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Benzimidazole-based dual dipeptidyl peptidase-4 and xanthine oxidase inhibitors.
Mental disorders comprise diverse human pathologies including depression, bipolar affective disorder, schizophrenia, and dementia that affect millions of people around the world. The causes of mental disorders are unclear but growing evidence suggests that oxidative stress and the purine/adenosine system play a key role in their development and progression. Xanthine oxidase (XO) is a flavoprotein enzyme essential for the catalysis of the oxidative hydroxylation of purines -hypoxanthine and xanthine- to generate uric acid. As a consequence of the oxidative reaction of XO, reactive oxygen species (ROS) such as superoxide and hydrogen peroxide are produced and, further, contribute to the pathogenesis of mental disorders. Altered XO activity has been associated with free radical-mediated neurotoxicity inducing cell damage and inflammation. Diverse studies reported a direct association between an increased activity of XO and diverse mental diseases including depression or schizophrenia. Small-molecule inhibitors, such as the well-known allopurinol, and dietary flavonoids, can modulate the XO activity and subsequent ROS production. In the present work, we review the available literature on XO inhibition by small molecules and their potential therapeutic application in mental disorders. In addition, we discuss the chemistry and molecular mechanism of XO inhibitors, as well as the use of structure-based and computational methods to design specific inhibitors with the capability of modulating XO activity.
Pulmonary hypertension (PH)is a progressive disorder characterized by alterations of the vascular structure and function in the lungs. Despite the success in its stabilisation by targeting pulmonary vascular tone and endothelial dysfunction, the prognosis remains poor and new therapeutic approaches via neglected macromolecular targets are needed. In the pathophysiology of PH the early stages of vascular remodelling are considered to be reversible, while endothelial to mesenchymal transition and proliferation/migration of fibroblasts play a critical role in staging the irreversible phase. Dipeptidyl peptidase-4 (DPP-4)/CD26 is present and active in the lungs and is expressed constitutively on lung fibroblasts, on which it exerts proliferative effects. Further, it is a marker of migrating fibroblasts and of their functional activation, including collagen synthesis and inflammatory cytokine secretion. Inhibiting DPP-4 improves the reversible phases of vascular dysfunction in PH, but is also highly likely to attenuate endothelial to mesenchymal transition and decrease the proliferation and migration of fibroblasts, preventing fibrosis and, consequently, should prolong or even inhibit entrance to the potentially irreversible phase of PH. Proposed mechanisms that support the multifaceted aspects of DPP-4 inhibition in terms of improving PH, involve pathways and mediators in pulmonary vascular and connective tissue remodelling. The latter are affected by the inhibition of this protease resulting in the synergistic beneficial antioxidative, anti-inflammatory and antifibrotic effects. We offer here an evidence-supported hypothesis that DPP-4 inhibitors are likely to be effective in the irreversible phase of remodelling in PH. Accordingly, we propose PH as a possible novel therapeutic indication for existing and new DPP-4 inhibitors.
Dipeptidyl peptidase‐4 (DPP‐4) cleaves N‐terminal dipeptides, with Pro, Ala or Ser at the penultimate position, and, in that way, modulates biological activity of certain polypeptides. Due to its ubiquitous distribution, many pathological processes are associated with altered DPP‐4 expression and activity. Besides the regulation of glucose metabolism, DPP‐4 also exhibits many other systemic effects, and the inhibition of its activity might lead to cardiovascular and renal protection. Mechanisms underlying these protective effects of DPP‐4 inhibition are ascribed to elevated bioavailability of its substrates, to impacts on mediators and signaling pathways that ameliorate cardiovascular and renal function through the suppression of oxidative stress, inflammation, fibrosis and apoptosis, improved endothelial function and tissue reparation. Inflammation contributes to and promotes progression of cardiovascular and renal disorders. Herein, we discuss cellular and molecular mechanisms mediating the anti‐inflammatory activity of clinically used DPP‐4 inhibitors in cardiovascular and renal protection.
