Inhibition of xanthine oxidase by the aldehyde oxidase inhibitor raloxifene: Implications for identifying molybdopterin nitrite reductases

Abstract

Sources of nitric oxide alternative to nitric oxide synthases are gaining significant traction as crucial mediators of vessel function under hypoxic inflammatory conditions. For example, capacity to catalyze the one electron reduction of nitrite ( NO2-) to •NO has been reported for hemoglobin, myoglobin and molybdopterin-containing enzymes including xanthine oxidoreductase (XOR) and aldehyde oxidase (AO). For XOR and AO, use of selective inhibition strategies is therefore crucial when attempting to assign relative contributions to nitrite-mediated •NO formation in cells and tissue. To this end, XOR inhibition has been accomplished with application of classic pyrazolopyrimidine-based inhibitors allo/oxypurinol or the newly FDA-approved XOR-specific inhibitor, Uloric® (febuxostat). Likewise, raloxifene, an estrogen receptor antagonist, has been identified as a potent (Ki = 1.0 nM) inhibitor of AO. Herein, we characterize the inhibition kinetics of raloxifene for XOR and describe the resultant effects on inhibiting XO-catalyzed •NO formation. Exposure of purified XO to raloxifene (PBS, pH 7.4) resulted in a dose-dependent (12.5–100 μM) inhibition of xanthine oxidation to uric acid. Dixon plot analysis revealed a competitive inhibition process with a Ki = 13 μM. This inhibitory process was more effective under acidic pH; similar to values encountered under hypoxic/inflammatory conditions. In addition, raloxifene also inhibited anoxic XO-catalyzed reduction of NO2- to •NO (EC50 = 64 μM). In contrast to having no effect on XO-catalyzed uric acid production, the AO inhibitor menadione demonstrated potent inhibition of XO-catalyzed NO2- reduction (EC50 = 60 nM); somewhat similar to the XO-specific inhibitor, febuxostat (EC50 = 4 nM). Importantly, febuxostat was found to be a very poor inhibitor of human AO (EC50 = 613 μM) suggesting its usefulness for validating XO-dependent contributions to NO2- reduction in biological systems. Combined, these data indicate care should be taken when choosing inhibition strategies as well as inhibitor concentrations when assigning relative NO2- reductase activity of AO and XOR.

A Novel Reaction Mediated by Human Aldehyde Oxidase: Amide Hydrolysis of GDC-0834

“…Whereas the oxidized hydroxymolybdenum species is capable of nucleophilic attack, as is evident by the nucleophilic aromatic substitution normally catalyzed by this reaction, we propose that Mo IV oxidative state is more suited for this reaction because of its higher electron density. This would require build-up of the Mo IV oxidative state in much the same way that AO-mediated reductions of compounds, such as nitrites, need a substrate to initially be at ASPET Journals on May 9, 2018 dmd.aspetjournals.org oxidized (Weidert et al, 2014). If this is the case, the substrate that is oxidized is presently unknown but could be an endogenous aldehyde or azaheterocyclic substrate in the cytosol.…”

Honey as an apitherapic product: its inhibitory effect on urease and xanthine oxidase

“…In general, ROS are formed through both physiological and non-physiological pathways. Some of the enzymes of myeloperoxidase, aldehyde oxidase, nitric oxide synthase and xanthine oxidase (XO) catalyze the formation of some ROS 5,6 . Many ROS are generated by XO to catalyze the oxidation of hypoxanthine into xanthine 7,8 .…”

In Vitro Oxidative Metabolism of 6-Mercaptopurine in Human Liver: Insights into the Role of the Molybdoflavoenzymes Aldehyde Oxidase, Xanthine Oxidase, and Xanthine Dehydrogenase

“…Raloxifene and febuxostat specifically inhibit drug metabolism by AO and XO, respectively, at concentrations up to 1 mM. (Obach, 2004;Weidert et al, 2014). Raloxifene inhibited the formation of 6TX by approximately 39%, whereas febuxostat demonstrated 67% inhibition (Fig.…”