| Autor: |
Gupta R; Division of Immunity and Pathogenesis, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL, United States., Netherton M; Division of Immunity and Pathogenesis, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL, United States., Byrd TF; Division of Infectious Diseases, Department of Internal Medicine, The University of New Mexico School of Medicine, Albuquerque, NM, United States., Rohde KH; Division of Immunity and Pathogenesis, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL, United States. |
| Abstrakt: |
Mycobacterium abscessus is a non-tuberculous mycobacterium that causes pulmonary and non-pulmonary infections. M. abscessus is resistant to many chemotherapeutic agents and the current treatment options show poor clinical outcomes. Thus, there is a dire need to find new antimicrobials effective at killing M. abscessus . Screening drug libraries to identify potential antimicrobials has been impeded by the lack of validated HTS assays for M. abscessus . In this study, we developed two 384-well high-throughput screening assays using fluorescent and bioluminescent reporter strains of M. abscessus for drug discovery. Optimization of inoculum size, incubation time and the volume-per-well based on Z-factor and signal intensity yielded two complementary, robust tools for M. abscessus drug discovery with Z-factor > 0.8. The MIC of known drugs, amikacin and clarithromycin, as determined by bioluminescence was in agreement with the published MIC values. A proof-of-concept screen of 2,093 natural product-inspired compounds was conducted using the 384-well bioluminescent assay to identify novel scaffolds active against M. abscessus. Five active "hit" compounds identified in this pilot screen were confirmed and characterized by a CFU assay and MIC determination. Overall, we developed and validated a 384-well screen that offers simple, sensitive and fast screening of compounds for activity against this emerging pathogen. To our knowledge, this is the first reporter-based high-throughput screening study aimed at M. abscessus drug discovery. |
