Novel Antibacterial Drugs Targeting DNA Repair Enzymes

描述（由申请人提供）：细菌感染后，宿主细胞释放破坏细菌DNA的化学物质。这种损害如果不加以修复，就会导致细菌死亡。基于重组的DNA修复蛋白，特别是RecBCD和AddAB解旋酶核酸酶，是一类新型抗菌药物的极好靶点，因为这些蛋白1)是DNA修复的主要途径所必需的，广泛分布于细菌中，但显然不存在于真核生物中，2)在几种不同病原体感染期间是特异性需要的，3)有助于诱导突变，导致对现有抗生素的耐药性。针对这些酶的药物应该能够自我限制细菌耐药性的进化。我们将首先在大肠杆菌细胞中筛选RecBCD的小分子抑制剂，使用一种简单、快速、灵敏的核酸酶活性测定方法。有趣的小分子将进一步测试在大肠杆菌中抑制recbcd介导的DNA修复和同源重组，以及在核酸酶和解旋酶实验中抑制纯化酶。我们将使用同样的方法从重要的胃病原体幽门螺杆菌中寻找AddAB的抑制剂。我们已经证明addAB缺失突变体的定植能力受损；因此，抑制AddAB活性可以限制细菌感染。这种方法应该使我们能够从其他致病菌中识别出针对类似酶的药物。这些药物将限制细菌耐药性的进化，并使传染病得到更有效的治疗。公共卫生相关性：我们的长期目标是发现和开发一类新的抗菌药物，这将提供一种新的手段来对抗细菌感染，面对细菌对许多目前使用的抗生素的耐药性。一旦被细菌感染，宿主细胞就会释放出破坏细菌DNA的化学物质。这种损害如果不加以修复，就会导致细菌死亡。基于重组的DNA修复蛋白是一类新型抗菌药物的极好靶点，因为这些蛋白的抑制剂会削弱DNA修复并导致细菌死亡。针对这些酶的药物也应该限制细菌耐药性的进化，并允许更有效地治疗传染病。

DESCRIPTION (provided by applicant): Upon bacterial infection, host cells release chemicals that damage the bacterial DNA. Such damage, if left unrepaired, leads to bacterial death. Recombination-based DNA repair proteins, specifically the RecBCD and AddAB helicase-nucleases, are excellent targets for a new class of anti- bacterial agents because these proteins 1) are required for the major pathway of DNA repair and are widely distributed in bacteria but apparently absent from eukaryotes, 2) are specifically required during infection by several diverse pathogens, and 3) contribute to induced mutation that causes resistance to existing antibiotics. Drugs against these enzymes should self-limit evolution of bacterial resistance. We will first screen for small-molecule inhibitors of RecBCD in Escherichia coli cells, using a simple, quick, and sensitive assay of nuclease activity in an easily grown E. coli strain. Interesting small molecules will be further tested for inhibition of RecBCD-mediated DNA repair and homologous recombination in E. coli and inhibition of purified enzyme in nuclease and helicase assays. We will use this same approach to look for inhibitors of AddAB from the important gastric pathogen Helicobacter pylori. We have shown that addAB deletion mutants have impaired colonization ability; thus, inhibiting AddAB activity should limit bacterial infections. This approach should allow us to identify drugs against similar enzymes from other pathogenic bacteria. These drugs should limit evolution of bacterial resistance and allow more effective treatment of infectious diseases. PUBLIC HEALTH RELEVANCE: Our long-term objective is to discover and develop a novel class of antibacterial drugs that will provide a new means to combat bacterial infections in the face of bacterial resistance to many currently used antibiotics. Upon bacterial infection, host cells release chemicals that damage the bacterial DNA. Such damage, if left unrepaired, leads to bacterial death. Recombination-based DNA repair proteins are excellent targets for a new class of anti-bacterial agents because inhibitors of these proteins will cripple DNA repair and lead to bacterial death. Drugs against these enzymes should also limit evolution of bacterial resistance and allow more effective treatment of infectious diseases.