title: Development of liquid chromatography/mass spectrometry based screening assay for PfTrxR inhibitors using relative quantitation of intact thioredoxin.
abstract: BACKGROUND
  Plasmodium falciparum (Pf) thioredoxin reductase (TrxR) catalyzes the reduction of thioredoxin disulfide (Trx-S(2)) to thioredoxin dithiol (Trx-(SH)(2)) that is essential for antioxidant defense mechanism and DNA synthesis in the parasite and is a validated drug target for new antimalarial agents.
  
  
  METHODS
  In this study, we have developed a liquid chromatography/mass spectrometry (LC/MS)-based functional assay to identify inhibitors of PfTrxR by quantifying the product formed (Trx-(SH)(2)) in the enzymatic reaction. Relative quantitation of the reaction product (intact Trx-(SH)(2)) was carried out using an Agilent 6520 QTOF mass spectrometer equipped with a positive mode electrospray ionization (ESI) source.
  
  
  RESULTS
  The calibration curve prepared for Trx-(SH)(2) at concentrations ranging from 1.8 to 116.5 µg/mL was linear (R(2) >0.998). The limit of detection (LOD) and limit of quantification (LOQ) of Trx-(SH)(2) were at 0.45 and 1.8 µg/mL respectively. To validate the developed functional assay we have screened reference compounds 1, 2 and 3 for their PfTrxR inhibitory activity and ten natural compounds (at 10 μM) which were earlier identified as ligands of PfTrxR by a UF-LC/MS based binding assay.
  
  
  CONCLUSIONS
  The developed LC/MS-based functional assay for identification of inhibitors of PfTrxR is a sensitive and reliable method that is also amendable for high-throughput format. This is the first representation of a relative quantitation of intact Trx-(SH)(2) using LC/MS.
authors: Department of Pharmacal Sciences, Harrison School of Pharmacy, Auburn University, Auburn, AL 36849, USA.
authors: Ranjith
authors: R
authors: Munigunti
authors: Angela I
authors: AI
authors: Calderón
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D055808:Drug Discovery; D004791:Enzyme Inhibitors / Q000032:analysis* / Q000494:pharmacology; D020128:Inhibitory Concentration 50; D057230:Limit of Detection; D016014:Linear Models; D013058:Mass Spectrometry / Q000379:methods*; D010963:Plasmodium falciparum / Q000201:enzymology*; D015203:Reproducibility of Results; D013438:Sulfhydryl Compounds / Q000032:analysis / Q000378:metabolism; D013880:Thioredoxin-Disulfide Reductase / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D013438:Sulfhydryl Compounds; D013880:Thioredoxin-Disulfide Reductase