important for validating isocitrate metabolising enzymes as drug targets. Therefore, my work is aimed at biochemical and biophysical studies on IDH and ICL and their regulation, including kinetic, mass spectrometry (MS), crystallography and nuclear magnetic resonance spectroscopy (NMR) studies, as well as early stage inhibitor development. Accordingly, I will be screening existing in-house libraries against these enzymes as well as inhibitor libraries provided by GSK. The potency, selectivity and binding-mode of hit molecules will be determined by biochemical, biophysical and crystallographic studies; and will further inform on chemical inhibitor optimisation. Potent inhibitors identified in my screens will be tested against mycobacteria cells at the Francis-Crick Institute. The results will yield a more detailed understanding of the role of the glyoxylate shunt and its regulation as well as yield inhibition information of the metabolism in mycobacteria. Alongside the development of inhibitors for Mtb IDH/ICL, analogous biochemical and biophysical studies will be carried out on human IDH2 and its variants which are involved in cancer (R140Q, R172K). These variants are reported to gain a neomorphic function to produce 2-hydroxyglutarate (2HG), an oncometabolite which is also produced by Mtb IDH(3). These investigations will support our understanding of the isocitrate metabolism in both tuberculosis and humans; and thus, could additionally pave the way to new cancer therapeutics.This project falls within the BBSRC research area "Combatting antimicrobial resistance" and aligns with the priorities to "Understand the fundamental microbiology of organisms with known resistance prevalence in order to understand how resistance develops and is maintained, and to develop mitigation strategies" and "Underpin the development of novel antimicrobials and alternatives to antimicrobials". It involves the University of Oxford, the Francis Crick Institute and GSK.