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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, Besra GS - PLoS ONE (2014)

Bottom Line: The human pathogen Mycobacterium tuberculosis is the causative agent of pulmonary tuberculosis (TB), a disease with high worldwide mortality rates.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.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom.

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.

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Induced Fit docked pose of compound 1 at the M. smegmatis AspS crystal structure.Only amino acids forming direct interactions with compound 1 are shown. Carbon atoms of AspS are colored light green except for carbons of resistance conferring mutant site residues, which are colored purple. Carbons of compound 1 are shown in orange; all other atoms are colored by atom type (Oxygen red, Nitrogen blue, Sulphur yellow, Chlorine dark green, Hydrogen white). Labels of residues participating in polar interactions are colored brown, labels of all other residues are in black. Polar interactions are indicated with dashed lines and are numbered. Distances between heavy atoms of the atoms groups participating in polar interactions are as follows: 1. 3.1 Å; 2. 3.8 Å; 3. 3.0 Å; 4. 3.2 Å; 5. 3.1 Å; 6. 3.7 Å.
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pone-0113568-g009: Induced Fit docked pose of compound 1 at the M. smegmatis AspS crystal structure.Only amino acids forming direct interactions with compound 1 are shown. Carbon atoms of AspS are colored light green except for carbons of resistance conferring mutant site residues, which are colored purple. Carbons of compound 1 are shown in orange; all other atoms are colored by atom type (Oxygen red, Nitrogen blue, Sulphur yellow, Chlorine dark green, Hydrogen white). Labels of residues participating in polar interactions are colored brown, labels of all other residues are in black. Polar interactions are indicated with dashed lines and are numbered. Distances between heavy atoms of the atoms groups participating in polar interactions are as follows: 1. 3.1 Å; 2. 3.8 Å; 3. 3.0 Å; 4. 3.2 Å; 5. 3.1 Å; 6. 3.7 Å.

Mentions: Using Induced Fit docking protocols we docked compound 1 into this pocket (Figure 9). The obtained docked pose predicts a number of favorable non-polar and polar contacts with residues lining the pocket as follows. The piperidine ring in compound 1 participates in non-polar interactions with P169 while the thiazolidinone moiety forms aromatic interactions with F514. The dichlorophenyl ring has non-polar/steric contacts with a number of residues: F451, L517, L199, F196, as well as T565 from the adjacent subunit. The side chain of T565 is in close proximity of the ligand’s phenyl ring (the closest ring carbon is at 3.8 Å from the threonine side chain methyl). Guided by the resistance conferring mutation sites that include this threonine, we generated the T565I AspS mutant structure in silico and attempted to re-dock compound 1 using the same protocols, however, no successfully docked poses could be obtained, consistently with the results of the resistant mutations study. Another resistance conferring mutant site corresponds to D174 which is the residue predicted to form hydrogen bonding interactions with the amide group of compound 1 (Figure 9). Re-docking compound 1 into the D174N mutant AspS structure resulted in a different positioning of the piperidine-amide that has no direct interaction with residue 174 (not shown). In this pose the distance between the amide groups of the N174 side chain and compound 1 is 8.3 Å. Thus, docked poses of compound 1 at the D174N mutant and the wild type M. smegmatis AspS structure are consistent with this residue being an important contributor to the binding of compound 1 at AspS. The docking hypothesis of compound 1 at the AspS crystal structure predicts several favorable polar contacts also, as follows. In addition to hydrogen bonding with D174, the amide group of compound 1 forms hydrogen bonding interactions with S167 and the backbone carbonyl of A172. The carbonyl of the thiazolidinone participates in hydrogen binding with T168 while the 2-chlorine substituent on the phenyl forms polar interactions with T165 and the backbone amine of R166. While F521 is not directly accessible to bound ligands, it is possible that the resistance-conferring mutation of F521 to Leu induces conformational changes that propagate to the proposed pocket. While we attempted to generate crystals of Ms-AspS bound to 1 and 2, we could not overcome poor solubility in the crystallization buffers. In the absence of co-crystallized ligand, the proposed docked pose of compound 1 provides a plausible model of its Asp-bound structure with a number of favorable polar and non-polar contacts as described here.


