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System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis
09:57

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Published on: April 5, 2017

Aptamer-mediated inhibition of Mycobacterium tuberculosis polyphosphate kinase 2

Ka To Shum1, Eric Lik Hang Lui, Sybil Cheuk Ki Wong

  • 1Department of Biochemistry, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong, China.

Biochemistry
|March 9, 2011
PubMed

Insights

Researchers developed DNA aptamers to inhibit Mycobacterium tuberculosis PPK2, a key enzyme in bacterial survival. A G-quadruplex aptamer showed potent and selective inhibition, offering a potential therapeutic strategy against tuberculosis.

Area of Science:

  • Microbiology
  • Biochemistry
  • Molecular Biology

Background:

  • Inorganic polyphosphate (polyP) is crucial for bacterial persistence, stress response, and virulence.
  • Polyphosphate kinase (PPK) enzymes regulate intracellular polyP metabolism.
  • Inhibiting PPK offers a strategy to disrupt polyP-dependent processes in pathogens.

Purpose of the Study:

  • Biochemically characterize Mycobacterium tuberculosis (MTB) PPK2.
  • Develop DNA aptamers to inhibit MTB PPK2 activity.
  • Investigate the inhibitory mechanism and specificity of aptamers.

Main Methods:

  • Biochemical assays to determine enzyme kinetics and catalytic rates.
  • Gel filtration chromatography for protein complex analysis.
  • Isothermal titration calorimetry for binding affinity and thermodynamics.
  • Circular dichroism for aptamer secondary structure determination.
  • Enzyme inhibition assays (IC50 determination) and kinetic analysis.
  • Mutational analysis to identify critical aptamer motifs.

Main Results:

  • MTB PPK2 exhibits a high polyP-dependent phosphorylation rate.
  • MTB PPK2 exists as an octamer.
  • A G-quadruplex DNA aptamer selectively binds MTB PPK2 with high affinity (Kd = 870 nM).
  • The aptamer noncompetitively inhibits MTB PPK2 with an IC50 of 40 nM.
  • The G-quadruplex motif is essential for inhibition.
  • The aptamer shows cross-inhibition against Vibrio cholerae PPK2.

Conclusions:

  • DNA aptamers, particularly G-quadruplex structures, are effective inhibitors of MTB PPK2.
  • The developed aptamer demonstrates high selectivity and potency.
  • This aptamer represents a promising lead for developing novel anti-tubercular therapeutics targeting PPK2.

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