Identification of natural products targeting new pathways in bacteria

描述（由申请人提供）：我们开发了一种新的方法来促进具有抗菌活性的天然产物的鉴定， 不同的作用机制（MOA）。这种方法，细菌细胞学分析（BCP），使用定量荧光显微镜来测量抗生素治疗对单个细胞的影响。靶向不同细胞途径和途径内不同步骤的抗生素产生独特的细胞学特征，允许在几小时内鉴定新化合物的可能MOA。我们现在已经开发出一种互补的方法，使我们能够识别抑制目前抗菌药物不靶向的蛋白质的分子。这种方法，我们称之为快速抑制分析（R.I.P.），需要快速的，可诱导的目标蛋白的消耗，然后进行细胞学分析。我们的初步数据表明，通过R.I.P.消耗药物靶点产生的细胞学效应与相应药物产生的效应相同。此外，目前的抗菌药物不靶向的必需蛋白质的消耗产生了新的细胞学特征，随后可用于鉴定抑制这些新靶点的分子。我们在此建议通过在大肠杆菌全基因组的基础上使用R.I.P.技术来更充分地发展R.I.P.技术。coli和B.枯草杆菌。这将创建一个与抑制基本细胞途径相关的综合参考谱集，这些途径不是当前抗菌药物的靶点。全基因组R.I.P.分析还将深入了解保守基因的功能，我们的初步数据表明，它将深入了解协调两个或多个生物合成途径的蛋白质，为未来的基础研究提供有趣的起点。然后，我们将使用这一更完整的参考数据集与BCP筛选一个独特的和多样化的收集天然产品提取物，以确定那些抑制这些新的药物靶点和杀死多药耐药 细菌然后，我们将与我们在麦地那基金会的合作者一起，纯化和表征我们最优先的铅分子（那些杀死多药耐药细菌的分子），目标是将它们推进毒性试验。

DESCRIPTION (provided by applicant): We developed a new approach to facilitate identification of natural products with antibacterial activities that rapidly discriminates between different mechanisms of action (MOA). This approach, bacterial cytological profiling (BCP), uses quantitative fluorescence microscopy to measure the effects of antibiotic treatment on individual cells. Antibiotics that target different cellular pathways and different steps within a pathway generate unique cytological profiles, allowing identification of the likely MOA of new compounds within a few hours. We have now developed a complimentary approach that will allow us to identify molecules that inhibit proteins that are not currently targeted by antibacterial drugs. Ths approach, which we call rapid inhibition profiling (R.I.P.), entails the rapid, inducible depletionof a target protein, followed by cytological profiling. Our preliminary data demonstrate that depletion of a drug target by R.I.P. produces cytological effects identical to those produced by the corresponding drug. Furthermore, depletion of essential proteins that are not targeted by current antibacterial drugs produces novel cytological profiles that can be subsequently used to identify molecules that inhibit these new targets. We here propose to more fully develop the R.I.P. technology by employing it on a genome wide basis in E. coli and B. subtilis. This will create a comprehensive reference set of profiles associated with the inhibition of essential cellular pathways that are not the targets of current antibacterial drugs. The genome-wide R.I.P. analysis will also provide insight into the function of conserved genes and our preliminary data suggests that it will provide insight into proteins that coordinate two or more biosynthetic pathways, providing interesting starting points for future basic research. We will then use this more complete reference data set with BCP to screen a unique and diverse collection of natural product extracts to identify those that inhibit these new drug targets and kill multidrug resistant bacteria. Together with our collaborators at Fundaci¿n Medina, we will then purify and characterize our highest priority lead molecules (those that kill multidrug resistant bacteria) wit a goal of advancing them into toxicity trials.