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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

Adenylate kinase release from M. smegmatis is compatible with high throughput screening.Scatter plot of raw data from an experiment in which alternating rows of a 96-well plate contained either streptomycin (2 μg/mL; 4X MIC) or carrier (H2O). The wells were processed as described in materials and methods. The Z’ factor was determined as described by Zhang et al. [12].
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pone.0129234.g002: Adenylate kinase release from M. smegmatis is compatible with high throughput screening.Scatter plot of raw data from an experiment in which alternating rows of a 96-well plate contained either streptomycin (2 μg/mL; 4X MIC) or carrier (H2O). The wells were processed as described in materials and methods. The Z’ factor was determined as described by Zhang et al. [12].

Mentions: In order to further validate the mycobacterial AK assay as an HTS platform, we determined the Z’ score for the assay in 96-well format with alternating columns of DMSO negative control (1%) and streptomycin (2 μg/mL) using the conditions described in the materials and methods. A scatter plot of the raw data from a representative screening plate is shown in Fig 2. The apparent sinusoidal variation is due to a small edge effect. The Z’ score for the depicted experiment was 0.87 and is well above the standard 0.5 cut-off for an HTS compatible assay [12]. Similar results were obtained in 384-well format with a Z’ of 0.76 (data not shown). Thus, the AK assay is an HTS compatible assay for the identification of molecules with bactericidal activity towards M. smegmatis.


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)

Adenylate kinase release from M. smegmatis is compatible with high throughput screening.Scatter plot of raw data from an experiment in which alternating rows of a 96-well plate contained either streptomycin (2 μg/mL; 4X MIC) or carrier (H2O). The wells were processed as described in materials and methods. The Z’ factor was determined as described by Zhang et al. [12].
© Copyright Policy
Related In: Results  -  Collection

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

pone.0129234.g002: Adenylate kinase release from M. smegmatis is compatible with high throughput screening.Scatter plot of raw data from an experiment in which alternating rows of a 96-well plate contained either streptomycin (2 μg/mL; 4X MIC) or carrier (H2O). The wells were processed as described in materials and methods. The Z’ factor was determined as described by Zhang et al. [12].
Mentions: In order to further validate the mycobacterial AK assay as an HTS platform, we determined the Z’ score for the assay in 96-well format with alternating columns of DMSO negative control (1%) and streptomycin (2 μg/mL) using the conditions described in the materials and methods. A scatter plot of the raw data from a representative screening plate is shown in Fig 2. The apparent sinusoidal variation is due to a small edge effect. The Z’ score for the depicted experiment was 0.87 and is well above the standard 0.5 cut-off for an HTS compatible assay [12]. Similar results were obtained in 384-well format with a Z’ of 0.76 (data not shown). Thus, the AK assay is an HTS compatible assay for the identification of molecules with bactericidal activity towards M. smegmatis.

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