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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 ISSN 1932-6203.

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Official URL: http://dx.doi.org/10.1371/journal.pone.0113568

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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
Subjects: R Medicine > RA Public aspects of medicine > RA0421 Public health. Hygiene. Preventive Medicine
Divisions: Faculty of Science, Engineering and Medicine > 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:
DateEvent
19 October 2014Published
13 October 2014Accepted
21 July 2014Submitted
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 (Creative Commons)
Date of first compliant deposit: 28 December 2015
Date of first compliant Open Access: 28 December 2015
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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