Biochemical and structural characterization of mycobacterial aspartyl-tRNA synthetase AspS, a promising TB drug target.
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 t...
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| Language: | English |
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Public Library of Science (PLoS)
2014-01-01
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| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0113568 |
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| author | Sudagar S Gurcha Veeraraghavan Usha Jonathan A G Cox Klaus Fütterer Katherine A Abrahams Apoorva Bhatt Luke J Alderwick Robert C Reynolds Nicholas J Loman VijayaShankar Nataraj Carlos Alemparte David Barros Adrian J Lloyd Lluis Ballell Judith V Hobrath Gurdyal S Besra |
| author_facet | Sudagar S Gurcha Veeraraghavan Usha Jonathan A G Cox Klaus Fütterer Katherine A Abrahams Apoorva Bhatt Luke J Alderwick Robert C Reynolds Nicholas J Loman VijayaShankar Nataraj Carlos Alemparte David Barros Adrian J Lloyd Lluis Ballell Judith V Hobrath Gurdyal S Besra |
| author_sort | Sudagar S Gurcha |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-d85fbdf303054892bbf5d1a8ee379a42 |
| institution | Kabale University |
| issn | 1932-6203 |
| language | English |
| publishDate | 2014-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj-art-d85fbdf303054892bbf5d1a8ee379a422025-08-20T03:46:11ZengPublic Library of Science (PLoS)PLoS ONE1932-62032014-01-01911e11356810.1371/journal.pone.0113568Biochemical and structural characterization of mycobacterial aspartyl-tRNA synthetase AspS, a promising TB drug target.Sudagar S GurchaVeeraraghavan UshaJonathan A G CoxKlaus FüttererKatherine A AbrahamsApoorva BhattLuke J AlderwickRobert C ReynoldsNicholas J LomanVijayaShankar NatarajCarlos AlemparteDavid BarrosAdrian J LloydLluis BallellJudith V HobrathGurdyal S BesraThe 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.https://doi.org/10.1371/journal.pone.0113568 |
| spellingShingle | Sudagar S Gurcha Veeraraghavan Usha Jonathan A G Cox Klaus Fütterer Katherine A Abrahams Apoorva Bhatt Luke J Alderwick Robert C Reynolds Nicholas J Loman VijayaShankar Nataraj Carlos Alemparte David Barros Adrian J Lloyd Lluis Ballell Judith V Hobrath Gurdyal S Besra Biochemical and structural characterization of mycobacterial aspartyl-tRNA synthetase AspS, a promising TB drug target. PLoS ONE |
| title | Biochemical and structural characterization of mycobacterial aspartyl-tRNA synthetase AspS, a promising TB drug target. |
| title_full | Biochemical and structural characterization of mycobacterial aspartyl-tRNA synthetase AspS, a promising TB drug target. |
| title_fullStr | Biochemical and structural characterization of mycobacterial aspartyl-tRNA synthetase AspS, a promising TB drug target. |
| title_full_unstemmed | Biochemical and structural characterization of mycobacterial aspartyl-tRNA synthetase AspS, a promising TB drug target. |
| title_short | Biochemical and structural characterization of mycobacterial aspartyl-tRNA synthetase AspS, a promising TB drug target. |
| title_sort | biochemical and structural characterization of mycobacterial aspartyl trna synthetase asps a promising tb drug target |
| url | https://doi.org/10.1371/journal.pone.0113568 |
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