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. 2010 Jan 29;17(1):86-97.
doi: 10.1016/j.chembiol.2009.12.014.

Methionine aminopeptidases from Mycobacterium tuberculosis as novel antimycobacterial targets

Omonike Olaleye  1 , Tirumalai R Raghunand, Shridhar Bhat, Jian He, Sandeep Tyagi, Gyanu Lamichhane, Peihua Gu, Jiangbing Zhou, Ying Zhang, Jacques Grosset, William R Bishai, Jun O Liu
Affiliations

Methionine aminopeptidases from Mycobacterium tuberculosis as novel antimycobacterial targets

Omonike Olaleye et al. Chem Biol. .

Abstract

Methionine aminopeptidase (MetAP) is a metalloprotease that removes the N-terminal methionine during protein synthesis. To assess the importance of the two MetAPs in Mycobacterium tuberculosis, we overexpressed and purified each of the MetAPs to near homogeneity and showed that both were active as MetAP enzymes in vitro. We screened a library of 175,000 compounds against MtMetAP1c and identified 2,3-dichloro-1,4-naphthoquinone class of compounds as inhibitors of both MtMetAPs. It was found that the MtMetAP inhibitors were active against replicating and aged nongrowing M. tuberculosis. Overexpression of either MtMetAP1a or MtMetAP1c in M. tuberculosis conferred resistance of bacterial cells to the inhibitors. Moreover, knockdown of MtMetAP1a, but not MtMetAP1c, resulted in decreased viability of M. tuberculosis. These results suggest that MtMetAP1a is a promising target for developing antituberculosis agents.

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Figures

Figure 1
Figure 1. Sequence Comparison of MtMetAP1a, MtMetAP1c, HsMetAP1 and EcMetAP1
The alignment was generated using ClustalW (www.ebi.ac.uk). Both MtMetAPs share a 33% similarity and the metal-chelating residues necessary for catalysis are conserved (*). MtMetAP1a and EcMetAP1 lack the N-terminal extension with a PXXPXP motif present in MtMetAP1c AND HsMetAP1 (underlined).
Figure 2
Figure 2. Purification and Kinetic Characterization of Recombinant MetAPs from M. tuberculosis
The recombinant MtMetAP1s were over-expressed in E. coli BL21 cells and purified by affinity chromatography as described the methods section. (A) PolyHis-tagged-MtMetAP1c (~32 kDa). (B) PolyHis-tagged-MtMetAP1a (~28 kDa). Lane 1, Molecular weight marker; Lane 2, un-induced whole cell lysate; Lane 3, induced cell lysate; Lane 4, purified polyHis-tagged MtMetAP1. The gel was stained with Coomassie blue. (C) Velocity versus substrate concentration plot for MtMetAP1a (triangles) and MtMetAP1c (squares). The kinetic constants were obtained by measuring enzyme activity at different substrate concentrations. The reactions were carried out in 96-well plates at room temperature and monitored at 405 nm on a UV-Vis spectrophotometer. The total volume of reaction was 100 µL (each reaction contains 40 mM HEPES (pH 7.5), 100 mM NaCl, 1 µM CoCl2, 100 µg/mL BSA, 0.1 U/mL ProAP, and 0–800 µM Met-Pro-pNA), 334 nM MtMetAP1c and 3.29 µM MtMetAP1a, respectively. The background hydrolysis was corrected. The data were from quadruplet experiments and were fitted against the Michealis-Menten equation: V = Vmax * [S]/(Km + [S]) using the Graphpad prism software for one-site binding hyperbola.
Figure 3
Figure 3. Overexpression of MtMetAP1a and MtMetAP1c Confer Resistance to Inhibitors
(A) Schematic representation of plasmids used for target validation. (i) Control plasmid pSCW35ΔsigF; (ii) Sense construct: pSCW35ΔsigF-(MtMetAP1); (iii) Anti-sense construct: pSCW35ΔsigF-(α-MtMetAP1). The MtMetAP genes were inserted downstream of the acetamide regulated promoter (Pace). (B and C) The expression of MtMetAP1a and MtMetAP1c mRNA in M. tuberculosis as determined by quantitative Real-Time RT-PCR. The levels of MtMetAP1a and MtMetAP1c were determined in M. tuberculosis strains transformed with vectors over-expressing the two genes in the sense (A-ii) and anti-sense (A-iii) orientation, respectively. The quantities of mRNA are shown as fold change compared to the expression in the wild-type with standard error from two independent experiments.
Figure 4
Figure 4. Target Validation of Naphthoquinone In vivo
M. tuberculosis knock-in strains containging MtMetAP1a, MtMetAP1c, or control expression plasmid were grown in liquid media in the presence (A) or absence (B) of 10 µg/mL Compound 4 and DMSO. Symbols: MtMetAP1a, diamonds; MtMetAP1c, squares; wild-type strain, stars; and sigma factor-F lacking mutant, triangles.
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