DMA 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 therapetuic 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 accumlated 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 stablized covalent complex will be studied. Sensitivity to topoisomerase I induced DNAlesion 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 suscepbility of bacteria to killing by trapped type IAtopoisomerase 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 represent 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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI069313-04
Application #
7541787
Study Section
Special Emphasis Panel (ZRG1-DDR-N (01))
Program Officer
Mukhopadhyay, Suman
Project Start
2006-02-01
Project End
2011-01-31
Budget Start
2009-02-01
Budget End
2010-01-31
Support Year
4
Fiscal Year
2009
Total Cost
$371,495
Indirect Cost
Name
New York Medical College
Department
Biochemistry
Type
Schools of Medicine
DUNS #
041907486
City
Valhalla
State
NY
Country
United States
Zip Code
10595
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Sandhaus, Shayna; Annamalai, Thirunavukkarasu; Welmaker, Greg et al. (2016) Small-Molecule Inhibitors Targeting Topoisomerase I as Novel Antituberculosis Agents. Antimicrob Agents Chemother 60:4028-36
Cheng, Bokun; Annamalai, Thirunavukkarasu; Sandhaus, Shayna et al. (2015) Inhibition of Zn(II) binding type IA topoisomerases by organomercury compounds and Hg(II). PLoS One 10:e0120022
Tse-Dinh, Yuk-Ching (2015) Targeting bacterial topoisomerase I to meet the challenge of finding new antibiotics. Future Med Chem 7:459-71

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