Screen for synthetic lethality in Mycobacterium tuberculosis

描述（由申请人提供）：结核病已被宣布为世卫组织全球卫生紧急事件，最近出现的多药耐药（MDR）和广泛耐药（XDR）结核病强调了对新结核病药物的迫切需求。我们假设HTS将识别化学致敏结核分枝杆菌的探针（M。结核病）现有的2-内酰胺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，其对M. TB.该提案寻求访问由分子库筛选中心网络（MLSCN）提供的HTS资源，其目标是鉴定致敏探针，鉴定其分子靶点，并找到候选药物作为进一步研究的潜在先导结构。提出了一种HTS就绪的基于荧光的全细胞筛选测定法，该测定法将利用毒性M。TB.与优化浓度的亚胺培南（IMI，代表2-内酰胺类）和克拉霉素（CLA，代表大环内酯类）组合。提出了以下目标，以进行有效的HTS评估的探针，是综合致命的M。TB.在2-内酰胺类和大环内酯类抗生素存在下，将M. tb./将辅助药物敏感性方案提交给指定的筛选中心，以重现、纯化和自动化相应的HTS试验，该试验将用于筛选MLSCN化合物文库中的致敏探针，提供技术支持，并进行二次试验，确认已鉴定探针的效价和特异性。 公共卫生相关性：结核病已被宣布为世界卫生组织的全球卫生紧急事件，最近出现的多药耐药（MDR）和广泛耐药（XDR）结核病强调了对新的结核病药物的迫切需要;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在治疗结核病方面没有临床用途，因为细菌渗透性差或微生物对药物的降解。我们的初步研究表明，特定的突变可以致敏结核分枝杆菌（M。结核病）青霉素和红霉素，从而支持的假设，新的药物可能会被确定，也将敏感的微生物对这些熟悉的抗生素类的活动。我们已经开发了一种微量滴定板测定法，以寻找能增强青霉素类和红霉素类关键成员抗M.结核病，我们建议将该检测转移到适当的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.