Identification of natural products targeting new pathways in bacteria

描述(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品的鉴定，这种方法可以快速区分 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衡量抗生素治疗对单个细胞的影响。针对不同细胞途径和途径中不同步骤的抗生素产生独特的细胞学特征，允许在几个小时内识别新化合物可能的MOA。我们现在已经开发出一种互补的方法，将使我们能够识别抑制蛋白质的分子，这些蛋白质目前不是抗菌药物的靶标。这种方法，我们称之为快速抑制谱(R.I.P.)，需要快速、可诱导地耗尽目标蛋白质，然后进行细胞学谱分析。我们的初步数据表明，R.I.P.耗尽药物靶点会产生与相应药物相同的细胞学效应。此外，耗尽当前抗菌药物没有靶点的必要蛋白质会产生新的细胞学特征，随后可以用来识别抑制这些新靶点的分子。我们在这里建议通过在大肠杆菌和枯草杆菌基因组的基础上应用RIP技术来更全面地开发RIP技术。这将创建一套与抑制关键细胞通路相关的全面参考集，这些途径不是当前抗菌药物的靶标。全基因组的RIP分析还将提供对保守基因功能的洞察，我们的初步数据表明，它将提供对协调两条或更多生物合成途径的蛋白质的洞察，为未来的基础研究提供有趣的起点。然后，我们将使用BCP的这个更完整的参考数据集来筛选独特和多样化的天然产品提取物集合，以识别那些抑制这些新药物靶点并杀死多药耐药的化合物 细菌。然后，我们将与我们在Fundacn Medina的合作者一起，提纯和表征我们最优先考虑的铅分子(那些杀死耐多药细菌的分子)，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.