Unveiling the anti-gout potential of terpenes isolated from Penicillium sp. supported by in silico studies

BackgroundClinically applied as anti-gout drugs, xanthine oxidoreductase (XOR) inhibitors, especially the potent, selective, non-purine-analog XOR inhibitors febuxostat and topiroxostat, exert organ-protective effects. We tested the hypothesis that preservation of tissue concentrations of high-energy phosphates, such as ATP and ADP, contributes to organ-protective effects through CE-TOFMS metabolomics.MethodsRats were subjected to 30 min of renal ischemia-reperfusion (I/R) injury 60 min after oral administration of 10 mg/kg febuxostat, 10 mg/kg topiroxostat, 50 mg/kg allopurinol, or vehicle.ResultsIn non-purine-analog XOR inhibitor-treated groups, renal concentrations of high-energy phosphates were greater before and after I/R injury, and renal adenine compounds were less depleted by I/R injury than in the vehicle and allopurinol groups. These findings were well in accordance with the proposed hypothesis that the recomposition of high-energy phosphates is promoted by non-purine-analog XOR inhibitors via the salvage pathway through blockade of hypoxanthine catabolism, whereas non-specific inhibitory effects of allopurinol on purine/pyrimidine enzymes impede this re-synthesis process.ConclusionsThis metabolic approach shed light on the physiology of the organ-protective effects of XOR inhibitors.

Global cerebral ischemia and reperfusion (I/R) often result in high mortality. Free radicals play an important role in global cerebral I/R. Xanthine oxidoreductase (XOR) inhibitors, such as allopurinol, have been reported to protect tissues from damage caused by reactive oxygen species (ROS) by inhibiting its production through XOR inhibition. The recently introduced XOR inhibitor febuxostat, which is a more potent inhibitor than allopurinol, is expected to decrease free radical production more effectively. Here, we analyzed the effects of allopurinol and febuxostat in decreasing global severe cerebral I/R damage in mice. Mice were divided into three groups: a placebo group, an allopurinol group, and a febuxostat group. Pathological examinations, which were performed in each group in the CA1 and CA2 regions of the hippocampus 4 days after I/R surgery, revealed that there was a decrease in the number of neuronal cells in the 14-min occlusion model in both regions and that drugs that were administered to prevent this damage were not effective. The enzymatic activity was extremely low in the mouse brain, and XOR could not be detected in the nonischemic and ischemic mice brains with western blot analyses. Thus, one of the reasons for the decreased effectiveness of XOR inhibitors in controlling severe whole-brain ischemia in a mouse model was the low levels of expression of XOR in the mouse brain.

Synthesis and bioevaluation of 1-phenylimidazole-4-carboxylic acid derivatives as novel xanthine oxidoreductase inhibitors

Xanthine oxidoreductase (XOR) inhibitors are used to treat gout, inhibiting uric acid production, which causes clinical symptoms. Commonly used XOR inhibitors are the small molecule febuxostat (Fbx) and the purine analogue allopurinol (Allo). Recent studies show that XOR inhibitors can reduce mature interleukin (IL)-1β production by activated macrophages. This effect is not due to reduced uric acid crystal formation, which can induce NLRP3 inflammasome activation, but an independent effect. Fbx and Allo have been used interchangeably in in vitro studies to highlight the role of XOR in pro-inflammatory macrophage function. Here, we analysed the effects of Fbx and Allo on pro-inflammatory macrophage signatures. Both XOR inhibitors maintain pro-inflammatory macrophage metabolic and phenotypic hallmarks. However, only Fbx reduces the activity of caspase-1 and the release of IL-1β by preventing inflammasome assembly in macrophages isolated from both mice and humans. Our study identified an Fbx-specific reduction in IL-1β production, which could be used clinically to reduce the deleterious effects of macrophage-derived IL-1β.

