Screen for synthetic lethality in Mycobacterium tuberculosis

描述(申请人提供)：结核病已被宣布为世卫组织全球卫生紧急情况，最近出现的多药耐药(MDR)和广泛耐药(XDR)结核病突显了对新的结核病药物的迫切需要。我们假设HTS将识别出对结核分枝杆菌(M.TB)具有化学敏感性的探针。对现有的2-内酰胺类和大环内酯类抗生素的活性。这种探针的鉴定可能导致类似于阿莫西林-克拉维酸的组合抗生素，它们对结核分枝杆菌具有效力。这项建议寻求访问分子图书馆筛选中心网络(MLSCN)提供的HTS资源，目的是识别敏化探针，识别其分子靶标，并寻找候选试剂作为潜在的先导结构进行进一步研究。一种HTS就绪的、基于荧光的全细胞筛选试验被提出，它将利用毒力强的结核分枝杆菌。与亚胺培南(IMI，代表2-内酰胺类)和克拉霉素(CLA，代表大环内酯类)的优化浓度相结合。提出了以下目标，以对对结核分枝杆菌具有合成致命性的探针进行有效的HTS评估。在2-内酰胺和大环内酯类抗生素存在的情况下：将结核分枝杆菌/佐剂药物敏感性方案转移到指定的筛选中心，以复制、小型化和自动化相应的HTS分析，该方法将用于筛选MLSCN化合物文库以增敏探针，提供技术支持，并进行二次检测，以确认所识别的探针的效力和特异性。 公共卫生相关性：结核病已被宣布为世卫组织全球卫生紧急情况，最近出现的多药耐药(MDR)和广泛耐药(XDR)结核病突显了对新的结核病药物的迫切需求；事实上，许多抗生素，如青霉素、红霉素和四环素家族的抗生素，由于细菌渗透性差或微生物对药物的降解，在临床上对治疗结核病没有用处。我们的初步研究表明，特定的突变可以使结核分枝杆菌(M.TB)增敏。对青霉素和红霉素的敏感性，从而支持了这样一种假设，即可能会发现也会使微生物对这些熟悉类别的抗生素的活性敏感的新药。我们已经开发了一种微量平板法来寻找能够放大青霉素类和红霉素类的关键成员对结核分枝杆菌的活性的化合物，并建议将该分析转移到适当的MLSCN实验室进行高通量筛选，以确定潜在的先导化合物。

DESCRIPTION (provided by applicant): Tuberculosis has been declared a WHO global health emergency, and the recent emergence of multidrug (MDR) and extensively drug resistant (XDR) TB has underscored the urgent need for new TB drugs. We hypothesize that HTS will identify probes which chemically sensitize Mycobacterium tuberculosis (M. tb.) to the activity of existing 2-lactam and macrolide antibiotics. The identification of such probes could lead to combination antibiotics, similar to amoxicillin-clavulanate, which have potency against M. tb. This proposal seeks access to the HTS resources provided by the Molecular Libraries Screening Center Network (MLSCN) with the goal of identifying sensitizing probes, identifying their molecular targets, and finding candidate agents as potential lead structures for further study. An HTS-ready, fluorescence-based whole cell screening assay is proposed that will utilize virulent M. tb. in combination with optimized concentrations of imipenem (IMI, representing the 2-lactam class) and clarithromycin (CLA, representing the macrolide class). The following aims are proposed to conduct an efficient HTS evaluation for probes that are synthetically lethal against M. tb. in the presence of 2-lactam and macrolide antibiotics: to transfer the M. tb./adjuvant drug susceptibility protocol to the designated screening center to reproduce, miniaturize, and automate a corresponding HTS assay that will be used to screen the MLSCN compound library for sensitizing probes, to provide technical support, and to conduct secondary assays confirming the potency and specificity of identified probes. PUBLIC HEALTH RELEVANCE: Tuberculosis has been declared a WHO global health emergency, and the recent emergence of multidrug (MDR) and extensively drug resistant (XDR) TB has underscored the urgent need for new TB drugs; indeed, many antibiotics such as those from the penicillin-, erythromycin-, and tetracycline-families are not clinically useful in the treatment of TB either because of poor bacterial penetration or drug degradation by the microbe. Our preliminary studies reveal that specific mutations can sensitize Mycobacterium tuberculosis (M. tb.) to penicillin and erythromycin, thus supporting the hypothesis that new drugs may be identified that would also sensitize the microbes to the activity of these familiar classes of antibiotics. We have developed a microtiter plate assay to seek compounds which amplify the activity of key members of the penicillin- and the erythromycin-class against M. tb., and we propose to transfer this assay to an appropriate MLSCN laboratory for high-throughput screening to identify potential lead compounds.