Identification of a novel xanthine oxidoreductase inhibitor for hyperuricemia treatment with high efficacy and safety profile.

Hyperuricemia is with growing incidence and of high risk to develop into gout and other metabolic diseases. The key enzyme catalyzing uric acid synthesis, xanthine oxidoreductase (XOR) is a vital target for anti-hyperuricemic drugs, while XOR inhibitors characterized as both potent and safe are currently in urgent need. In this study, a novel small molecule compound, CC15009, was identified as a specific XOR inhibitor. CC15009 exerted strongest in vitro XOR inhibitory activity among current XOR inhibitors. It also showed favorable dose-dependent uric acid-lowering effects in two different XOR substrate-induced hyperuricemic mouse models, which was significantly superior than the current first-line drug, allopurinol. Mechanically, the direct binding of CC15009 against XOR was confirmed by molecular docking and SPR analysis. The inhibition mode was competitive and reversible. Besides, the potential antioxidant activity of CC15009 was indicated by its strong inhibitory activity against the oxidized isoform of XOR, which reduced ROS generation as the byproduct. Regarding the safety concerns of current XOR inhibitors, especially in cardiovascular risks, the safety of CC15009 was comprehensively evaluated. No significant abnormality was observed in the acute, subacute toxicity tests and mini-AMES test. Notably, there was no obvious inhibition of CC15009 against cardiac ion channels, including hERG, Nav1.5, Cav1.2 at the concentration of 30 µM, indicating its lower cardiovascular risk. Taken together, our results supported CC15009 as a candidate of high efficacy and safety profile to treat hyperuricemia through direct XOR inhibition.

Topiroxostat improves glomerulosclerosis in type 2 diabetic Nagoya Shibata Yasuda mice with early diabetic kidney disease.

Reactive oxygen species production might be prevented by xanthine oxidoreductase (XOR) inhibitors, which can cause glomerulosclerosis. We aimed to investigate whether topiroxostat, an XOR inhibitor, prevents diabetic kidney disease development in mice. Six-week-old control Institute of Cancer Research (ICR) mice and type 2 diabetic Nagoya Shibata Yasuda (NSY) mice were divided into the ICR group (ICR mice which received a lard-containing high-fat diet [HFD] based on the AIN-93G diet), NSY control group (NSY mice which received the same aforementioned diet), and NSY + topiroxostat group (NSY mice which received the same aforementioned diet with addition of 0.0012% topiroxostat). After 20 weeks, plasma biomarkers, XOR activity and oxidative stress levels, which were assessed using malondialdehyde (MDA), were measured through enzyme-linked immunosorbent assay or enzymatic methods. Renal pathology was evaluated using periodic acid-Schiff staining. Redox gene and protein expression were determined using RT-qPCR and western blotting, respectively. Plasma XOR activity was lower in NSY mice treated with topiroxostat than those without. Plasma cystatin C and creatinine levels did not differ between the ICR and NSY control groups or between the NSY control and NSY + topiroxostat groups. The NSY + topiroxostat group showed a smaller mesangial area than the NSY control group. The mRNA expression of Sod3, Prdx1, Gpx2, and Gpx3 was higher in the NSY + topiroxostat group than in the NSY control group. Renal MDA levels were lower in the NSY + topiroxostat group than in the NSY control group. Topiroxostat can reduce glomerulosclerosis, and the reduction is associated with renal oxidative markers.

Significance and amplification methods of the purine salvage pathway in human brain cells.

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.

[Uric Acid Metabolism, Uric Acid Transporters and Dysuricemia].

