Xanthine oxidoreductase (XOR) is a metalloenzyme that catalyzes the final steps in purine metabolism by converting hypoxanthine to xanthine and then uric acid. Allopurinol, an analog of hypoxanthine, is widely used as an anti-gout drug, as XOR-mediated metabolism of allopurinol to oxypurinol leads to oxypurinol rotation in the enzyme active site and reduction of the molybdenum Mo(VI) active center to Mo(IV), inhibiting subsequent urate production. However, when oxypurinol is administered directly to a mouse model of hyperuricemia, it yields a weaker urate-lowering effect than allopurinol. To better understand its mechanism of inhibition and inform patient dosing strategies, here we performed kinetic and structural analyses of the inhibitory activity of oxypurinol. Our results demonstrated that oxypurinol was less effective than allopurinol both in vivo and in vitro. We show that upon reoxidation to Mo(VI), oxypurinol binding is greatly weakened, and reduction by xanthine, hypoxanthine, or allopurinol is required for reformation of the inhibitor-enzyme complex. In addition, we show oxypurinol only weakly inhibits the conversion of hypoxanthine to xanthine and is therefore unlikely to effect feedback inhibition of de novo purine synthesis. Furthermore, we observed weak allosteric inhibition of purine nucleoside phosphorylase by oxypurinol, which has potentially adverse effects for patients. Considering these results, we propose the single-dose method currently used to treat hyperuricemia can result in unnecessarily high levels of allopurinol. While the short half-life of allopurinol in blood suggests that oxypurinol is responsible for enzyme inhibition, we anticipate multiple, smaller doses of allopurinol would reduce the total allopurinol patient load.

Xanthine oxidoreductase (XOR) is an enzyme that catalyzes the two-step reaction from hypoxanthine to xanthine and from xanthine to uric acid in purine metabolism. XOR generally carries dehydrogenase activity (XDH) but is converted into an oxidase (XO) under various pathophysiologic conditions. The complex structure and enzymatic function of XOR have been well investigated by mutagenesis studies of mammalian XOR and structural analysis of XOR–inhibitor interactions. Three XOR inhibitors are currently used as hyperuricemia and gout therapeutics but are also expected to have potential effects other than uric acid reduction, such as suppressing XO–generating reactive oxygen species. Isolated XOR deficiency, xanthinuria type I, is a good model of the metabolic effects of XOR inhibitors. It is characterized by hypouricemia, markedly decreased uric acid excretion, and increased serum and urinary xanthine concentrations, with no clinically significant symptoms. The pathogenesis and relationship between mutations and XOR activity in xanthinuria are useful for elucidating the biological role of XOR and the details of the XOR reaction process. In this review, we aim to contribute to the basic science and clinical aspects of XOR by linking the mutations in xanthinuria to structural studies, in order to understand the function and reaction mechanism of XOR in vivo.

Previous studies suggest that uric acid or reactive oxygen species, products of xanthine oxidoreductase (XOR), may associate with neurodegenerative diseases. However, neither relationship has ever been firmly established. Here, we analyzed human brain samples, obtained under protocols approved by research ethics committees, and found no expression of XOR and only low levels of uric acid in various regions of the brain. In the absence of XOR, hypoxanthine will be preserved and available for incorporation into the purine salvage pathway. To clarify the importance of salvage in the brain, we tested using human-induced pluripotent stem cell-derived neuronal cells. Stable isotope analyses showed that the purine salvage pathway was more effective for ATP synthesis than purine de novo synthesis. Blood uric acid levels were related to the intracellular adenylate pool (ATP + ADP + AMP), and reduced levels of this pool result in lower uric acid levels. XOR inhibitors are related to extracellular hypoxanthine levels available for uptake into the purine salvage pathway by inhibiting the oxidation of hypoxanthine to xanthine and uric acid in various organs where XOR is present and can prevent further decreases in the intracellular adenylate pool under stress. Furthermore, adding precursors of the pentose phosphate pathway enhanced hypoxanthine uptake, indicating that purine salvage is activated by phosphoribosyl pyrophosphate replenishment. These findings resolve previous contradictions regarding XOR products and provide new insights into clinical studies. It is suggested that therapeutic strategies maximizing maintenance of intracellular adenylate levels may effectively treat pathological conditions associated with ischemia and energy depletion.

Xanthine oxidoreductase (XOR) is the only enzyme responsible for uric acid production and is essential for preventing gout. While XOR inhibitors effectively reduce serum urate levels, they also influence purine salvage and de novo pathways, as well as energy metabolism, raising concerns about metabolic adaptation and rebound effects upon treatment discontinuation. In this review, we outline the fundamental regulatory mechanisms of purine metabolism and summarize the mechanisms of action of XOR inhibitors and their associated metabolic effects with reference to XOR deficiency, type I xanthinuria. Furthermore, we discuss the impact of discontinuing XOR inhibitors and examine their potential for rebound.