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

SS Gurcha, V Usha, JA Cox, K Fütterer, KA Abrahams, A Bhatt, LJ Alderwick, RC Reynolds, NJ Loman, V Nataraj, C Alemparte, D Barros, AJ Lloyd, L Ballell, JV Hobrath, GS Besra

Research output: Contribution to journalArticle

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.
Original languageEnglish
Article numbere113568
JournalPLoS ONE
Volume9
Issue number11
DOIs
Publication statusPublished - 19 Nov 2014

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Aspartate-tRNA Ligase
RNA, Transfer, Asp
Mycobacterium tuberculosis
Viperidae
Polymorphism
tuberculosis
Mycobacterium smegmatis
Mycobacterium bovis BCG
Mycobacterium bovis
Tuberculosis
Nucleotides
Genes
minimum inhibitory concentration
drugs
mutants
single nucleotide polymorphism
Pathogens
Transfer RNA
Cell culture
Genetic Promoter Regions

Bibliographical note

© 2014 Gurcha et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Cite this

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 ; Besra, GS. / Biochemical and structural characterization of mycobacterial aspartyl-tRNA synthetase AspS, a promising TB drug target. In: PLoS ONE. 2014 ; Vol. 9, No. 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 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 {\AA}.",
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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 & Besra, GS 2014, 'Biochemical and structural characterization of mycobacterial aspartyl-tRNA synthetase AspS, a promising TB drug target.', PLoS ONE, vol. 9, no. 11, e113568. https://doi.org/10.1371/journal.pone.0113568

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; Besra, GS.

In: PLoS ONE, Vol. 9, No. 11, e113568, 19.11.2014.

Research output: Contribution to journalArticle

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T1 - Biochemical and structural characterization of mycobacterial aspartyl-tRNA synthetase AspS, a promising TB drug target.

AU - Gurcha, SS

AU - Usha, V

AU - Cox, JA

AU - Fütterer, K

AU - Abrahams, KA

AU - Bhatt, A

AU - Alderwick, LJ

AU - Reynolds, RC

AU - Loman, NJ

AU - Nataraj, V

AU - Alemparte, C

AU - Barros, D

AU - Lloyd, AJ

AU - Ballell, L

AU - Hobrath, JV

AU - Besra, GS

N1 - © 2014 Gurcha et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

PY - 2014/11/19

Y1 - 2014/11/19

N2 - 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.

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