Serum urate levels are determined by the balance between uric acid production and uric acid excretion capacity from the kidneys and intestinal tract. Dysuricemia, including hyperuricemia and hypouricemia, develops when the balance shifts towards an increase or a decrease in the uric acid pool. Hyperuricemia is mostly a multifactorial genetic disorder involving several disease susceptibility genes and environmental factors. Hypouricemia, on the other hand, is caused by genetic abnormalities. The main genes involved in dysuricemia are xanthine oxidoreductase, an enzyme that produces uric acid, and the urate transporters urate transporter 1/solute carrier family 22 member 12 (URAT1/SLC22A12), glucose transporter 9/solute carrier family 2 member 9 (GLUT9/SLC2A9) and ATP binding cassette subfamily G member 2 (ABCG2). Deficiency of xanthine oxidoreductase results in xanthinuria, a rare disease with marked hypouricemia. Xanthinuria can be due to a single deficiency of xanthine oxidoreductase or in combination with aldehyde oxidase deficiency as well. The latter is caused by a deficiency in molybdenum cofactor sulfurase, which is responsible for adding sulphur atoms to the molybdenum cofactor required for xanthine oxidoreductase and aldehyde oxidase to exert their action. URAT1/SLC22A12 and GLUT9/SLC2A9 are involved in urate reabsorption and their deficiency leads to renal hypouricemia, a condition that is common in Japanese due to URAT1/SLC22A12 deficiency. On the other hand, ABCG2 is involved in the secretion of urate, and many Japanese have single nucleotide polymorphisms that result in its reduced function, leading to hyperuricemia. In particular, severe dysfunction of ABCG2 leads to hyperuricemia with reduced extrarenal excretion.

Xanthine oxidoreductase inhibition ameliorates high glucose-induced glomerular endothelial injury by activating AMPK through the purine salvage pathway.

Xanthine oxidoreductase (XOR) contributes to reactive oxygen species production. We investigated the cytoprotective mechanisms of XOR inhibition against high glucose (HG)-induced glomerular endothelial injury, which involves activation of the AMP-activated protein kinase (AMPK). Human glomerular endothelial cells (GECs) exposed to HG were subjected to febuxostat treatment for 48 h and the expressions of AMPK and its associated signaling pathways were evaluated. HG-treated GECs were increased xanthine oxidase/xanthine dehydrogenase levels and decreased intracellular AMP/ATP ratio, and these effects were reversed by febuxostat treatment. Febuxostat enhanced the phosphorylation of AMPK, the activation of peroxisome proliferator-activated receptor (PPAR)-gamma coactivator (PGC)-1α and PPAR-α and suppressed the phosphorylation of forkhead box O (FoxO)3a in HG-treated GECs. Febuxostat also decreased nicotinamide adenine dinucleotide phosphate oxidase (Nox)1, Nox2, and Nox4 expressions; enhanced superoxide dismutase activity; and decreased malondialdehyde levels in HG-treated GECs. The knockdown of AMPK inhibited PGC-1α-FoxO3a signaling and negated the antioxidant effects of febuxostat in HG-treated GECs. Despite febuxostat administration, the knockdown of hypoxanthine phosphoribosyl transferase 1 (HPRT1) also inhibited AMPK-PGC-1α-FoxO3a in HG-treated GECs. XOR inhibition alleviates oxidative stress by activating AMPK-PGC-1α-FoxO3a signaling through the HPRT1-dependent purine salvage pathway in GECs exposed to HG conditions.

Design, synthesis, and biological evaluation of 3-phenyl substituted pyridine derivatives as potential dual inhibitors of XOR and URAT1.

Xanthine oxidoreductase (XOR) and uric acid transporter 1 (URAT1) are two most widely studied targets involved in production and reabsorption of uric acid, respectively. Marketed drugs almost target XOR or URAT1, but sometimes, single agents might not achieve aim of lowering uric acid to ideal value in clinic. Thus, therapeutic strategies of combining XOR inhibitors with uricosuric drugs were proposed and implemented. Based on our initial work of virtual screening, A and B were potential hits for dual-targeted inhibitors on XOR/URAT1. By docking A/B with XOR/URAT1 respectively, compounds I1-7 were designed to get different degree of inhibition effect on XOR and URAT1, and I7 showed the best inhibitory effect on XOR (IC50 = 0.037 ± 0.001 µM) and URAT1 (IC50 = 546.70 ± 32.60 µM). Further docking research on I7 with XOR/URAT1 led to the design of compounds II with the significantly improved inhibitory activity on XOR and URAT1, such as II11 and II15. Especially, for II15, the IC50 of XOR is 0.006 ± 0.000 µM, superior to that of febuxostat (IC50 = 0.008 ± 0.000 µM), IC50 of URAT1 is 12.90 ± 2.30 µM, superior to that of benzbromarone (IC50 = 27.04 ± 2.55 µM). In acute hyperuricemia mouse model, II15 showed significant uric acid lowering effect. The results suggest that II15 had good inhibitory effect on XOR/URAT1, with the possibility for further investigation in in-vivo models of hyperuricemia.