Hyperuricemia has been shown to associate with high incidence of cardiovascular or kidney disease [1,2] and the beneficial effect of the treatment with allopurinol has been shown in hyperuricemic chronic kidney disease (CKD) patients [3]. Moreover, recent investigations have demonstrated that monosodium urate crystals activate natural immune system through its binding to Toll-like receptors (TLRs) and nucleotide binding and oligomerization domain-like receptors (NLRs) [4]. These indicate that xanthine oxidoreductase (XOR) inhibitor is a good candidate for renoprotective agents. However, there is evidence that, in contrast to the pro-inflammatory action of monosodium urate crystals, soluble form of uric acid in plasma acts as a major anti-oxidant in body [5]. Furthermore, allopurinol has been speculated to have nonspecific action on purine nucleotide metabolism pathway, because it is generated as purine nucleotide derivative. Thus, presently it is unclear whether treating non-symptomatic hyperuricemic subject with XOR inhibitor has beneficial effects on the progression of cardiovascular or kidney disease.

Uric acid is the end product of purine metabolism in humans. Hyperuricemia is a metabolic disease caused by the increased formation or reduced excretion of serum uric acid (SUA). Alterations in SUA homeostasis have been linked to a number of diseases, and hyperuricemia is the major etiologic factor of gout and has been correlated with metabolic syndrome, cardiovascular disease, diabetes, hypertension, and renal disease. Oxidative stress is usually defined as an imbalance between free radicals and antioxidants in our body and is considered to be one of the main causes of cell damage and the development of disease. Studies have demonstrated that hyperuricemia is closely related to the generation of reactive oxygen species (ROS). In the human body, xanthine oxidoreductase (XOR) catalyzes the oxidative hydroxylation of hypoxanthine to xanthine to uric acid, with the accompanying production of ROS. Therefore, XOR is considered a drug target for the treatment of hyperuricemia and gout. In this review, we discuss the mechanisms of uric acid transport and the development of hyperuricemia, emphasizing the role of oxidative stress in the occurrence and development of hyperuricemia. We also summarize recent advances and new discoveries in XOR inhibitors.

Background and AimsThere are two types of serum uric acid‐lowering agents, the xanthine oxidoreductase (XO) inhibitor and non‐XO inhibitor. We investigated whether febuxostat, XO inhibitor, could produce more favorable effects on coronary endothelial function (CEF) and renal function than benzbromarone, non‐XO inhibitor, in hyperuricemic coronary artery disease (CAD) patients.MethodsWe divided 21 hyperuricemic patients with stenting for left anterior descending (LAD) or left circumflex (LCX) artery into patients started on febuxostat (F group) and those on benzbromarone (B group). After 8 months, all patients underwent CEF evaluations (acetylcholine provocation test) and optical coherence tomography (OCT) for non‐culprit vessels (e.g. if patients received LAD stenting, we evaluated LCX). We compared the diameter ratio induced by acetylcholine and baseline (CEF ratio), thin‐cap fibroatheroma and calcified plaque by OCT, uric acid, oxidative stress biomarkers, and renal function including estimated glomerular filtration rate (eGFR) between F and B groups. Creatinine 2 days after stenting was measured to evaluate contrast‐induced nephropathy (CIN).ResultsChange of eGFR was significantly lower in F group (n= 11) than B group over 8 months while the other parameters including CEF ratio were similar. F group showed favorable effects for CIN.ConclusionIn conclusion, 8‐months of febuxostat, XO inhibitor, does not significantly protect CEF but can protect the renal function including CIN in hyperuricemic patients with CAD compared to benzbromarone, non‐XO inhibitor.

Renoprotective effect of the xanthine oxidoreductase inhibitor Topiroxostat under decreased angiotensin II type 1a receptor expression

The nucleotide-binding oligomerization domain–like receptor family, pyrin domain–containing 3 (NLRP3) inflammasome mediates caspase-1 activation and IL-1β processing and is implicated in autoinflammatory as well as other chronic inflammatory diseases. Recent studies have demonstrated that xanthine oxidoreductase (XOR) inhibition attenuated IL-1β secretion in activated macrophages, but the detailed mechanism of inhibition remains unclear. In this study, we report that febuxostat, an inhibitor of XOR, suppressed NLRP3 inflammasome-mediated IL-1β secretion and cell death by two mechanisms: in a mitochondrial ROS (mitoROS)-dependent and mitoROS-independent manner. MitoROS-independent effects of febuxostat were mediated by an increase of intracellular ATP and improved mitochondrial energetics via the activation of purine salvage pathway. Our findings suggest that cellular bioenergetics are important in regulating NLRP3 activation, and XOR inhibition may be clinically relevant in NLRP3-related inflammatory diseases.

