Targeting Biotin Metabolism in Mycobacterium Tuberculosis

总结 结核分枝杆菌（Mtb）是结核病（TB）的主要病原体，感染超过三分之一的 目前，单一病原体是导致传染病死亡的主要原因。结核分枝杆菌需要生物素 并重新合成这种必需的辅因子。在使用遗传方法的初步研究中， 我们已经表明生物素生物合成和连接对于小鼠中的Mtb感染是必需的。我们合成了一种 生物素蛋白连接酶的选择性纳摩尔抑制剂，称为Bio-AMS，其靶向生物素蛋白酶 连接酶（BPL）负责将生物素连接到生物素依赖性酶上。我们还确定了 天然产物酸霉素，其靶向于BioB催化的生物素生物合成的最后一步。然而，生物- AMS和酸霉素在药物处置特性方面有缺陷，导致清除迅速，体积小， 分布和有限的口服生物利用度。我们对它们的机制的了解也存在空白 与其他结核病药物联合使用时的耐药性和活性本申请的目的是：1） 通过优化ADME（吸收， 分布、代谢和消除）特性和药代动力学参数转化为可行的临床前 候选人，2）更深入地阐明Mtb的作用机制和耐药性，3）确定 安全性特征和潜在的药物-药物相互作用，以及4）确定与其他TB药物的相互作用（即， 协同作用）。我们将通过追求三个具体目标来实现本申请的总体目标。在目标1中， 我们将进行一个迭代的基于结构的Bio-AMS和酸性霉素的药物化学计划， 使用以下组合同时优化药代动力学（PK）参数和全细胞活性： 方法包括简化、结构简化和引入构象约束。在aim中 2，我们将进行生化和细胞研究，以评估酶抑制，靶向接合，细胞 积累和针对Mtb以及耐药菌株的全细胞活性。抗性世代 菌株随后进行全基因组测序将用于表征潜在的耐药机制 并确定电阻频率。最后，各种一线和二线结核病的联合研究 将开展药物研究，以评估协同作用的潜力。在目标3中，Bio-AMS和酸霉素类似物将 在体内进行评估，以确定其完整的药代动力学参数，目的是改善 分布容积（Vd）、固有清除率（CL）和生物利用度（F）。我们将评估化合物 对照一组试验（hERG、β-内酰胺酶抑制、艾姆斯致突变性），以确保安全性和选择性。体内 将使用急性和慢性结核病感染的小鼠模型进行疗效研究

SUMMARY Mycobacterium tuberculosis (Mtb), the principal etiological agent of tuberculosis (TB), infects over one-third of humanity and is now the leading cause of infectious disease mortality by a single pathogen. Mtb requires biotin for survival and synthesizes this essential cofactor de novo. In preliminary studies using a genetic approach, we have shown biotin biosynthesis and ligation are essential for Mtb infection in mice. We have synthesized a selective nanomolar inhibitor of biotin protein ligase termed Bio-AMS that targets the enzyme biotin protein ligase (BPL,) responsible for the ligation of biotin onto biotin-dependent enzymes. We have also identified the natural product acidomycin, which targets the final step of biotin biosynthesis catalyze by BioB. However, Bio- AMS and acidomycin have liabilities in their drug disposition properties leading to rapid clearance, poor volume of distribution, and limited oral bioavailability. There are also gaps in our knowledge regarding their mechanism of resistance and activity when combined with other TB drugs. The objectives of this application are: 1) to develop our lead compounds Bio-AMS and acidomycin through the optimization of their ADME (absorption, distribution, metabolism and elimination) properties and pharmacokinetic parameters into viable preclinical candidates, 2) to more deeply illuminate the mechanism of action and resistance in Mtb, 3) to determine the safety profile and potential drug-drug interactions, and 4) to identify interactive effects with other TB drugs (i.e. synergy). We will accomplish the overall objectives of this application by pursuing three specific aims. In aim 1, we will carry out an iterative structure-based medicinal chemistry program of Bio-AMS and acidomycin to concurrently optimize pharmacokinetic (PK) parameters and whole-cell activity using a combination of approaches including fluorination, structural simplification, and introduction of conformation constraints. In aim 2, we will perform biochemical and cellular studies to evaluate enzyme inhibition, target engagement, cellular accumulation, and whole-cell activity against Mtb as well as drug-resistant strains. Generation of resistant strains followed by whole-genome sequencing will be used to characterize potential resistance mechanisms and determine the resistance frequency. Finally, combination studies with various first and second-line TB drugs will be undertaken to assess potential for synergy. In aim 3, the Bio-AMS and acidomycin analogues will be assessed in vivo to determine their complete pharmacokinetic parameters with a goal to improve on the volume of distribution (Vd), intrinsic clearance (CL), and bioavailability (F). We will evaluate compounds against a panel of assays (hERG, CYP inhibition, Ames mutagenicity) to ensure safety and selectivity. In vivo efficacy studies will be done using murine models of acute and chronic TB infection