Kidney Failure Secondary to Hereditary Xanthinuria due to a Homozygous Deletion of the XDH Gene in the Absence of Overt Kidney Stone Disease.

Hereditary xanthinuria (HXAN) is a rare metabolic disorder that results from mutations in either the xanthine dehydrogenase (XDH) or the molybdenum cofactor sulfurase genes (MOCOS), respectively defining HXAN type I and type II. Hypouricemia, hypouricosuria, and abnormally high plasma and urine levels of xanthine, causing susceptibility to xanthine nephrolithiasis and deposition of xanthine crystals in tissues, are the metabolic hallmarks of HXAN. Several pathogenic variants in the XDH gene have so far been identified in patients with HXAN type I, but the clinical phenotype associated with the whole deletion of the human XDH gene is unknown. Herein, we report the case of a woman diagnosed with HXAN, whose molecular genetic testing revealed a homozygous microdeletion involving the XDH gene. Distinctive features of her medical history were the diagnosis of arterial hypertension and microalbuminuria at 22 years of age; a single pregnancy at the age of 25, complicated by proteinuria and transient kidney function deterioration in the third trimester; unexplained severe hypouricemia incidentally discovered during pregnancy; inability to breastfeed her newborn daughter due to primary agalactia; chronic kidney disease (CKD) stage 3 diagnosed at age 35; and progression to end-stage kidney disease over the next 12 years. Protocol noninvasive laboratory and imaging investigation was not informative as to the cause of CKD. This is the first description of the clinical phenotype associated with a natural knockout of the human XDH gene. Despite the lack of kidney histopathology data, the striking similarities with the phenotypes exhibited by comparable murine models validate the latter as useful sources of mechanistic insights for the pathogenesis of the human disease, supporting the hypothesis that the absence of xanthine dehydrogenase activity might represent a susceptibility factor for chronic tubulointerstitial nephritis, even in patients without kidney stones.

Neutrophils and NADPH Oxidases Are Major Contributors to Mild but Not Severe Ischemic Acute Kidney Injury in Mice.

Upregulation of free radical-generating NADPH oxidases (NOX), xanthine oxidoreductase (XOR), and neutrophil infiltration-induced, NOX2-mediated respiratory burst contribute to renal ischemia-reperfusion injury (IRI), but their roles may depend on the severity of IRI. We investigated the role of NOX, XOR, and neutrophils in developing IRI of various severities. C57BL/6 and Mcl-1ΔMyelo neutrophil-deficient mice were used. Oxidases were silenced by RNA interference (RNAi) or pharmacologically inhibited. Kidney function, morphology, immunohistochemistry and mRNA expression were assessed. After reperfusion, the expression of NOX enzymes and XOR increased until 6 h and from 15 h, respectively, while neutrophil infiltration was prominent from 3 h. NOX4 and XOR silencing or pharmacological XOR inhibition did not protect the kidney from IRI. Attenuation of NOX enzyme-induced oxidative stress by apocynin and neutrophil deficiency improved kidney function and ameliorated morphological damage after mild but not moderate/severe IRI. The IR-induced postischemic renal functional impairment (BUN, Lcn-2), tubular necrosis score, inflammation (TNF-α, F4/80), and decreases in the antioxidant enzyme (GPx3) mRNA expression were attenuated by both apocynin and neutrophil deficiency. Inhibition of NOX enzyme-induced oxidative stress or the lack of infiltration by NOX2-expressing neutrophils can attenuate reperfusion injury after mild but not moderate/severe renal IR.