Design, synthesis, and pharmacological and pharmacokinetic evaluation of 3-phenyl-5-pyridyl-1,2,4-triazole derivatives as xanthine oxidoreductase inhibitors

BackgroundGout flare due to rapid urate reduction after initiating urate-lowering therapy (ULT) is one of the major issues in the therapy, which impairs patients’ quality of life and adherence. For...

Secondary metabolites are organic compounds with complex chemical structures and diverse physiological functions. Secondary metabolites include antibiotics, pigments, and other bioactive compounds. Many of these compounds have important agricultural and medical applications. Microorganisms, especially actinomycetes and filamentous fungi, are noted as a rich source of bioactive secondary metabolites. Typically, each species produces several antibiotics, with the profile being species-specific. Secondary metabolites are synthesized from their precursors through multistep biosynthetic pathways. In general, the genes governing the biosynthesis of secondary metabolites are clustered together, and an increasing number of gene clusters responsible for the biosynthesis of secondary metabolites have been discovered. The availability of clusters has accelerated functional investigations of biosynthetic pathways of secondary metabolites. A thorough understanding of the enzymatic process is required for metabolic engineering to improve production of secondary metabolites and for combinatorial biosynthesis to generate novel compounds or derivatives. Elucidation of the biosynthetic process requires knowledge from diverse disciplines, including bioinformatics, chemistry, and genetics. Secondary metabolites are generally produced during the stationary phase of growth in microorganisms. The biosynthesis of secondary metabolites is a complex process involving cascade regulations, and these regulatory mechanisms have been investigated extensively at the transcriptional level. However, regulation could also occur at the pre-transcriptional and/or post-transcriptional levels. Pretranscriptional regulation occurs primarily at the chromatin level (epigenetic regulation), while post-transcriptional regulation is achieved via small non-coding RNAs (sRNAs) and protein degradation machinery. Though still in its infancy, some interesting progress has been made in this field [1,2]. As antibiotics are the most important of the secondary metabolites, we will focus on antibiotics hereafter. The alarming rise in emergence and prevalence of antibiotic resistance poses a major threat to human healthcare. It is clear that novel antibiotics are urgently needed to combat this problem. However, the supply of new antibiotics has declined in the last decade [3]. To reverse this trend, several strategies have been devised to find or create new antibiotics, which we describe in detail below.

BackgroundWhether uric acid (UA)-lowering therapy (ULT) is effective in reducing the progression of renal dysfunction in patients with chronic kidney disease (CKD) remains controversial. Since several advances have been made in therapies for hyperuricemia, including novel xanthine oxidoreductase (XOR) inhibitors, we conducted a systematic review to clarify the effectiveness of ULT in preserving renal function among CKD patients.MethodsIn this systematic review, the MEDLINE database was searched up to June 2019. We included complete randomized controlled trials comparing renal events between adult non-dialyzed CKD patients, defined as estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 m2, with and without ULTs. Changes in eGFR were expressed as the mean difference (MD). The incidence of dichotomous outcomes was expressed as a risk ratio. This review was performed using the predefined protocol published in PROSPERO (CRD 42019140346).ResultsEleven studies with 4277 CKD patients were included. Drugs used in the intervention groups of all studies were XOR inhibitors (allopurinol, febuxostat or topiroxostat). Although patients with ULT tended to show superior preservation of eGFR as compared to those without ULT, no significant differences were identified (MD, 2.52; 95% confidence interval, − 0.15 to 5.18). In subgroup analysis, the use of allopurinol was associated with superior preservation of eGFR, whereas the newer XOR inhibitors, febuxostat and topiroxostat, showed no significant effects on eGFR changes. Neither incidence of end-stage kidney disease nor treatment-emergent adverse events differed significantly between groups.ConclusionsThe present systematic review and meta-analysis suggested that CKD patients with ULT tend to show superior eGFR preservation as compared to patients without ULT, but further studies are needed to verify the renoprotective effects of ULT.

