title: A New Screen for Tuberculosis Drug Candidates Utilizing a Luciferase-Expressing Recombinant Mycobacterium bovis Bacillus Calmette-Guéren.
abstract: Tuberculosis (TB) is a serious infectious disease caused by a bacterial pathogen. Mortality from tuberculosis was estimated at 1.5 million deaths worldwide in 2013. Development of new TB drugs is needed to not only to shorten the medication period but also to treat multi-drug resistant and extensively drug-resistant TB. Mycobacterium tuberculosis (Mtb) grows slowly and only multiplies once or twice per day. Therefore, conventional drug screening takes more than 3 weeks. Additionally, a biosafety level-3 (BSL-3) facility is required. Thus, we developed a new screening method to identify TB drug candidates by utilizing luciferase-expressing recombinant Mycobacterium bovis bacillus Calmette-Guéren (rBCG). Using this method, we identified several candidates in 4 days in a non-BSL-3 facility. We screened 10,080 individual crude extracts derived from Actinomyces and Streptomyces and identified 137 extracts which possessed suppressive activity to the luciferase of rBCG. Among them, 41 compounds inhibited the growth of both Mtb H37Rv and the extensively drug-resistant Mtb (XDR-Mtb) strains. We purified the active substance of the 1904-1 extract, which possessed strong activity toward rBCG, Mtb H37Rv, and XDR-Mtb but was harmless to the host eukaryotic cells. The MIC of this substance was 0.13 μg/ml, 0.5 μg/ml, and 2.0-7.5 μg/ml against rBCG, H37Rv, and 2 XDR-strains, respectively. Its efficacy was specific to acid-fast bacterium except for the Mycobacterium avium intracellular complex. Mass spectrometry and nuclear magnetic resonance analyses revealed that the active substance of 1904-1 was cyclomarin A. To confirm the mode of action of the 1904-1-derived compound, resistant BCG clones were used. Whole genome DNA sequence analysis showed that these clones contained a mutation in the clpc gene which encodes caseinolytic protein, an essential component of an ATP-dependent proteinase, and the likely target of the active substance of 1904-1. Our method provides a rapid and convenient screen to identify an anti-mycobacterial drug.
authors.affiliation: Department of Bacteriology, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan.
authors.forename: Yuriko
authors.initials: Y
authors.lastname: Ozeki
authors.affiliation: Department of Microbiology, Institute of Microbial Chemistry, Microbial Chemistry Research Foundation, Tokyo, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Igarashi
authors.affiliation: Graduate School of Sciences, Osaka City University, Osaka, Japan.
authors.forename: Matsumi
authors.initials: M
authors.lastname: Doe
authors.affiliation: Bacteriology Division, Osaka Prefectural Institute of Public Health, Osaka, Japan.
authors.forename: Aki
authors.initials: A
authors.lastname: Tamaru
authors.affiliation: Department of Microbiology, Institute of Microbial Chemistry, Microbial Chemistry Research Foundation, Tokyo, Japan.
authors.forename: Naoko
authors.initials: N
authors.lastname: Kinoshita
authors.affiliation: Division of Bioenvironmental Science, Frontier Science Research Center, University of Miyazaki, Miyazaki, Japan.
authors.forename: Yoshitoshi
authors.initials: Y
authors.lastname: Ogura
authors.affiliation: Department of Microbiology, Kobe Institute of Health, Kobe, Japan.
authors.forename: Tomotada
authors.initials: T
authors.lastname: Iwamoto
authors.affiliation: Department of Microbiology, Institute of Microbial Chemistry, Microbial Chemistry Research Foundation, Tokyo, Japan.
authors.forename: Ryuichi
authors.initials: R
authors.lastname: Sawa
authors.affiliation: Department of Microbiology, Institute of Microbial Chemistry, Microbial Chemistry Research Foundation, Tokyo, Japan.
authors.forename: Maya
authors.initials: M
authors.lastname: Umekita
authors.affiliation: Department of Bacteriology, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan.|Department of Microbiology and Immunology, Faculty of Pharmacy, Suez Canal University, Ismailia, Egypt.
authors.forename: Shymaa
authors.initials: S
authors.lastname: Enany
authors.affiliation: Toneyama Institute for Tuberculosis Research, Osaka City University Medical School, Osaka, Japan.
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Nishiuchi
authors.affiliation: Food Hygiene and Environmental Health Division of Applied Life Science, Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, Kyoto, Japan.
authors.forename: Mayuko
authors.initials: M
authors.lastname: Osada-Oka
authors.affiliation: Division of Bioenvironmental Science, Frontier Science Research Center, University of Miyazaki, Miyazaki, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Hayashi
authors.affiliation: Department of Bacteriology, Osaka City University Graduate School of Medicine, Osaka, Japan.
authors.forename: Mamiko
authors.initials: M
authors.lastname: Niki
authors.affiliation: Department of Bacteriology, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan.
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Tateishi
authors.affiliation: Department of Microbiology, Institute of Microbial Chemistry, Microbial Chemistry Research Foundation, Tokyo, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Hatano
authors.affiliation: Department of Bacteriology, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan.
authors.forename: Sohkichi
authors.initials: S
authors.lastname: Matsumoto
mesh_terms: D000190:Actinomyces; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D000995:Antitubercular Agents / Q000494:pharmacology*; D001500:BCG Vaccine / Q000737:chemistry*; D045744:Cell Line, Tumor; D017077:Culture Media, Conditioned; D015195:Drug Design; D004353:Drug Evaluation, Preclinical; D054908:Extensively Drug-Resistant Tuberculosis / Q000188:drug therapy; D006801:Humans; D008156:Luciferases / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008810:Mice, Inbred C57BL; D008826:Microbial Sensitivity Tests / Q000379:methods*; D015269:Mycobacterium avium Complex / Q000235:genetics; D009163:Mycobacterium bovis / Q000235:genetics*; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D009842:Oligopeptides / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D013302:Streptomyces
chemical_list: D000995:Antitubercular Agents; D001500:BCG Vaccine; D017077:Culture Media, Conditioned; D009842:Oligopeptides; C459019:cyclomarin A; D000255:Adenosine Triphosphate; D008156:Luciferases