Targeting Biotin Metabolism in Mycobacterium Tuberculosis

摘要 结核分枝杆菌(Mtb)是结核病(TB)的主要病原体，感染了超过三分之一的 目前已成为单一病原体导致传染病死亡的主要原因。结核分枝杆菌需要生物素 以求生存并合成这种重要的从头开始的辅因子。在使用遗传学方法的初步研究中， 我们已经证明生物素的生物合成和连接在小鼠结核分枝杆菌感染中是必不可少的。我们已经合成了一种 靶向生物素蛋白连接酶的选择性纳米分子生物素蛋白连接酶抑制剂 连接酶(BPL)，负责将生物素连接到依赖生物素的酶上。我们还确定了 天然产物酸性霉素，目标是生物素生物合成的最后一步，由Biob催化。然而，Bio- AMS和阿霉素类药物在药物处置性质上存在缺陷，导致清除快、容量小 分布的局限性和有限的口服生物利用度。我们对它们的机制也有认识上的空白 当与其他结核病药物联合使用时，耐药性和活性的变化。本申请的目标是：1) 开发我们的先导化合物Bio-AMS和阿霉素ADME(吸收， 分布、代谢和消除)性质和药代动力学参数进入活性临床前 候选人，2)更深入地阐明结核分枝杆菌的作用和抗性机制，3)确定 安全性概况和潜在的药物-药物相互作用，以及4)确定与其他结核病药物的相互作用效果(即 协同效应)。我们将通过追求三个具体目标来实现此应用程序的总体目标。在目标1中， 我们将开展基于Bio-AMS和阿霉素的迭代结构药物化学计划，以 同时优化药代动力学(PK)参数和全细胞活性 方法包括氟化、结构简化和引入构象约束。在AIM 2、我们将进行生化和细胞研究，以评估酶抑制、靶标结合、细胞 对结核分枝杆菌和耐药菌株的累积和全细胞活性。抗病世代 随后进行全基因组测序的菌株将被用于表征潜在的耐药性机制。 并确定电阻频率。最后，与各种一线和二线结核病的联合研究 将进行药物开发，以评估协同增效的潜力。在目标3中，Bio-AMS和阿霉素类似物将 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 分布体积(Vd)、固有清除量(CL)和生物利用度(F)。我们将评估化合物 对一系列检测(HERG、CYP抑制、Ames致突变性)进行检测，以确保安全性和选择性。活体内 疗效研究将使用急性和慢性结核病感染的小鼠模型进行

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