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A High Throughput Screening Assay for Anti-Mycobacterial Small Molecules Based on Adenylate Kinase Release as a Reporter of Cell Lysis.

Forbes L, Ebsworth-Mojica K, DiDone L, Li SG, Freundlich JS, Connell N, Dunman PM, Krysan DJ - PLoS ONE (2015)

Bottom Line: Mycobacterium tuberculosis (Mtb) is well-established to be one of the most important bacterial pathogens for which new antimicrobial therapies are needed.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology/Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York, 14642, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus

Screen of diversity library with M. smegmatis identifies molecules with activity against both M. smegmatis and M. tuberculosis.The set of molecules identified in a screen of the ChemBridge Diversity Set performed as Materials and methods. The structure of each hit is shown along with the Chembridge ID number. The MIC for the compound against M. smegmatis is listed first and is followed by the MIC against M. tuberculosis. The boxes highlight molecules which have MIC against M. smegmatis that is ≥4 folder higher than against M. tuberculosis.
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pone.0129234.g004: Screen of diversity library with M. smegmatis identifies molecules with activity against both M. smegmatis and M. tuberculosis.The set of molecules identified in a screen of the ChemBridge Diversity Set performed as Materials and methods. The structure of each hit is shown along with the Chembridge ID number. The MIC for the compound against M. smegmatis is listed first and is followed by the MIC against M. tuberculosis. The boxes highlight molecules which have MIC against M. smegmatis that is ≥4 folder higher than against M. tuberculosis.

Mentions: A set of 25 molecules met the hit criteria which corresponded to a hit rate of 0.125%. All 25 hits were subsequently confirmed to induce increased AK release relative to mock treated controls and their structures are shown in Fig 4. Next, we determined the MIC for each hit against M. smegmatis using a standard microdilution growth assay. As shown in Fig 4, the molecules that generated increased AK release showed a range of activities by MIC testing (8 to >264 μg/mL).


A High Throughput Screening Assay for Anti-Mycobacterial Small Molecules Based on Adenylate Kinase Release as a Reporter of Cell Lysis.

Forbes L, Ebsworth-Mojica K, DiDone L, Li SG, Freundlich JS, Connell N, Dunman PM, Krysan DJ - PLoS ONE (2015)

Screen of diversity library with M. smegmatis identifies molecules with activity against both M. smegmatis and M. tuberculosis.The set of molecules identified in a screen of the ChemBridge Diversity Set performed as Materials and methods. The structure of each hit is shown along with the Chembridge ID number. The MIC for the compound against M. smegmatis is listed first and is followed by the MIC against M. tuberculosis. The boxes highlight molecules which have MIC against M. smegmatis that is ≥4 folder higher than against M. tuberculosis.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4476654&req=5

pone.0129234.g004: Screen of diversity library with M. smegmatis identifies molecules with activity against both M. smegmatis and M. tuberculosis.The set of molecules identified in a screen of the ChemBridge Diversity Set performed as Materials and methods. The structure of each hit is shown along with the Chembridge ID number. The MIC for the compound against M. smegmatis is listed first and is followed by the MIC against M. tuberculosis. The boxes highlight molecules which have MIC against M. smegmatis that is ≥4 folder higher than against M. tuberculosis.
Mentions: A set of 25 molecules met the hit criteria which corresponded to a hit rate of 0.125%. All 25 hits were subsequently confirmed to induce increased AK release relative to mock treated controls and their structures are shown in Fig 4. Next, we determined the MIC for each hit against M. smegmatis using a standard microdilution growth assay. As shown in Fig 4, the molecules that generated increased AK release showed a range of activities by MIC testing (8 to >264 μg/mL).

Bottom Line: Mycobacterium tuberculosis (Mtb) is well-established to be one of the most important bacterial pathogens for which new antimicrobial therapies are needed.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology/Immunology, University of Rochester School of Medicine and Dentistry, Rochester, New York, 14642, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus