Bacterial cell killing topoisomerase I--DNA lesion

描述(申请人提供)：DNA拓扑异构酶是参与DNA复制、转录和重组的普遍存在的酶。众所周知，被切割的DNA与II型或IB型DNA拓扑异构酶之间形成的共价复合体被治疗剂捕获后，可以杀死癌细胞或细菌细胞。IA型DNA拓扑异构酶是一类很有前途的新型治疗药物靶点，但针对IA型DNA拓扑异构酶的分子尚未发现。我们已经确定了鼠疫杆菌拓扑异构酶I的一个突变体，由于与切割的DNA形成的共价复合体的稳定，该突变体在大肠杆菌中表达时可以导致广泛的杀伤。这是首次证明了由IA型DNA拓扑异构酶形成的累积共价复合体对细菌细胞的杀伤作用。该项目的具体目标是：1.将在哈佛医学院的NSRB设施中利用高通量测试来筛选可用于小分子的150,000种化合物，这些化合物将导致重组鼠疫杆菌拓扑异构酶I形成的共价复合体的积累。HITS将以体外拓扑异构酶分析和细菌细胞杀灭为特征，并与NSRB的药物化学家合作进一步开发。2.为了探讨稳定型IA型DNA拓扑异构酶共价复合体对细菌细胞的杀灭机制，本实验研究了表达突变的鼠疫杆菌拓扑异构酶I形成稳定型共价复合体的大肠杆菌。比较不同生长条件下细胞对拓扑异构酶I诱导DNA损伤的敏感性，以确定DNA复制和蛋白质合成在细胞杀伤机制中的作用。3.为了确定影响细菌对捕获的IA型拓扑异构酶裂解复合体的敏感性的其他因素，我们将研究具有重组和修复途径突变的大肠杆菌。多拷贝质粒中的大肠杆菌基因组文库将用于鉴定在较高水平表达时可对拓扑异构酶I介导的细胞杀伤产生抵抗力的蛋白质。将进行转座子突变，以筛选对细胞杀伤具有更高敏感性或抵抗力的突变株。 对所有常见抗生素产生抗药性的病原菌的出现对公共卫生构成了严峻的挑战。未来使用细菌病原体的恐怖袭击可能涉及对当前抗生素具有抗药性的制剂。这项研究有可能导致发现一类新的抗生素。

DESCRIPTION (provided by applicant): DNA topoisomerases are ubiquitous enzymes involved in DNA replication, transcription and recombination. It is well known that trapping of covalent complexes formed between cleaved DNA and type II or type IB DNA topoisomerases by therapeutic agents leads to the killing of cancer or bacterial cells. The class of type IA DNA topoisomerases is a promising target for novel therapeutic agents, but molecules targeting type IA DNA topoisomerases have not been discovered. We have identified a mutant of Yersinia pestis topoisomerase I that can result in extensive killing when expressed in E. coli cells due to the stabilization of the covalent complex with cleaved DNA. This is the first demonstration of bacterial cell killing from accumulated covalent complex formed by a type IA DNA topoisomerase. The specific aims of the project are: 1. A high-through-put assay will be utilized at the NSRB facility at Harvard Medical School to screen the 150,000 compounds available for small molecules that will result in accumulation of covalent complex formed by recombinant Yersinia pestis topoisomerase I. Hits will be characterized by in vitro topoisomerase assay and bacterial cell killing and further developed in collaboration with medicinal chemists at NSRB. 2. To investigate the mechanism of bacterial cell killing by stabilized type IA DNA topoisomerase covalent complex, E. coli expressing the mutant Y. pestis topoisomerase I that forms the stabilized covalent complex will be studied. Sensitivity to topoisomerase I induced DNA lesion will be compared under different growth conditions to determine the role of DNA replication and protein synthesis in the cell killing mechanism. 3. To identify other factors influencing the susceptibility of bacteria to killing by trapped type IA topoisomerase cleaved complex, E. coli strains with mutations in recombination and repair pathways will be studied. An E. coli genomic library in a multi-copy plasmid will be used to identify proteins that when expressed in a higher level, can confer resistance to topoisomerase I mediated cell killing. Transposon mutagenesis will be carried out to screen for mutants with increased sensitivity or resistance to the cell killing. The emergence of pathogenic bacteria resistant to all common antibiotics represents a critical challenge in public health. Future terrorist attacks employing bacterial pathogens could involve agents resistant to current antibiotics. This research has the potential to lead to the discovery of a novel class of antibiotics.