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

摘要: Rationale Plasmodium falciparum (Pf) thioredoxin reductase (TrxR) catalyzes the reduction of disulfide (Trx-S(2)) to dithiol (Trx-(SH)(2)) that is essential for antioxidant defense mechanism and DNA synthesis in parasite a validated drug target new antimalarial agents. Methods In this study, we have developed liquid chromatography/mass spectrometry (LC/MS)-based functional assay identify inhibitors PfTrxR by quantifying product formed enzymatic reaction. Relative quantitation reaction (intact Trx-(SH)(2)) was carried out using an Agilent 6520 QTOF mass spectrometer equipped with positive mode electrospray ionization (ESI) source. Results The calibration curve prepared Trx-(SH)(2) at concentrations ranging from 1.8 116.5 µg/mL linear (R(2) >0.998). limit detection (LOD) quantification (LOQ) were 0.45 respectively. To validate screened reference compounds 1, 2 3 their inhibitory activity ten natural (at 10 μM) which earlier identified as ligands UF-LC/MS based binding assay. Conclusions LC/MS-based identification sensitive reliable method also amendable high-throughput format. This first representation relative intact LC/MS.