The presence of xanthine dehydrogenase is crucial for the maturation of the rat kidneys.

The development of the kidney involves essential cellular processes, such as cell proliferation and differentiation, which are led by interactions between multiple signaling pathways. Xanthine dehydrogenase (XDH) catalyzes the reaction producing uric acid in the purine catabolism, which plays a multifaceted role in cellular metabolism. Our previous study revealed that the genetic ablation of the Xdh gene in rats leads to smaller kidneys, kidney damage, decline of renal functions, and failure to thrive. Rats, unlike humans, continue their kidney development postnatally. Therefore, we explored whether XDH plays a critical role in kidney development using SS-/- rats during postnatal development phase. XDH expression was significantly increased from postnatal day 5 to 15 in wild-type but not homozygote rat kidneys. The transcriptomic profile of renal tissue revealed several dysregulated pathways due to the lack of Xdh expression with the remodeling in inflammasome, purinergic signaling, and redox homeostasis. Further analysis suggested that lack of Xdh affects kidney development, likely via dysregulation of epidermal growth factor and its downstream STAT3 signaling. The present study showed that Xdh is essential for kidney maturation. Our data, alongside the previous research, suggests that loss of Xdh function leads to developmental issues, rendering them vulnerable to kidney diseases in adulthood.

Xanthine Oxidoreductase in the Pathogenesis of Endothelial Dysfunction: An Update.

Xanthine oxidoreductase (XOR) is a rate-limiting enzyme in the formation of uric acid (UA) and is involved in the generation of reactive oxygen species (ROS). Overproduction of ROS has been linked to the pathogenesis of hypertension, atherosclerosis, and cardiovascular disease, with multiple studies over the last 30 years demonstrating that XOR inhibition is beneficial. The involvement of XOR and its constituents in the advancement of chronic inflammation and ROS, which are responsible for endothelial dysfunction, is the focus of this evidence-based review. An overabundance of XOR products and ROS appears to drive the inflammatory response, resulting in significant endothelium damage. It has also been demonstrated that XOR activity and ED are connected. Diabetes, hypertension, and cardiovascular disease are all associated with endothelial dysfunction. ROS mainly modifies the activity of vascular cells and can be important in normal vascular physiology as well as the development of vascular disease. Suppressing XOR activity appears to decrease endothelial dysfunction, probably because it lessens the generation of reactive oxygen species and the oxidative stress brought on by XOR. Although there has long been a link between higher vascular XOR activity and worse clinical outcomes, new research suggests a different picture in which positive results are mediated by XOR enzymatic activity. Here in this study, we aimed to review the association between XOR and vascular endothelial dysfunction. The prevention and treatment approaches against vascular endothelial dysfunction in atherosclerotic disease.

Hypoxia-Mediated Upregulation of Xanthine Oxidoreductase Causes DNA Damage of Colonic Epithelial Cells in Colitis.

Xanthine oxidoreductase (XOR) serves as the primary source of hydrogen peroxide and superoxide anions in the intestinal mucosa. However, its specific contribution to the progression of colonic disease remains unclear. In this study, we investigated the role of XOR in ulcerative colitis (UC) and attempted to identify the underlying mechanisms. We used the dextran sulfate sodium (DSS)-induced mouse model to mimic UC and observed that XOR inhibitors, allopurinol and diphenyleneiodonium sulfate (DPI), significantly alleviated UC in mice. In addition, treatment with cobalt chloride (CoCl2) and 1% O2 increased the expression of XOR and induced DNA oxidative damage in colonic epithelial cells. Furthermore, we identified that XOR accumulation in the nucleus may directly cause DNA oxidative damage and regulates HIF1α protein levels. In addition, allopurinol effectively protected colon epithelial cells from CoCl2-induced DNA damage. Altogether, our data provided evidence that XOR could induce DNA damage under hypoxic conditions, indicating a significant role of XOR in the initiation and early development of colitis-associated colorectal cancer (CAC).