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 - PLoS ONE (2014)

Induced Fit docked pose of compound 1 at the M. smegmatis AspS crystal structure.Only amino acids forming direct interactions with compound 1 are shown. Carbon atoms of AspS are colored light green except for carbons of resistance conferring mutant site residues, which are colored purple. Carbons of compound 1 are shown in orange; all other atoms are colored by atom type (Oxygen red, Nitrogen blue, Sulphur yellow, Chlorine dark green, Hydrogen white). Labels of residues participating in polar interactions are colored brown, labels of all other residues are in black. Polar interactions are indicated with dashed lines and are numbered. Distances between heavy atoms of the atoms groups participating in polar interactions are as follows: 1. 3.1 Å; 2. 3.8 Å; 3. 3.0 Å; 4. 3.2 Å; 5. 3.1 Å; 6. 3.7 Å.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4237437&req=5

pone-0113568-g009: Induced Fit docked pose of compound 1 at the M. smegmatis AspS crystal structure.Only amino acids forming direct interactions with compound 1 are shown. Carbon atoms of AspS are colored light green except for carbons of resistance conferring mutant site residues, which are colored purple. Carbons of compound 1 are shown in orange; all other atoms are colored by atom type (Oxygen red, Nitrogen blue, Sulphur yellow, Chlorine dark green, Hydrogen white). Labels of residues participating in polar interactions are colored brown, labels of all other residues are in black. Polar interactions are indicated with dashed lines and are numbered. Distances between heavy atoms of the atoms groups participating in polar interactions are as follows: 1. 3.1 Å; 2. 3.8 Å; 3. 3.0 Å; 4. 3.2 Å; 5. 3.1 Å; 6. 3.7 Å.
Mentions: Using Induced Fit docking protocols we docked compound 1 into this pocket (Figure 9). The obtained docked pose predicts a number of favorable non-polar and polar contacts with residues lining the pocket as follows. The piperidine ring in compound 1 participates in non-polar interactions with P169 while the thiazolidinone moiety forms aromatic interactions with F514. The dichlorophenyl ring has non-polar/steric contacts with a number of residues: F451, L517, L199, F196, as well as T565 from the adjacent subunit. The side chain of T565 is in close proximity of the ligand’s phenyl ring (the closest ring carbon is at 3.8 Å from the threonine side chain methyl). Guided by the resistance conferring mutation sites that include this threonine, we generated the T565I AspS mutant structure in silico and attempted to re-dock compound 1 using the same protocols, however, no successfully docked poses could be obtained, consistently with the results of the resistant mutations study. Another resistance conferring mutant site corresponds to D174 which is the residue predicted to form hydrogen bonding interactions with the amide group of compound 1 (Figure 9). Re-docking compound 1 into the D174N mutant AspS structure resulted in a different positioning of the piperidine-amide that has no direct interaction with residue 174 (not shown). In this pose the distance between the amide groups of the N174 side chain and compound 1 is 8.3 Å. Thus, docked poses of compound 1 at the D174N mutant and the wild type M. smegmatis AspS structure are consistent with this residue being an important contributor to the binding of compound 1 at AspS. The docking hypothesis of compound 1 at the AspS crystal structure predicts several favorable polar contacts also, as follows. In addition to hydrogen bonding with D174, the amide group of compound 1 forms hydrogen bonding interactions with S167 and the backbone carbonyl of A172. The carbonyl of the thiazolidinone participates in hydrogen binding with T168 while the 2-chlorine substituent on the phenyl forms polar interactions with T165 and the backbone amine of R166. While F521 is not directly accessible to bound ligands, it is possible that the resistance-conferring mutation of F521 to Leu induces conformational changes that propagate to the proposed pocket. While we attempted to generate crystals of Ms-AspS bound to 1 and 2, we could not overcome poor solubility in the crystallization buffers. In the absence of co-crystallized ligand, the proposed docked pose of compound 1 provides a plausible model of its Asp-bound structure with a number of favorable polar and non-polar contacts as described here.

Bottom Line: The human pathogen Mycobacterium tuberculosis is the causative agent of pulmonary tuberculosis (TB), a disease with high worldwide mortality rates.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.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom.

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.

Show MeSH
Related in: MedlinePlus