Development of New Therapeutics Against Bacillus Anthracis

Petrobactin是炭疽杆菌感染和致病所需的铁载体，是 一种由非核糖体多肽合成酶(NRPS)衍生的新铁络合剂家族 独立路径。汉纳实验室的初步研究表明，遗传基因座 (AS/？)炭疽杆菌在巨噬细胞中的生长需要负责拍动蛋白的生物合成 以及对小鼠的毒力。最近，我们揭示了铁载体的生物合成基础。 并对这三种“早期”酶进行了详细的生化研究，包括 Asb途径。对于这个竞争延续的GLRCE项目，我们建议进行研究，以确认 剩余的两种Asb酶(AsbB、AsbF)的催化功能和机制，并发展强劲 生物合成抑制物的功能分析。在这项工作中，高吞吐量 密歇根大学中心的筛选、化学多样性和化学信息学能力 将利用化学基因组学来发现和开发新的抗感染药物 靶向这些非NRPS铁载体合成途径。具体目标包括： 1.继续确定AsbF、Asba、AsbB的高效力化学和天然产物抑制剂 来自对体外高通量筛选的点击(与化学中心合作 基因组学(CCG，U-M)，并通过体外生化和体内实验表征其疗效 生长抑制研究[与密歇根大学菲尔·汉纳博士合作]。 2.研究AsbF作为3-脱氢莽草酸的生化机制 3，4-二羟基苯甲酸前体生物合成的脱水酶。继续评估 AsbB的生化作用，包括底物耐受性和动力学参数。追求 对6种Asb蛋白进行了结晶学分析，为下一步的抑制剂研究提供依据 设计研究[与Andrzej Joachimiak博士合作]。

Petrobactin, a siderophore required for Bacillus anthracis infection and pathogenesis is a member of a new family of iron-chelating agents derived by a non-ribosomal peptide synthetase (NRPS) independent pathway. Initial studies in the Hanna laboratory demonstrated that the genetic locus (as/?) responsible for petrobactin biosynthesis is required for growth of B. anthracis in macrophages and virulence in mice. More recently, we have revealed the biosynthetic basis for siderophore assembly, and have pursued detailed biochemical studies on the three "early" enzymes comprising the Asb pathway. For this competing continuation GLRCE project, we propose studies to confirm the catalytic function and mechanism of two remaining Asb enzymes (AsbB, AsbF), and develop robust functional assays to identify inhibitors of petrobactin biosynthesis. In this work, high throughput screening, chemical diversity, and chemoinformatic capabilities of the University of Michigan Center for Chemical Genomics will be harnessed in order to discover and develop new anti-infective agents targeting these NRPS-independent siderophore synthesis pathways. The specific objectives include: 1. Continue to identify highly potent chemical and natural product inhibitors of AsbF, AsbA, AsbB from hits against in vitro high throughput screens (Collaboration with the Center for Chemical Genomics (CCG), U-M) and characterize their efficacy through in vitro biochemical and in vivo growth inhibition studies [Collaboration with Dr. Phil Hanna, U-M]. 2. Characterize the biochemical mechanism of AsbF, shown to function as a 3-dehydroshikimate dehydratase for 3,4-dihydroxybenzoic acid precursor biosynthesis. Continue to assess the biochemical role of AsbB, including substrate tolerance and kinetic parameters. Pursue crystallographic analysis on each of the six Asb proteins to provide the basis for future inhibitor design studies [Collaboration with Dr. Andrzej Joachimiak, ANL].