In Anopheles gambiae, the most efficient vector of the malaria parasite Plasmodium falciparum, 3-hydroxykynurenine is endowed with a toxic potential. In adult mosquitoes, the excess of 3-hydroxykynurenine is removed by a specific transaminase (3-hydroxykynurenine transaminase, 3-HKT) which converts the compound into the more stable xanthurenic acid. Interfering with 3-hydroxykynurenine metabolism in A. gambiae is a potential approach for the development of transmission-blocking drugs and insecticides. Hence, the aims of this work were to develop and validate a new LC-MS/MS method for the evaluation of A. gambiae 3-hydroxykynurenine transaminase (Ag-HKT) activity and the determination of the potency of inhibitors of the enzyme. We set up a LC-MS/MS based enzymatic assay for the determination of kinetic constants values of the recombinant Ag-HKT enzyme and for the evaluation of Ag-HKT inhibition by a known protein inhibitor used as reference and a newly synthesized compound. The chromatographic separation was performed in a gradient mode on a Phenomenex Synergi Polar-RP (150 mm × 2.0, 4 μm) with methanol and water containing both 0.2% formic acid. Mass spectrometric detection was achieved with an ion trap equipped with an ESI source, in positive ionization scan, operating in SRM mode. The LC-MS/MS method was validated in terms of selectivity, linearity, precision and accuracy.
Keywords: Ag-HKT; Anopheles gambiae; Enzyme assay; Kynurenine pathway; LC-MS/MS.
Copyright © 2019 Elsevier B.V. All rights reserved.