Development of liquid chromatography/mass spectrometry based screening assay for PfTrxR inhibitors using relative quantitation of intact thioredoxin

• Published in: Rapid communications in mass spectrometry 2012-09, Vol.26 (17), p.2051-2056
• Main Authors: Munigunti, Ranjith, Calderón, Angela I.
• Format: Article
• Language: English
• Subjects: Analytical chemistry; Assaying; Binding; Chromatography; Chromatography, Liquid - methods; Drug Discovery; Enzyme Inhibitors - analysis; Enzyme Inhibitors - pharmacology; Inhibitors; Inhibitory Concentration 50; Ionization; Limit of Detection; Linear Models; Liquid chromatography; Mass spectrometry; Mass Spectrometry - methods; Plasmodium falciparum; Plasmodium falciparum - enzymology; Reductases; Reproducibility of Results; Reproduction; Sulfhydryl Compounds - analysis; Sulfhydryl Compounds - metabolism; Thioredoxin-Disulfide Reductase - antagonists & inhibitors; Thioredoxin-Disulfide Reductase - metabolism
• ISSN: 0951-4198; 1097-0231
• DOI: 10.1002/rcm.6316

RATIONALE Plasmodium falciparum (Pf) thioredoxin reductase (TrxR) catalyzes the reduction of thioredoxin disulfide (Trx–S2) 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 (R2 >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. Copyright © 2012 John Wiley & Sons, Ltd.