Journal article
A High Throughput Screening Assay for Anti-Mycobacterial Small Molecules Based on Adenylate Kinase Release as a Reporter of Cell Lysis
PloS one, Vol.10(6), pp.e0129234-e0129234
2015
DOI: 10.1371/journal.pone.0129234
PMCID: PMC4476654
PMID: 26098625
Abstract
Mycobacterium tuberculosis (Mtb) is well-established to be one of the most important bacterial pathogens for which new antimicrobial therapies are needed. Herein, we describe the development of a high throughput screening assay for the identification of molecules that are bactericidal against Mycobacteria. The assay utilizes the release of the intracellular enzyme adenylate kinase into the culture medium as a reporter of mycobacterial cell death. We demonstrate that the assay is selective for mycobactericidal molecules and detects anti-mycobacterial activity at concentrations below the minimum inhibitory concentration of many molecules. Thus, the AK assay is more sensitive than traditional growth assays. We have validated the AK assay in the HTS setting using the Mtb surrogate organism M. smegmatis and libraries of FDA approved drugs as well as a commercially available Diversity set. The screen of the FDA-approved library demonstrated that the AK assay is able to identify the vast majority of drugs with known mycobactericidal activity. Importantly, our screen of the Diversity set revealed that the increased sensitivity of the AK assay increases the ability of M. smegmatis-based screens to detect molecules with relatively poor activity against M. smegmatis but good to excellent activity against Mtb.
Details
- Title: Subtitle
- A High Throughput Screening Assay for Anti-Mycobacterial Small Molecules Based on Adenylate Kinase Release as a Reporter of Cell Lysis
- Creators
- Lauren Forbes - Department of Microbiology/Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York, 14642, United States of AmericaKatherine Ebsworth-Mojica - Department of Pediatrics, University of Rochester School of Medicine and Dentistry, Rochester, New York, 14642, United States of AmericaLouis DiDone - Department of Pediatrics, University of Rochester School of Medicine and Dentistry, Rochester, New York, 14642, United States of AmericaShao-Gang Li - Department of Pharmacology, Rutgers University, Newark, New Jersey, 07103, United States of AmericaJoel S Freundlich - Department of Pharmacology, Rutgers University, Newark, New Jersey, 07103, United States of America; Department of Physiology, Rutgers University, Newark, New Jersey, 07103, United States of America; Department of Medicine, Center for Emerging and Re-emerging Pathogens, Rutgers University, Newark, New Jersey, 07103, United States of AmericaNancy Connell - Department of Physiology, Rutgers University, Newark, New Jersey, 07103, United States of America; Department of Medicine, Center for Emerging and Re-emerging Pathogens, Rutgers University, Newark, New Jersey, 07103, United States of AmericaPaul M Dunman - Department of Microbiology/Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York, 14642, United States of AmericaDamian J Krysan - Department of Microbiology/Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York, 14642, United States of America; Department of Pediatrics, University of Rochester School of Medicine and Dentistry, Rochester, New York, 14642, United States of America
- Resource Type
- Journal article
- Publication Details
- PloS one, Vol.10(6), pp.e0129234-e0129234
- DOI
- 10.1371/journal.pone.0129234
- PMID
- 26098625
- PMCID
- PMC4476654
- NLM abbreviation
- PLoS One
- ISSN
- 1932-6203
- eISSN
- 1932-6203
- Publisher
- Public Library of Science
- Grant note
- 5R01 AI103507 / NIAID NIH HHS R01 AI103507 / NIAID NIH HHS
- Language
- English
- Date published
- 2015
- Academic Unit
- Microbiology and Immunology; Stead Family Department of Pediatrics; Infectious Disease (Pediatrics)
- Record Identifier
- 9984093366902771
Metrics
31 Record Views