Xanthine oxidoreductase (XOR) could contribute to the pathogenesis of metabolic syndrome through the oxidative stress and the inflammatory response induced by XOR-derived reactive oxygen species and uric acid. Hyperuricemia is strongly linked to hypertension, insulin resistance, obesity and hypertriglyceridemia. The serum level of XOR is correlated to triglyceride/high density lipoprotein cholesterol ratio, fasting glycemia, fasting insulinemia and insulin resistance index. Increased activity of endothelium-linked XOR may promote hypertension. In addition, XOR is implicated in pre-adipocyte differentiation and adipogenesis. XOR and uric acid play a role in cell transformation and proliferation as well as in the progression and metastatic process. Collected evidences confirm the contribution of XOR and uric acid in metabolic syndrome. However, in some circumstances XOR and uric acid may have anti-oxidant protective outcomes. The dual-face role of both XOR and uric acid explains the contradictory results obtained with XOR inhibitors and suggests caution in their therapeutic use.
Diabetes mellitus is a medical condition characterized by the body's loss of control over blood sugar. The frequency of diagnosed cases and consequential increases in medical costs makes it a rapidly growing chronic disease that threatens human health worldwide. In addition, its unnerving statistical projections are perilous to both the economy of the nation and man's life expectancy. Type-I and type-II diabetes are the two clinical forms of diabetes mellitus. Type-II diabetes mellitus (T2DM) is illustrated by the abnormality of glucose homeostasis in the body, resulting in hyperglycemia. Although significant research attention has been devoted to the development of diabetes regimens, which demonstrates success in lowering blood glucose levels, their efficacies are unsustainable due to undesirable side effects such as weight gain and hypoglycemia. Over the years, heterocyclic scaffolds have been the basis of anti-diabetic chemotherapies; hence, in this review we consolidate the use of bioactive scaffolds, which have been evaluated for their biological response as inhibitors against their respective anti-diabetic molecular targets over the past five years (2012-2017). Our investigation reveals a diverse target set which includes; protein tyrosine phosphatase 1 B (PTP1B), dipeptidly peptidase-4 (DPP-4), free fatty acid receptors 1 (FFAR1), G protein-coupled receptors (GPCR), peroxisome proliferator activated receptor-γ (PPARγ), sodium glucose co-transporter-2 (SGLT2), α-glucosidase, aldose reductase, glycogen phosphorylase (GP), fructose-1,6-bisphosphatase (FBPase), glucagon receptor (GCGr) and phosphoenolpyruvate carboxykinase (PEPCK). This review offers a medium on which future drug design and development toward diabetes management may be modelled (i.e. optimization via structural derivatization), as many of the drug candidates highlighted show promise as an effective anti-diabetic chemotherapy.
Background: Pharmacologic treatments for type 2 diabetes are based upon increasing insulin availability and improving sensitivity to insulin. Nowadays, glucagon like peptide-1 (GLP-1) based therapies aims at glucose control through DPP-4 inhibitors. DPP-4 is a transmembrane glycoprotein belongs to prolyl oligopeptidase family, with the specificity of removing X-Pro or X-Ala dipeptides from the N-terminus of polypeptides. GLP-1 effect by stimulating glucose-dependent insulin release from the pancreatic islets, inhibit inappropriate post-meal glucagon release and slow gastric emptying promoting leaky gut. The current study investigated DPP-4 inhibitory activity of catechin, isolated from Withania somnifera (WS), for ethnopharmacological treatment of type 2 diabetes and aimed to increase availability of GLP-1and sensitivity to insulin. Materials and Methods: Young and matured fresh roots, leaves, and fruits of WS plant extract were considered and were systematically evaluated for DPP-4 inhibitory activity using in vitro method, enzyme kinetics, phytochemical analysis, RP-HPLC, LCMS and 1H and 13C NMR method and structure-activity relationship (SAR) studies. Results: In this study, methanol (100% and 80%) extracts of WS matured root exhibited maximum DPP-4 inhibitory activity when compared to other extracts. The maximum DPP-4 inhibitory activity was found in 100% methanol extract of matured root. Phytobioactive was purified by RP-HPLC. The compound purified was found to be flavonoid and was characterized (LCMS, 1H and 13C NMR studies), identified as catechin. Auxiliary, molecular docking was performed using Ligand Fit method using PatchDock package. The study revealed the binding affinity of catechin with DPP-4 to be -6.601 kcal/mol with 13 hydrogen interactions with the receptor and was very similar to the standard potent blockers withaferin A and others (cuscohygrine, scopoletin, sitoindoside IV, tropine), further confirming its hyperglycemic potency. Conclusion: The study reveals that, 100% methanol extract of WS matured roots contains the compound- catechin, which exhibits DPP-4 inhibitory activity resulting in increased level of bioactive GLP-1 and GIP. In this background, we concluded that the WS will be a better source for further development as new antidiabetic drugs.