Mechanistic insights into iron-sulfur clusters and flavin oxidation of a novel xanthine oxidoreductase from Sulfobacillus acidophilus TPY.

Xanthine oxidoreductase (XOR) catalyzes the oxidation of purines (hypoxanthine and xanthine) to uric acid. XOR is widely used in various therapeutic and biotechnological applications. In this study, we characterized the biophysical and mechanistic properties of a novel bacterial XOR from Sulfobacillus acidophilus TPY (SaXOR). Our results showed that SaXOR is a heterotrimer consisting of three subunits, namely XoA, XoB, and XoC, which denote the molybdenum cofactor (Moco), 2Fe-2S, and FAD-binding domains, respectively. XoC was found to be stable when co-expressed with XoB, forming an XoBC complex. Furthermore, we prepared a fusion of XoB and XoC via a flexible linker (fusXoBC) and evaluated its function in comparison to that of XoBC. Spectroscopic analysis revealed that XoB harbors two 2Fe-2S clusters, whereas XoC bears a single-bound FAD cofactor. Electron transfer from reduced forms of XoC, XoBC, and fusXoBC to molecular oxygen (O2 ) during oxidative half-reaction yielded no flavin semiquinones, implying ultrafast single-electron transfer from 2Fe-2Sred to FAD. In the presence of XoA, XoBC and fusXoBC exhibited comparable XoA affinity and exploited a shared overall mechanism. Nonetheless, the linkage may accelerate the two-step, single-electron transfer cascade from 2Fe-2Sred to FAD while augmenting protein stability. Collectively, our findings provide novel insights into SaXOR properties and oxidation mechanisms divergent from prior mammalian and bacterial XOR paradigms.

Uric acid formation is driven by crosstalk between skeletal muscle and other cell types.

Hyperuricemia is implicated in numerous pathologies, but the mechanisms underlying uric acid production are poorly understood. Using a combination of mouse studies, cell culture studies, and human serum samples, we sought to determine the cellular source of uric acid. In mice, fasting and glucocorticoid treatment increased serum uric acid and uric acid release from ex vivo-incubated skeletal muscle. In vitro, glucocorticoids and the transcription factor FoxO3 increased purine nucleotide degradation and purine release from differentiated muscle cells, which coincided with the transcriptional upregulation of AMP deaminase 3, a rate-limiting enzyme in adenine nucleotide degradation. Heavy isotope tracing during coculture experiments revealed that oxidation of muscle purines to uric acid required their transfer from muscle cells to a cell type that expresses xanthine oxidoreductase, such as endothelial cells. Last, in healthy women, matched for age and body composition, serum uric acid was greater in individuals scoring below average on standard physical function assessments. Together, these studies reveal skeletal muscle purine degradation is an underlying driver of uric acid production, with the final step of uric acid production occurring primarily in a nonmuscle cell type. This suggests that skeletal muscle fiber purine degradation may represent a therapeutic target to reduce serum uric acid and treat numerous pathologies.

Diurnal Variations in Serum Uric Acid, Xanthine, and Xanthine Oxidoreductase Activity in Male Patients with Coronary Artery Disease.

Hyperuricemia is influenced by diet and can cause gout. Whether it is a potential risk factor for cardiovascular disease (CVD) remains controversial, and the mechanism is unclear. Similar to CVDs, gout attacks occur more frequently in the morning and at night. A possible reason for this is the diurnal variation in uric acid (UA), However, scientific data regarding this variation in patients with CVD are not available. Thus, we aimed to investigate diurnal variations in serum levels of UA and plasma levels of xanthine, hypoxanthine, and xanthine oxidoreductase (XOR) activity, which were measured at 18:00, 6:00, and 12:00 in male patients with coronary artery disease. Thirty eligible patients participated in the study. UA and xanthine levels significantly increased from 18:00 to 6:00 but significantly decreased from 6:00 to 12:00. By contrast, XOR activity significantly increased both from 18:00 to 6:00 and 6:00 to 12:00. Furthermore, the rates of increase in UA and xanthine levels from night to morning were significantly and positively correlated. In conclusion, UA and xanthine showed similar diurnal variations, whereas XOR activity showed different diurnal variations. The morning UA surge could be due to UA production. The mechanism involved XOR activity, but other factors were also considered.

