Using new modalities of synthetic-lethal interactions to find alternative antibacterial drug targets

7.项目摘要/摘要 该计划旨在为革兰氏阴性抗菌素寻找新的窄谱分子靶标。 通过利用合成-致命的相互作用。在过去的一个世纪里，天然产物的发现工作产生了 超过70%的临床抗生素；然而，令人沮丧的是，随着发现的步伐 下降，耐药率正在迅速增长。人类病原体伯克霍尔德氏菌也是如此。 类鼻疽，类鼻疽病的病原体，在世界部分地区是地方病或高度地方病。我们 建议使用泰兰伯克霍尔德氏菌，一种非致病性的革兰氏阴性细菌，也是B。 为发现具有新机制的抗生素，研究合成-致死相互作用 行动的一部分。合成致死性是指两个不单独致命的基因的致命性失活。我们是 使用类似的术语来指代两个原本无毒的小分子之间的相互作用(在使用的剂量下) 来创造一种合成-致命的组合。为此，我们已经证明了低剂量的抗生素 可以引起细菌次级代谢的变化，包括叶酸生物合成蛋白的上调 甲氧苄氨嘧啶(TMP)治疗。此外，foe2的缺失不会对生长造成缺陷，但对 存在其他非致命性剂量的TMP，表明FolE2的丢失和对TMP的部分抑制 二氢叶酸还原酶被TMP创造了一个合成致命的场景。因此，我们假设一个小的 FolE2的分子抑制剂将是我们抗菌库中的一个有价值的工具，使我们能够使用更低的 增加抗生素的剂量，从而增加其治疗窗口(目标1)。构建转座子突变体文库 将使我们能够研究TMP或其他小剂量抗生素结合功能丧失的影响 整个基因组中的突变，进一步探索相互作用，使我们能够发现潜在的新的 目标(目标2)。我们对细菌合成致死性的表征将为开发 不仅针对伯克霍尔德氏菌，而且对广泛的人类病原体使用一种全新的 接近。

7. Project Summary/Abstract The proposed project seeks to discover new narrow spectrum molecular targets for Gram-negative antimicrobials by exploiting synthetic-lethal interactions. Natural products discovery efforts in the past century have produced over 70% of the current collection of clinical antibiotics; however, frustratingly, as the pace of discovery has declined, the rate of resistance is rapidly growing. This is also the case for the human pathogen Burkholderia pseudomallei, the causative agent of melioidosis, which is endemic or hyperendemic in parts of the world. We propose to use Burkholderia thailandensis , a nonpathogenic Gram-negative bacterium and a model strain for B. pseudomallei , to investigate the synthetic-lethal interactions for the discovery of antibiotics with new mechanisms of action. Synthetic lethality refers to a lethal inactivation of two genes which are not individually lethal. We are using a similar terminology to refer to the interaction of two otherwise non-toxic small molecules (at doses used) to create a synthetic-lethal combination. To this end, we have already demonstrated that low doses of antibiotics can induce changes in bacterial secondary metabolism, including upregulation of the folate biosynthetic protein FolE2 by trimethoprim (TMP). In addition, the deletion of folE2 is not growth-defective, but is lethal in the presence of otherwise nonlethal doses of TMP, suggesting that loss of FolE2 and partial inhibition of dihydrofolate reductase by TMP create a synthetic-lethal scenario. Therefore, we hypothesize that a small molecule inhibitor of FolE2 would represent a valuable tool in our antimicrobial arsenal, allowing us to use lower doses of antibiotics, thereby increasing their therapeutic window (Aim 1). Creating a library of transposon mutants would allow us to study the effects of TMP or other low-dose antibiotics in combination with loss of function mutations across the entire genome, further probing interactions that would enable us to discover potential new targets (Aim 2). Our characterization of bacterial synthetic lethality would lay the groundwork for development of targeted therapies for not just Burkholderia, but a broad spectrum of human pathogens using an entirely new approach.