Molecular target of a novel broad-spectrum antiviral and antibacterial compound

 描述（由申请人提供）：新的抗感染策略的重要性怎么强调都不过分。由以前未知的感染原引起的新病症的出现，加上已知和新型耐抗生素病原体的迅速增加，导致了治疗选择的危机，需要新的思维方式。解决这个问题的一种方法是开发可以解决问题的各个方面的新型抗感染化合物。我们的实验室已经鉴定并表征了一种新型的基于硫脲的小分子，称为ST-669，其对多种病毒以及细胞内细菌衣原体和贝氏柯克斯体具有低微摩尔活性。病毒对 ST-669作用包括牛痘（EC-50 = 0.25微摩尔）、埃博拉、流感、HIV-1、登革热、脑心肌炎病毒和长曲棍球病毒。（EC-50 = 0.27微摩尔）。本项目的总体目标是确定ST-669的作用机制。该化合物仅在灵长类来源的细胞中具有活性，并且活性对用放线菌酮处理细胞敏感。围绕ST- 669活性收集的数据使我们假设该化合物通过上调宿主蛋白质或代谢途径起作用。因此，我们的两个目标使用互补的方法来敲除（目标1）或敲低（目标2）人源细胞中所有可能的基因，并寻找消除化合物抑制作用的单个突变。在目标1中，将使用逆转录病毒基因陷阱方法随机突变单倍体人细胞系，并筛选收集的突变群体以消除ST-669活性。在目标2中，我们将在高通量筛选策略中使用全局siRNA文库来敲低个体蛋白质丰度，并检查经处理的细胞是否缺乏ST-669活性。在实验室常规使用的高通量试验中，将在该筛选方法中使用衣原体生长。将对每项试验进行二次筛选，并检查所得数据的重复性和不一致性。这些结果将使我们的实验室能够开发假设驱动的实验，以阐明ST-669的特定作用机制，并设计在未来小分子筛选中靶向该途径的方法。这些实验还可以确定细胞用于控制非常不同的细胞内病原体感染的新工具。

 DESCRIPTION (provided by applicant): The importance of new anti-infective strategies cannot be overstated. Emergence of new conditions caused by previously unknown infectious agents, in combination with the rapid increase in known and novel antimicrobial-resistant pathogens, has led to crises in therapeutic options that require new ways of thinking. One approach to this problem is the development of novel anti-infective compounds that can address aspects of the problem. Our laboratories have identified and characterized a novel thiourea-based small molecule, termed ST-669 that has low-micromolar activity against a wide variety of viruses as well as the intracellular bacteria Chlamydia and Coxiella burnetii. Viruses sensitive to the action of ST-669 include vaccinia (EC-50 = 0.25 micromolar), ebola, influenza, HIV-1, dengue, encephalomyocarditis virus, and Lacrosse virus. (EC-50 = 0.27 micromolar). The overall goal of this project is to identify the mechanism of action of ST-669. The compound is active only in cells of primate origin and activity is sensitive to treatment of cells with cycloheximide. The collected data surrounding ST- 669 activity lead us to hypothesize that the compound works by upregulating a host protein or metabolic pathway. Therefore, our two aims use complementary approaches to knock out (Aim 1) or knock down (Aim 2) all possible genes in cells of human origin and looking for individual mutations that abrogate the inhibitory effects of the compound. In Aim 1, a haploid human cell line will be randomly mutated using a retroviral gene trap approach and the collected mutated populations screened for elimination of ST-669 activity. In Aim 2, we will use a global siRNA library in a high throughput screening strategy to knock down individual protein abundance, and examine treated cells for lack of ST-669 activity. Growth of Chlamydia caviae will be used in this screening approach, in a high throughput assay routinely used in the laboratory. Secondary screens for each assay will be conducted and the resulting data examined for consistencies and inconsistencies. These results will position our laboratories to develop hypothesis-driven experiments to elucidate the specific mechanism of action of ST-669, and to design approaches to target this pathway in future small molecule screens. These experiments may also identify novel tools used by cells to control infections by very different groups of intracellular pathogens.