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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 library of FDA-approved library validates the adenylate kinase assay and indicates it is more sensitive than growth-based assays.A. Scatter plot of raw data from primary screen of the Selleck FDA-approved drug library. The cut-off for hit identification (2-fold increase in AK activity) is indicated by the solid line. B. Table of representative hits from screen that have MIC values above the concentrations at which the screen was performed, indicating that the adenylate kinase assay is more sensitive than growth assays.
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pone.0129234.g003: Screen of library of FDA-approved library validates the adenylate kinase assay and indicates it is more sensitive than growth-based assays.A. Scatter plot of raw data from primary screen of the Selleck FDA-approved drug library. The cut-off for hit identification (2-fold increase in AK activity) is indicated by the solid line. B. Table of representative hits from screen that have MIC values above the concentrations at which the screen was performed, indicating that the adenylate kinase assay is more sensitive than growth assays.

Mentions: To evaluate the performance of the AK assay for HTS with M. smegmatis, we initially screened the Selleck library of 1163 FDA-approved/off-patent drugs. The library was selected for this pilot screening because it contains a large number of antibacterial agents with well-established anti-mycobacterial activity including fluoroquinolones, rifampin derivatives, streptomycin and other aminoglycosides [13]. As such, the library allowed us to assess both the sensitivity and specificity of the AK assay in the HTS setting. We carried out the screen in 96-well format using the procedure described above for the Z’-score determination. The final concentration of the drugs was 5 μM with <1% DMSO per well. Positive (streptomycin at 2X MIC) and negative control wells (DMSO only) were processed during the screen and gave the expected signal-to-noise (7.4 fold increase in AK relative to untreated). The cut-off for determination of a hit was a 2-fold increase in AK signal relative to DMSO-only wells and a total of 62 hits were identified in the primary screen (hit rate ~5.3%). An example of a representative plate from the screen is shown in Fig 3A. As observed with smaller scale assays, the background was uniformly low across the plates and hits were readily identified. All 62 hits identified in the primary screen were re-tested at the screening concentration of 5 μM and 55/62 (89%) were confirmed to induce >2-fold increase in AK release. There was no identifiable pattern to the false positives expect all showed less than a 2.5-fold increase in AK.


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 library of FDA-approved library validates the adenylate kinase assay and indicates it is more sensitive than growth-based assays.A. Scatter plot of raw data from primary screen of the Selleck FDA-approved drug library. The cut-off for hit identification (2-fold increase in AK activity) is indicated by the solid line. B. Table of representative hits from screen that have MIC values above the concentrations at which the screen was performed, indicating that the adenylate kinase assay is more sensitive than growth assays.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0129234.g003: Screen of library of FDA-approved library validates the adenylate kinase assay and indicates it is more sensitive than growth-based assays.A. Scatter plot of raw data from primary screen of the Selleck FDA-approved drug library. The cut-off for hit identification (2-fold increase in AK activity) is indicated by the solid line. B. Table of representative hits from screen that have MIC values above the concentrations at which the screen was performed, indicating that the adenylate kinase assay is more sensitive than growth assays.
Mentions: To evaluate the performance of the AK assay for HTS with M. smegmatis, we initially screened the Selleck library of 1163 FDA-approved/off-patent drugs. The library was selected for this pilot screening because it contains a large number of antibacterial agents with well-established anti-mycobacterial activity including fluoroquinolones, rifampin derivatives, streptomycin and other aminoglycosides [13]. As such, the library allowed us to assess both the sensitivity and specificity of the AK assay in the HTS setting. We carried out the screen in 96-well format using the procedure described above for the Z’-score determination. The final concentration of the drugs was 5 μM with <1% DMSO per well. Positive (streptomycin at 2X MIC) and negative control wells (DMSO only) were processed during the screen and gave the expected signal-to-noise (7.4 fold increase in AK relative to untreated). The cut-off for determination of a hit was a 2-fold increase in AK signal relative to DMSO-only wells and a total of 62 hits were identified in the primary screen (hit rate ~5.3%). An example of a representative plate from the screen is shown in Fig 3A. As observed with smaller scale assays, the background was uniformly low across the plates and hits were readily identified. All 62 hits identified in the primary screen were re-tested at the screening concentration of 5 μM and 55/62 (89%) were confirmed to induce >2-fold increase in AK release. There was no identifiable pattern to the false positives expect all showed less than a 2.5-fold increase in AK.

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