title: Biochemical and structural characterization of mycobacterial aspartyl-tRNA synthetase AspS, a promising TB drug target.
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.
authors.affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom.
authors.forename: Sudagar S
authors.initials: SS
authors.lastname: Gurcha
authors.affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom.
authors.forename: Veeraraghavan
authors.initials: V
authors.lastname: Usha
authors.affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom.
authors.forename: Jonathan A G
authors.initials: JA
authors.lastname: Cox
authors.affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom.
authors.forename: Klaus
authors.initials: K
authors.lastname: Fütterer
authors.affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom.
authors.forename: Katherine A
authors.initials: KA
authors.lastname: Abrahams
authors.affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom.
authors.forename: Apoorva
authors.initials: A
authors.lastname: Bhatt
authors.affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom.
authors.forename: Luke J
authors.initials: LJ
authors.lastname: Alderwick
authors.affiliation: Department of Chemistry, University of Alabama at Birmingham, College of Arts and Sciences, 1530 3rd Avenue South, Birmingham, Alabama, 35294-1240, United States of America.
authors.forename: Robert C
authors.initials: RC
authors.lastname: Reynolds
authors.affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Loman
authors.affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom.
authors.forename: VijayaShankar
authors.initials: V
authors.lastname: Nataraj
authors.affiliation: Diseases of the Developing World, GlaxoSmithKline, Severo Ochoa 2, 28760, Tres Cantos, Madrid, Spain.
authors.forename: Carlos
authors.initials: C
authors.lastname: Alemparte
authors.affiliation: Diseases of the Developing World, GlaxoSmithKline, Severo Ochoa 2, 28760, Tres Cantos, Madrid, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Barros
authors.affiliation: Department of Life Sciences, University of Warwick, Coventry, CV4 7AL, United Kingdom.
authors.forename: Adrian J
authors.initials: AJ
authors.lastname: Lloyd
authors.affiliation: Diseases of the Developing World, GlaxoSmithKline, Severo Ochoa 2, 28760, Tres Cantos, Madrid, Spain.
authors.forename: Lluis
authors.initials: L
authors.lastname: Ballell
authors.affiliation: Organic Chemistry Department, Southern Research Institute, Birmingham, Alabama, 35205, United States of America.
authors.forename: Judith V
authors.initials: JV
authors.lastname: Hobrath
authors.affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom.
authors.forename: Gurdyal S
authors.initials: GS
authors.lastname: Besra
mesh_terms: D000595:Amino Acid Sequence; D000995:Antitubercular Agents / Q000494:pharmacology* / Q000627:therapeutic use; D001226:Aspartate-tRNA Ligase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D019281:Dimerization; D024901:Drug Resistance, Multiple, Bacterial / Q000187:drug effects; D006801:Humans; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009163:Mycobacterium bovis / Q000187:drug effects* / Q000201:enzymology; D020102:Mycobacterium smegmatis / Q000187:drug effects / Q000201:enzymology; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000201:enzymology; D010880:Piperidines / Q000737:chemistry / Q000494:pharmacology* / Q000627:therapeutic use; D020641:Polymorphism, Single Nucleotide; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013844:Thiazoles / Q000737:chemistry / Q000494:pharmacology* / Q000627:therapeutic use; D014397:Tuberculosis, Pulmonary / Q000188:drug therapy / Q000473:pathology
chemical_list: D000995:Antitubercular Agents; D010880:Piperidines; D011993:Recombinant Fusion Proteins; D013844:Thiazoles; D001226:Aspartate-tRNA Ligase