Bacterial pathogens cause devastating diseases, and it is imperative to improve our ability to treat bacterial infections. Among the most potent drugs in current use are the fluoroquinolone antibiotics, which trap the normally transient covalent intermediate formed by DNA gyrase with DNA, to create local DNA damage that is cytotoxic. In addition to gyrase, which is a type 2A topoisomerase, most bacteria also contain a structurally distinct type 1A topoisomerase, Topoisomerase 1 (Topo I). Like DNA gyrase, Topo I forms a transient covalent DNA adduct and should in principle be an excellent drug target, but no drugs currently target Topo I. Discovery and optimization of drugs that target Topo I and other topoisomerases has been limited by the lack of a mechanism-based assay for induction of topoisomerase-DNA adducts. We recently developed an assay, the RADAR assay (Rapid Assay of the DNA Adduct Response), that quantifies the covalent topoisomerase-DNA adducts formed as signature DNA damage in cells treated with topoisomerase poisons. We hypothesize that the RADAR assay will be useful for identifying new drugs that function by this mechanism, and for quantifying the potency of topoisomerase poisons in current use. The goal of this application is to validate the RADAR assay for screening compound libraries for drugs that poison bacterial topoisomerases. To achieve this, we will address current challenges in three aims. We will (1) show that DNA adducts formed by M. tuberculosis Topo I are toxic in mycobacteria and can be detected by the RADAR assay; (2) adapt and optimize the RADAR assay for high throughput screening; and (3) validate the RADAR assay for drug discovery by screening the TB Alliance TB Active Collection library. Impact: Validation of the RADAR assay will enable its application to discovery of new mechanism- based drugs to treat TB and a wide range of other infectious diseases.

Public Health Relevance

Some of the most potent drugs currently used to treat infectious disease function by trapping essential intermediates in DNA metabolism, to create DNA damage that kills cells. We will apply a new assay to quantifying these toxic intermediates, in experiments designed to develop a general approach for optimizing current drugs and discovering new ones.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21AI123501-02
Application #
9404004
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Boyce, Jim P
Project Start
2016-12-20
Project End
2018-11-30
Budget Start
2017-12-01
Budget End
2018-11-30
Support Year
2
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of Washington
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195