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Biochemical and structural characterization of mycobacterial aspartyl-tRNA synthetase AspS, a promising TB drug target
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Gurcha, Sudagar S., Usha, Veeraraghavan, Cox, Jonathan A. G., Fütterer, Klaus, Abrahams, Katherine A., Bhatt, Apoorva, Alderwick, Luke J., Reynolds, Robert C., Loman, Nicholas J., Nataraj, VijayaShankar, Alemparte, Carlos, Barros, David, Lloyd, Adrian J., Ballell, Lluis, Hobrath, Judith V. and Besra, Gurdyal S. (2014) Biochemical and structural characterization of mycobacterial aspartyl-tRNA synthetase AspS, a promising TB drug target. PLoS One, Volume 9 (Number 11). Article number e113568. doi:10.1371/journal.pone.0113568
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Official URL: http://dx.doi.org/10.1371/journal.pone.0113568
Abstract
The human pathogen Mycobacterium tuberculosis is the causative agent of pulmonary tuberculosis (TB), a disease with high worldwide mortality rates. Current treatment programs are under significant threat from multi-drug and extensively-drug resistant strains of M. tuberculosis, and it is essential to identify new inhibitors and their targets. We generated spontaneous resistant mutants in Mycobacterium bovis BCG in the presence of 10× the minimum inhibitory concentration (MIC) of compound 1, a previously identified potent inhibitor of mycobacterial growth in culture. Whole genome sequencing of two resistant mutants revealed in one case a single nucleotide polymorphism in the gene aspS at 535GAC>535AAC (D179N), while in the second mutant a single nucleotide polymorphism was identified upstream of the aspS promoter region. We probed whole cell target engagement by overexpressing either M. bovis BCG aspS or Mycobacterium smegmatis aspS, which resulted in a ten-fold and greater than ten-fold increase, respectively, of the MIC against compound 1. To analyse the impact of inhibitor 1 on M. tuberculosis AspS (Mt-AspS) activity we over-expressed, purified and characterised the kinetics of this enzyme using a robust tRNA-independent assay adapted to a high-throughput screening format. Finally, to aid hit-to-lead optimization, the crystal structure of apo M. smegmatis AspS was determined to a resolution of 2.4 Å.
| Item Type: | Journal Article | ||||||||
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| Subjects: | R Medicine > RA Public aspects of medicine > RA0421 Public health. Hygiene. Preventive Medicine | ||||||||
| Divisions: | Faculty of Science > Life Sciences (2010- ) | ||||||||
| Library of Congress Subject Headings (LCSH): | Tuberculosis -- Microbiology, Mycobacterium tuberculosis, Bacterial genetics | ||||||||
| Journal or Publication Title: | PLoS One | ||||||||
| Publisher: | Public library of science | ||||||||
| ISSN: | 1932-6203 | ||||||||
| Official Date: | 19 October 2014 | ||||||||
| Dates: |
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| Volume: | Volume 9 | ||||||||
| Number: | Number 11 | ||||||||
| Number of Pages: | 14 | ||||||||
| Article Number: | Article number e113568 | ||||||||
| DOI: | 10.1371/journal.pone.0113568 | ||||||||
| Status: | Peer Reviewed | ||||||||
| Publication Status: | Published | ||||||||
| Access rights to Published version: | Open Access | ||||||||
| Funder: | Royal Society (Great Britain). Wolfson Research Merit Award (RSWRMA), Medical Research Council (Great Britain) (MRC), Seventh Framework Programme (European Commission) (FP7), Wellcome Trust (London, England) | ||||||||
| Grant number: | 261378 (FP7) |
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