title: Bioluminescent Reporters for Rapid Mechanism of Action Assessment in Tuberculosis Drug Discovery.
abstract: The tuberculosis (TB) drug discovery pipeline is fueled by compounds identified in whole-cell screens against the causative agent, Mycobacterium tuberculosis Phenotypic screening enables the selection of molecules that inhibit essential cellular functions in live, intact bacilli grown under a chosen in vitro condition. However, deducing the mechanism of action (MOA), which is important to avoid promiscuous targets, often requires significant biological resources in a lengthy process that risks decoupling medicinal chemistry and biology efforts. Therefore, there is a need to develop methods enabling rapid MOA assessment of putative "actives" for triage decisions. Here, we describe a modified version of a bioluminescence reporter assay that allows nondestructive detection of compounds targeting either of two macromolecular processes in M. tuberculosis: cell wall biosynthesis or maintenance of DNA integrity. Coupling the luxCDABE operon from Photorhabdus luminescens to mycobacterial promoters driving expression of the iniBAC operon (PiniB-LUX) or the DNA damage-inducible genes, recA (PrecA-LUX) or radA (PradA-LUX), provided quantitative detection in real time of compounds triggering expression of any of these promoters over an extended 10- to 12-day incubation. Testing against known anti-TB agents confirmed the specificity of each reporter in registering the MOA of the applied antibiotic in M. tuberculosis, independent of bactericidal or bacteriostatic activity. Moreover, profiles obtained for experimental compounds indicated the potential to infer complex MOAs in which multiple cellular processes are disrupted. These results demonstrate the utility of the reporters for early triage of compounds based on the provisional MOA and suggest their application to investigate polypharmacology in known and experimental anti-TB agents.
authors: MRC/NHLS/UCT Molecular Mycobacteriology Research Unit, DST/NRF Centre of Excellence for Biomedical Tuberculosis Research, Department of Pathology, University of Cape Town, Cape Town, South Africa.
authors: Krupa
authors: K
authors: Naran
authors: MRC/NHLS/UCT Molecular Mycobacteriology Research Unit, DST/NRF Centre of Excellence for Biomedical Tuberculosis Research, Department of Pathology, University of Cape Town, Cape Town, South Africa.
authors: Atica
authors: A
authors: Moosa
authors: Tuberculosis Research Section, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.|Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa.
authors: Clifton E
authors: CE
authors: Barry
authors: Tuberculosis Research Section, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors: Helena I M
authors: HI
authors: Boshoff
authors: MRC/NHLS/UCT Molecular Mycobacteriology Research Unit, DST/NRF Centre of Excellence for Biomedical Tuberculosis Research, Department of Pathology, University of Cape Town, Cape Town, South Africa.|Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa.
authors: Valerie
authors: V
authors: Mizrahi
authors: MRC/NHLS/UCT Molecular Mycobacteriology Research Unit, DST/NRF Centre of Excellence for Biomedical Tuberculosis Research, Department of Pathology, University of Cape Town, Cape Town, South Africa digby.warner@uct.ac.za.|Institute of Infectious Disease and Molecular Medicine, University of Cape Town, Cape Town, South Africa.
authors: Digby F
authors: 0000-0002-4146-0930
authors: DF
authors: Warner
mesh_terms: D000995:Antitubercular Agents / Q000737:chemistry / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002473:Cell Wall / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D055808:Drug Discovery; D017930:Genes, Reporter; D057166:High-Throughput Screening Assays; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008163:Luminescent Measurements; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D009876:Operon; D020637:Photorhabdus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C100901:RadA protein, bacteria; D008156:Luciferases; D011938:Rec A Recombinases