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

7.项目总结/摘要 拟议的项目旨在发现革兰氏阴性抗菌剂的新窄谱分子靶标 通过利用合成致命的相互作用。在过去的世纪中，天然产物的发现努力已经产生了 超过70%的当前临床抗生素集合;然而，令人沮丧的是，随着发现的步伐 下降，耐药率迅速上升。人类病原体伯克霍尔德氏菌也是如此 假鼻疽是类鼻疽病的病原体，类鼻疽病在世界部分地区是地方病或高地方病。我们 建议使用Burkholderia thailandensis，一种非致病性革兰氏阴性细菌和B的模式菌株。 类鼻疽，研究合成-致死相互作用，以发现具有新机制的抗生素 的行动。合成致死性是指两个单独不致死的基因的致死性失活。我们 使用类似的术语来指两种其他无毒小分子的相互作用（在所用剂量下） 来制造一种合成致命组合为此，我们已经证明，低剂量的抗生素 可诱导细菌次级代谢的变化，包括叶酸生物合成蛋白的上调 FolE 2通过甲氧苄啶（TMP）。此外，folE 2的缺失不是生长缺陷的，但在肿瘤中是致命的。 存在其他非致死剂量的TMP，这表明FolE 2的丧失和部分抑制 二氢叶酸还原酶通过TMP产生合成致死情况。因此，我们假设一个小的 FolE 2的分子抑制剂将是我们抗菌药物库中的一个有价值的工具，使我们能够使用更低的剂量。 剂量的抗生素，从而增加其治疗窗口（目标1）。创建转座子突变体库 这将使我们能够研究TMP或其他低剂量抗生素与功能丧失联合使用的效果 整个基因组的突变，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，而且还使用一种全新的 approach.

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.