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Biochemical and structural characterization of mycobacterial aspartyl-tRNA synthetase AspS, a promising TB drug target.

Gurcha, SS, Usha, V, Cox, JA, Fütterer, K, Abrahams, KA, Bhatt, A, Alderwick, LJ, Reynolds, RC, Loman, NJ, Nataraj, V , Alemparte, C, Barros, D, Lloyd, AJ, Ballell, L, Hobrath, JV and Besra, GS (2014) Biochemical and structural characterization of mycobacterial aspartyl-tRNA synthetase AspS, a promising TB drug target. PLoS One, 9 (11).

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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 (535)GAC>(535)AAC (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: Article
Authors :
NameEmailORCID
Gurcha, SSUNSPECIFIEDUNSPECIFIED
Usha, VUNSPECIFIEDUNSPECIFIED
Cox, JAUNSPECIFIEDUNSPECIFIED
Fütterer, KUNSPECIFIEDUNSPECIFIED
Abrahams, KAUNSPECIFIEDUNSPECIFIED
Bhatt, AUNSPECIFIEDUNSPECIFIED
Alderwick, LJUNSPECIFIEDUNSPECIFIED
Reynolds, RCUNSPECIFIEDUNSPECIFIED
Loman, NJUNSPECIFIEDUNSPECIFIED
Nataraj, Vv.nataraj@surrey.ac.ukUNSPECIFIED
Alemparte, CUNSPECIFIEDUNSPECIFIED
Barros, DUNSPECIFIEDUNSPECIFIED
Lloyd, AJUNSPECIFIEDUNSPECIFIED
Ballell, LUNSPECIFIEDUNSPECIFIED
Hobrath, JVUNSPECIFIEDUNSPECIFIED
Besra, GSUNSPECIFIEDUNSPECIFIED
Date : 2014
Identification Number : 10.1371/journal.pone.0113568
Uncontrolled Keywords : Amino Acid Sequence, Antitubercular Agents, Aspartate-tRNA Ligase, Cloning, Molecular, Crystallography, X-Ray, Dimerization, Drug Resistance, Multiple, Bacterial, Humans, Microbial Sensitivity Tests, Molecular Sequence Data, Mycobacterium bovis, Mycobacterium smegmatis, Mycobacterium tuberculosis, Piperidines, Polymorphism, Single Nucleotide, Protein Binding, Recombinant Fusion Proteins, Sequence Alignment, Sequence Analysis, DNA, Sequence Homology, Amino Acid, Thiazoles, Tuberculosis, Pulmonary
Related URLs :
Depositing User : Symplectic Elements
Date Deposited : 17 May 2017 10:29
Last Modified : 17 May 2017 10:29
URI: http://epubs.surrey.ac.uk/id/eprint/827960

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