Fast and low-cost evaluation of hydroxykynurenine activity

Abstract

The enzyme 3-hydroxykynurenine transaminase (HKT) acts as an important enzyme in tryptophan catabolism of disease-carrier insects, e.g. Aedes aegypti and Anopheles gambiae. HKT is a detoxification enzyme that converts 3-hydroxykynurenine (a precursor for reactive nitrogen and oxygen species) into xanthurenic acid (stable and nontoxic compound). We have previously synthesized eleven new oxadiazole derivatives and demonstrated their noncompetitive inhibitory activity towards HKT from A. aegypti (https://doi.org/10.1016/j.bmc.2019.115252). These findings are presented in a research paper accompanying the present technical report on a new assay to overcome the fact that the substrate and product of the HKT-catalyzed reaction exhibit maximum absorption at very near wavelength (370 and 369 nm, respectively). The methods previously described in the literature rely on chromatographic separation prior to absorbance quantification, which limits their use for inhibitor screening. Due to HKT attractive features as a molecular target for larvicidal compounds, we report herein a new, faster and affordable methodology to evaluate the enzymatic activity of recombinant HKT, and therefore allow for the fast screening of potential HKT inhibitors via absorbance spectrophotometer. The advantages of the proposed methodology to previously described ones are:•It is faster and cheaper than HPLC-based assays because it does not require the use of chromatography columns and solvents to separate reaction components;•It uses of 96-well plates, enabling the simultaneous quantification of samples;•It can be applied to all transaminases that have xanthurenic acid as a product.

Keywords: HKT activity; Inhibition assay; Kynurenine pathway.

Graphical abstract

Fig. 1

Calibration curve used for quantification of biochemical and inhibition data. Each sample contains 2 mM sodium pyruvate, 40 µM PLP, 10 mM FeCl₃·6H₂O in 0.1 M HCl and variable concentration of XA (0–2 mM) in a final volume of 200 µL.

Fig. 2

The progress curve (A), pH evaluation in buffers Tris pH 6,7 and 8, HEPES pH 7.5 and Borate pH 9 (B), temperature evaluation (C), the non-linear regression curve of inhibition data from the compound sodium 4-[3-(p-tolyl)−1,2,4-oxadiazol-5-yl]butanoate to estimate its IC₅₀(D) and a picture of the plate after a experiment (E).