title: A High Throughput Screening Assay for Anti-Mycobacterial Small Molecules Based on Adenylate Kinase Release as a Reporter of Cell Lysis.
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.
authors.affiliation: Department of Microbiology/Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York, 14642, United States of America.
authors.forename: Lauren
authors.initials: L
authors.lastname: Forbes
authors.affiliation: Department of Pediatrics, University of Rochester School of Medicine and Dentistry, Rochester, New York, 14642, United States of America.
authors.forename: Katherine
authors.initials: K
authors.lastname: Ebsworth-Mojica
authors.affiliation: Department of Pediatrics, University of Rochester School of Medicine and Dentistry, Rochester, New York, 14642, United States of America.
authors.forename: Louis
authors.initials: L
authors.lastname: DiDone
authors.affiliation: Department of Pharmacology, Rutgers University, Newark, New Jersey, 07103, United States of America.
authors.forename: Shao-Gang
authors.initials: SG
authors.lastname: Li
authors.affiliation: 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 America.
authors.forename: Joel S
authors.initials: JS
authors.lastname: Freundlich
authors.affiliation: 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 America.
authors.forename: Nancy
authors.initials: N
authors.lastname: Connell
authors.affiliation: Department of Microbiology/Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York, 14642, United States of America.
authors.forename: Paul M
authors.initials: PM
authors.lastname: Dunman
authors.affiliation: 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.
authors.forename: Damian J
authors.initials: DJ
authors.lastname: Krysan
mesh_terms: D000262:Adenylyl Cyclases / Q000737:chemistry* / Q000378:metabolism; D000995:Antitubercular Agents / Q000737:chemistry / Q000494:pharmacology*; D055808:Drug Discovery / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods*; D009161:Mycobacterium / Q000187:drug effects*; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D000995:Antitubercular Agents; D054852:Small Molecule Libraries; D000262:Adenylyl Cyclases