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

DOI: 10.1371/journal.pone.0113568
PMID: 25409504

Authors: Sudagar S. Gurcha (University of Birmingham) , Veeraraghavan Usha (University of Birmingham) , Jonathan A. G. Cox (University of Birmingham) , Klaus Futterer (University of Birmingham) , K Abrahams (University of Birmingham) , Apoorva Bhatt (University of Birmingham) , Luke Alderwick (University of Birmingham) , Robert C. Reynolds (University of Alabama at Birmingham) , Nicholas J. Loman (University of Birmingham) , Vijayashankar Nataraj (University of Birmingham) , Carlos Alemparte (GlaxoSmithKline) , David Barros (GlaxoSmithKline) , Adrian J. Lloyd (University of Warwick) , Lluis Ballell (GlaxoSmithKline) , Judith V. Hobrath (Southern Research Institute (USA)) , Gurdyal S. Besra (University of Birmingham)
Co-authored by industrial partner: Yes

Type: Journal Paper
Journal: Plos One , VOL 9 (11)

State: Published (Approved)
Published: November 2014
Diamond Proposal Number(s): 10369

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.

Journal Keywords: Mycobacterium tuberculosis; Mycobacterium bovis; Crystal structure; Library screening; Reverse transcriptase-polymerase chain reaction; Aminoacyl-tRNA synthetases; Polymerase chain reaction; Single nucleotide polymorphisms

Diamond Keywords: Tuberculosis (TB); Bacteria

Subject Areas: Biology and Bio-materials, Medicine, Chemistry


Instruments: I04-Macromolecular Crystallography

Other Facilities: Home source was used in addition to data sets recorded at Diamond.

Added On: 27/03/2015 17:20

Documents:
file-4.pdf

Discipline Tags:

Pathogens Infectious Diseases Health & Wellbeing Biochemistry Chemistry Structural biology Drug Discovery Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)