Development, validation and application of a 96-well enzymatic assay based on LC-ESI-MS/MS quantification for the screening of selective inhibitors against Mycobacterium tuberculosis purine nucleoside phosphorylase.

  title={Development, validation and application of a 96-well enzymatic assay based on LC-ESI-MS/MS quantification for the screening of selective inhibitors against Mycobacterium tuberculosis purine nucleoside phosphorylase.},
  author={Giulia Cattaneo and Daniela Ubiali and Enrica Calleri and Marco Rabuffetti and Georg C H{\"o}fner and Klaus T. Wanner and Marcela Cristina de Moraes and Leonardo K. Martinelli and Di{\'o}genes Santiago Santos and Giovanna Speranza and Gabriella Massolini},
  journal={Analytica chimica acta},
Mycobacterium tuberculosis (Mtb) purine nucleoside phosphorylase (PNP, EC has been identified as a target for the development of specific inhibitors with potential antimycobacterial activity. We hereby described the development and validation of a new 96-well LC-ESI-MS/MS method to assess the inhibition activity of nucleoside analogues towards MtbPNP and the human PNP (HsPNP). Enzyme activity was determined by monitoring the phosphorolysis of inosine (Ino) to hypoxanthine (Hpx). The… 
6 Citations
Development of a UHPLC-MS method for inhibitor screening against α-L-1,3-fucosidase
UHPLC-MS has been utilized to develop a simple, sensitive, and accurate assay for enzyme kinetics and inhibition studies of AFU3, a member of the AFU family, and the bioanalytical quantitative screening method was validated according to US-FDA guidance.
Dual-target inhibitor screening against thrombin and factor Xa simultaneously by mass spectrometry.
The results indicated that the dual-target assay by UHPLC-MS/MS analysis could be used as a reliable method for screening anticoagulant agents.
On-flow magnetic particle activity assay for the screening of human purine nucleoside phosphorylase inhibitors.
Inhibition studies conducted with a fourth generation immucillin derivative were employed as proof of concept to validate the use of the HsPNP-MPs assays for screening purposes and showed that both presented assays can be applied to selectively recognizing and characterizing HspnP inhibitors.
Global mapping of protein–metabolite interactions in Saccharomyces cerevisiae reveals that Ser-Leu dipeptide regulates phosphoglycerate kinase activity
A biochemical approach named PROMIS was applied to address the complexity of the protein–small molecule interactome in the model yeast Saccharomyces cerevisiae and provided a unique dataset, which can be queried for interactions between 74 small molecules and 3982 proteins using a user-friendly interface.
Metabolomics: current application and prospects in crop production
This review focuses on plant improvement, which is a driver for improved food security and development is needed to increase the understanding of microbial metabolites in order to develop bioproducts that will increase growth and eventual yield, protecting plants from pathogens and in the process providing nutritious food for the teeming populace.
Instrumentation Applied to Metabolomic Analysis


Capillary bioreactors based on human purine nucleoside phosphorylase: a new approach for ligands identification and characterization.
This new approach is a valuable tool to PNP ligand screening, since it directly measures the hypoxanthine released by inosine phosphorolysis, thus furnishing more reliable results than those one used in a coupled enzymatic spectrophotometric assay.
Mass spectrometry for enzyme assays and inhibitor screening: an emerging application in pharmaceutical research.
  • K. Greis
  • Chemistry, Medicine
    Mass spectrometry reviews
  • 2007
How mass spectrometry for studying enzymes activity has progressed from simple qualitative questions to quantitative measures of enzyme activity and kinetics and then as a tool for rapidly screening inhibitory compounds as an alternative to current methods of high throughput drug screening is captured.
Immobilized purine nucleoside phosphorylase from Aeromonas hydrophila as an on-line enzyme reactor for biocatalytic applications.
  • E. Calleri, D. Ubiali, +5 authors G. Massolini
  • Chemistry, Medicine
    Journal of chromatography. B, Analytical technologies in the biomedical and life sciences
  • 2014
Comparison with the soluble enzyme showed that the AhPNP-based bioreactor is reliable as the same ranking order, with respect to the standard activity assay, was obtained.
Immobilized purine nucleoside phosphorylase from Schistosoma mansoni for specific inhibition studies
Use of an SmPNP immobilized enzyme reactor (IMER) for inhibitor-screening assays was demonstrated, and the method had high selectivity and specificity compared with screening by use of the free enzyme by the Kalckar method, and furnished results without the need for verification of the absence of false positives.
Developing a Collection of Immobilized Nucleoside Phosphorylases for the Preparation of Nucleoside Analogues: Enzymatic Synthesis of Arabinosyladenine and 2,3-Dideoxyinosine
The use of nucleoside phosphorylases (NPs; EC 2.4.2.n) represents a convenient alternative to the chemical route for the synthesis of natural and modified nucleosides. We purified four recombinantly
Extending matrix-assisted laser desorption/ionization triple quadrupole mass spectrometry enzyme screening assays to targets with small molecule substrates.
Data demonstrate that a MALDI-QqQMS-based readout platform is amenable to measuring small molecule substrates and products and offers significant advantages over current HTS methods in terms of speed, sensitivity, reproducibility and reagent costs.
Ultrahigh performance liquid chromatography-triple quadrupole mass spectrometry inhibitors fishing assay: a novel method for simultaneously screening of xanthine oxidase inhibitor and superoxide anion scavenger in a single analysis.
An ultrahigh performance liquid chromatography and triple quadrupole mass spectrometry (UHPLC-TQ-MS) method of higher accuracy and speed that combines screening of superoxide anion scavenger and XOD inhibitor in a single analysis by adding WST-1 to the enzymatic reaction.
Crystal structure and molecular dynamics studies of purine nucleoside phosphorylase from Mycobacterium tuberculosis associated with acyclovir.
Findings contribute to the search of new specific inhibitors for MtPNP, since peculiarities between the mycobacterial and human enzyme binding sites have been identified, making a structural-based drug design feasible.
Inhibitors of purine nucleoside phosphorylase, C(8) and C(5') substitutions.
Abstract The C(8) and C(5') positions of base and nucleoside substrates of human erythrocytic purine nucleoside phosphorylase (PNP) are promising sites for the development of an inhibitor of this
Cloning, overexpression, and purification of functional human purine nucleoside phosphorylase.
PCR amplification of human PNP from a liver cDNA library, cloning, expression in Escherichia coli host, purification, and activity measurement of homogeneous enzyme are described.