Adenine phosphoribosyltransferase (APRT) deficiency is a rare, autosomal recessive disorder characterized by urinary excretion of the poorly soluble 2,8-dihydroxyadenine (DHA), leading to kidney stones and chronic kidney disease. Treatment with the xanthine oxidoreductase (XOR) inhibitors allopurinol and febuxostat reduces DHA production. DHA and adenine were measured in 122 paired plasma and urine samples from 26 individuals with confirmed APRT deficiency, using ultra-performance liquid chromatography-tandem mass spectrometry assays. The relationship between plasma DHA and adenine concentrations, urine DHA-to-creatinine (DHA/Cr) and adenine-to-creatinine (adenine/Cr) ratios, and age and estimated glomerular filtration rate (eGFR) was evaluatedin a subset of 87 paired plasma and urine samples from 23 individuals using Spearman's rank correlation. Allopurinol and febuxostat treatment reduced plasma DHA, with the median (range) concentration decreasing from 249 (123-1315) ng/mL in untreated individuals to below the limit of detection in those receiving higher doses. Plasma adenine increased during XOR inhibitor treatment. In untreated individuals, a strong negative correlation was observed between plasma DHA and eGFR (rs = -0.74, p < 0.0001). Plasma DHA correlated with urine DHA/Cr ratio in individuals treated with allopurinol or febuxostat (rs = 0.65, p < 0.0001), while no significant correlation was observed in untreated individuals (rs = -0.26, p = 0.14). Treatment with XOR inhibitors effectively reduces the plasma concentration and urinary excretion of DHA. The strong correlation between plasma DHA and eGFR, combined with the lack of correlation between plasma DHA and urine DHA/Cr ratio in untreated individuals, suggests that plasma DHA may be a more reliable marker of systemic DHA burden.

Background and Aims Adenine phosphoribosyltransferase (APRT) deficiency is a rare disorder of purine metabolism, characterized by urinary excretion of the poorly soluble 2,8-dihydroxyadenine (DHA), leading to nephrolithiasis and chronic kidney disease (CKD). Treatment with xanthine oxidoreductase (XOR) inhibitor, allopurinol or febuxostat, reduces DHA excretion and slows or halts CKD progression. The aim of the study was to optimize and validate an ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS)-based assay for simultaneous quantification of DHA, adenine, allopurinol, oxypurinol and febuxostat in human plasma, for monitoring of pharmacotherapy in patients with APRT deficiency. Method The UPLC-MS/MS-based assay was optimized employing the chemometric approach design of experiments, using the software Modde Pro 13 (Sartorius, Umeå, Sweden). The assay was validated and used for absolute quantification of DHA, adenine, allopurinol, oxypurinol and febuxostat in plasma samples from both untreated and treated APRTd patients and healthy controls. Results Accuracy and precision were within ±15% for all analytes, and the analytes were stable in plasma for 12 months at −80°C. The median (range) concentration of DHA in plasma samples from six APRT deficiency patients was 204.0 (187.2–315.8) ng/mL when they were untreated and 69.2 (below the limit of quantification [BLQ]–131.6) ng/mL when treated with allopurinol (400 mg/day), and BLQ when febuxostat (80 mg/day) was the therapeutic agent. The median (range) plasma adenine concentration was 238.1 (218–502) ng/mL in the untreated patients, and 561.1 (418–2104) and 856.0 (726–1711) ng/mL in those on treatment with allopurinol and febuxostat, respectively. DHA was not detected in plasma samples from healthy controls. The median (range) plasma concentration of allopurinol, oxypurinol and febuxostat in patients receiving XOR inhibitor therapy was 1657 (BLQ-3266) ng/mL, 8102 (4778–34906) ng/mL and 109 (BLQ-1657) ng/mL, respectively. Conclusion The UPLC-MS/MS-based assay provides accurate and precise quantification of DHA, adenine, allopurinol, oxypurinol and febuxostat in human plasma. Measurement of plasma DHA is being implemented as a diagnostic test for APRT deficiency and the full assay for monitoring of XOR inhibitor treatment in patients with the disorder.