Herein it is reported the development and application of two chromatographic assays for the measurement of the activity of 3-Hydroxyanthranilate-3,4-dioxygenase (3HAO). Such an enzyme converts 3-Hydroxyanthranilic acid (3HAA) to 2-amino-3-carboxymuconic semialdehyde (ACMS), which undergo a spontaneous, non-enzymatic cyclization to produce quinolinic acid (QUIN). The enzyme activity was measured by quantitation of the sub-strate consumption over time either with spectrophotometric (UV) or mass spectrometric (MS) detection upon reversed-phase chromatographic separation. MS detection resulted more selective and sensitive, but less accurate and precise. However, both methods have sufficient sensitivity to allow the measurement of enzyme activity with consistent results compared to literature data. Since MS detection allowed less sample consumption it was used to calculate the kinetics parameters (i.e., Vmax and Kd) of recombinant 3HAO. Another MS-based method was then developed to measure the amount of QUIN produced, revealing an incomplete conversion of 3HAA to QUIN. As suggested by previous studies, the enzyme activity was apparently sensitive to the redox state of the enzyme thiols. In fact, thiol reducing agents such as dithiothreitol (DTT) and glutathione (GSH), can alter the enzyme activity although the investigation on the exact mechanism involved in such effect was beyond the scope of the research. Interestingly, edaravone (EDA) induced an in vitro suppression of QUIN production through direct, competitive 3HAO inhibition. EDA is a molecule approved for the treatment of amyotrophic lateral sclerosis (ALS), a neurodegenerative disease associated with an increase of QUIN concentrations in both serum and ce-rebrospinal fluid. Although EDA was reported to mitigate ALS progression its mode of action is still largely unknown. Some studies reported antioxidant and radical scavenger properties of EDA, but none confirm a direct activity as 3HAO enzyme inhibitor. Since QUIN is reported to be a neurotoxic metabolite, 3HAO inhibition can contribute to the beneficial effect of EDA in ALS, although such a mechanism must be then confirmed in vivo. However, EDA might be a convenient scaffold for the design of selective 3HAO inhibitors with potential ap-plications in ALS treatment.
File in questo prodotto:
Non ci sono file associati a questo prodotto.