Topoisomerases are ubiquitous enzymes that enable vital cellular processes by overcoming topological barriers in DNA. Bacterial topoisomerase I affects global and local DNA supercoiling influencing DNA replication, transcription, recombination and repair. Small molecules that target bacterial topoisomerase I specifically during its catalytic cycle would be very useful tools for studying the dynamics of DNA supercoiling and its relationship with bacterial physiology. There is also an urgent need to identify new leads for development of novel antibacterial drugs to combat multi-drug resistant pathogens. The lack of drugs in the pipeline for gram-negative bacteria is especially critical. Yersinia pestis is the causative agent for plague, and multi-drug resistant Y. pestis has the potential to be used as a weapon in future terrorist attacks. Trapping of covalent DNA cleavage intermediate formed by bacterial topoisomerase I has been shown lead to rapid bacterial cell death. Newly identified small molecules that can enhance the accumulation of bacterial topoisomerase I DNA cleavage products should be effective bactericidal agents, similar to fluoroquinolones that target bacterial type IIA topoisomerases, but would not be affected by the prevalent fluoroquinolone resistance. In this proposed project, an in vitro high throughput assay will be developed utilizing a fluorescence assay to detect small molecules that can enhance the formation of DNA cleavage products by recombinant Y. pestis topoisomerase I. During the first year of the project, the following assay parameters will be optimized: (i) oligonucleotide substrate sequence, and the choice of modifications for fluorescence detection of cleavage product;(ii) incubation conditions that are compatible with the HTS setting to achieve maximum sensitivity of signal detection. Signal-to background ratio, coefficient of variation, between-plate and day-today variations will be determined against a small set of compounds obtained from NIH in the 384 well format. In addition, the assay will be tested against 5,000 to 10,000 compounds in a pilot screen at the HTS facility at the NERCE screening facility associated with Harvard Medical School. Secondary topoisomerase relaxation and cleavage activity assays are already in place. Current in vivo counterscreens will be further optimized to improve on selecting leads that specifically target topoisomerase I enzyme in bacteria. Y. pestis topoisomerase I shares extensive sequence homology with topoisomerase I from E. coli and other gram negative bacteria. It is expected that the molecules identified for Y. pestis topoisomerase I could be used for targeting topoisomerase I in E. coli and many gram negative pathogens. The results from the assay development and configuration should allow a fast track submission for screening under the MLPCN to establish collaboration with the National Screening Center in the second year.

Public Health Relevance

In this project, an in vitro high throughput assay will be developed to identify for the first time, novel compounds targeting bacterial topoisomerase I specifically. The proposed work would address the need for development of new antibacterial therapeutics to combat the serious public health problem of multi- resistant gram negative bacterial pathogens, some of which could potentially be used in bioterrorist attacks.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Exploratory/Developmental Grants (R21)
Project #
3R21NS067592-01A1S1
Application #
8234706
Study Section
Special Emphasis Panel (ZRG1-BST-J (51))
Program Officer
Scheideler, Mark A
Project Start
2010-06-01
Project End
2012-05-31
Budget Start
2010-06-01
Budget End
2012-05-31
Support Year
1
Fiscal Year
2011
Total Cost
$39,750
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
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
Cheng, Bokun; Cao, Shugeng; Vasquez, Victor et al. (2013) Identification of anziaic acid, a lichen depside from Hypotrachyna sp., as a new topoisomerase poison inhibitor. PLoS One 8:e60770