Hemin and iron increase synthesis and trigger export of xanthine oxidoreductase from hepatocytes to the circulation.

We recently reported a previously unknown salutary role for xanthine oxidoreductase (XOR) in intravascular heme overload whereby hepatocellular export of XOR to the circulation was identified as a seminal step in affording protection. However, the cellular signaling and export mechanisms underpinning this process were not identified. Here, we present novel data showing hepatocytes upregulate XOR expression/protein abundance and actively release it to the extracellular compartment following exposure to hemopexin-bound hemin, hemin or free iron. For example, murine (AML-12 cells) hepatocytes treated with hemin (10 µM) exported XOR to the medium in the absence of cell death or loss of membrane integrity (2.0 ± 1.0 vs 16 ± 9 µU/mL p < 0.0001). The path of exocytosis was found to be noncanonical as pretreatment of the hepatocytes with Vaculin-1, a lysosomal trafficking inhibitor, and not Brefeldin A inhibited XOR release and promoted intracellular XOR accumulation (84 ± 17 vs 24 ± 8 hemin vs 5 ± 3 control µU/mg). Interestingly, free iron (Fe2+ and Fe3+) induced similar upregulation and release of XOR compared to hemin. Conversely, concomitant treatment with hemin and the classic transition metal chelator DTPA (20 µM) or uric acid completely blocked XOR release (p < 0.01). Our previously published time course showed XOR release from hepatocytes likely required transcriptional upregulation. As such, we determined that both Sp1 and NF-kB were acutely activated by hemin treatment (∼2-fold > controls for both, p < 0.05) and that silencing either or TLR4 with siRNA prevented hemin-induced XOR upregulation (p < 0.01). Finally, to confirm direct action of these transcription factors on the Xdh gene, chromatin immunoprecipitation was performed indicating that hemin significantly enriched (∼5-fold) both Sp1 and NF-kB near the transcription start site. In summary, our study identified a previously unknown pathway by which XOR is upregulated via SP1/NF-kB and subsequently exported to the extracellular environment. This is, to our knowledge, the very first study to demonstrate mechanistically that XOR can be specifically targeted for export as the seminal step in a compensatory response to heme/Fe overload.

Design, synthesis, and evaluation of the in vitro activity of novel dual inhibitors of XOR and URAT1 containing a benzoic acid group.

Xanthine oxidoreductase (XOR) and uric acid transporter 1 (URAT1) are involved in the production and reabsorption of uric acid, respectively. However, the currently available individual XOR- or URAT1-targeted drugs have limited efficacy. Thus, strategies for combining XOR inhibitors with uricosuric drugs have been developed. Previous virtual screening identified Compounds 1-5 as hits for the potential dual inhibition of XOR/URAT1. Nevertheless, in vitro experiments yielded unsatisfactory results. The first round of optimization work on those hits was performed, and two series of compounds were designed and synthesized. Compounds of the A series exerted moderate inhibitory effects on URAT1, but extremely weak inhibitory effects on XOR. Compounds of the B series exerted strong inhibitory effects on both XOR and URAT1. B5 exhibited the greatest inhibitory activity, with similar inhibitory effects on XOR and URAT1. The half maximal inhibitory concentration (IC50 ) of XOR was 0.012 ± 0.001 µM, equivalent to that of febuxostat (IC50 = 0.010 ± 0.001 µM). The IC50 of URAT1 was 30.24 ± 3.46 µM, equivalent to that of benzbromarone (IC50 = 24.89 ± 7.53 µM). Through this optimization, the in vitro activity of most compounds of the A and B series against XOR and URAT1 was significantly improved versus that of the hits. Compound B5 should be further investigated.