In recent years, peptides derived from a variety of dietary proteins have been reported to exhibit inhibitory activity against the dipeptidyl-peptidase IV (DPP-IV) enzyme, a target in the management of type 2 diabetes. While much attention has been given to the production and identification of peptides with DPP-IV inhibitory activity from food proteins, particularly dairy proteins, little is known on the bioavailability of these molecules. In this study, the stability and transport of five previously identified milk-derived peptides (LKPTPEGDL, LPYPY, IPIQY, IPI and WR) and a whey protein isolate (WPI) digest with DPP-IV-inhibitory activity were investigated using Caco-2 cell monolayers as a model system for human intestinal absorption. Even though a small percentage (ranging from 0.05% for LPYPY to 0.47% for WR) of the bioactive peptides added to the apical side was able to cross the monolayer intact, all five peptides investigated were susceptible to peptidase action during the transport study. Conversely, only minor changes to the WPI digest composition were observed. Determination of the DPP-IV inhibitory activity of the peptides and amino acids identified in the apical and basolateral solutions showed that most degradation products were less effective at inhibiting DPP-IV than the peptide they originated from. Findings from this research suggest that the susceptibility of food-derived DPP-IV inhibitory peptides to degradation by intestinal brush border membrane enzymes may alter their biological activity in vivo. Further research should be conducted to enhance the bioavailability of DPP-IV inhibitory peptides.
The association between uric acid (UA) on one side and systemic hypertension (Htn), dyslipidemia, glucose intolerance, overweight, fatty liver, renal disease and cardiovascular disease (CVD) on the other side is well recognized. However, the causal relationship between UA and these different clinical problems is still debatable. The recent years have witnessed hundreds of experimental and clinical trials that favored the opinion that UA is a probable player in the pathogenesis of these disease entities. These studies disclosed the strong association between hyperuricemia and metabolic syndrome (MS), obesity, Htn, type 2 diabetes mellitus (DM), non-alcoholic fatty liver disease, hypertriglyceridemia, acute kidney injury, chronic kidney disease (CKD), coronary heart disease (CHD), heart failure and increased mortality among cardiac and CKD patients. The association between UA and nephrolithiasis or preeclampsia is a non-debatable association. Recent experimental trials have disclosed different changes in enzyme activities induced by UA. Nitric oxide (NO) synthase, adenosine monophosphate kinase (AMPK), adenosine monophosphate dehydrogenase (AMPD), and nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase are affected by UA. These changes in enzymatic activities can lead to the observed biochemical and pathological changes associated with UA. The recent experimental, clinical, interventional, and epidemiologic trials favor the concept of a causative role of UA in pathogenesis of MS, renal, and CVDs.
We investigated the effects of teneligliptin on uric acid metabolism in male Wistar rats and 3T3-L1 adipocytes. The rats were fed with a normal chow diet (NCD) or a 60% high-fat diet (HFD) with or without teneligliptin for 4 weeks. The plasma uric acid level was not significantly different between the control and teneligliptin groups under the NCD condition. However, the plasma uric acid level was significantly decreased in the HFD-fed teneligliptin treated rats compared to the HFD-fed control rats.The expression levels of xanthine dehydrogenase (Xdh) mRNA in liver and epididymal adipose tissue of NCD-fed rats were not altered by teneligliptin treatment. On the other hand, Xdh expression was reduced significantly in the epididymal adipose tissue of the HFD-fed teneligliptin treated rats compared with that of HFD-fed control rats, whereas Xdh expression in liver did not change significantly in either group. Furthermore, teneligliptin significantly decreased Xdh expression in 3T3-L1 adipocytes. DPP-4 treatment significantly increased Xdh expression in 3T3-L1 adipocytes.With DPP-4 pretreatment, teneligliptin significantly
decreased Xdh mRNA expression compared to the DPP-4-treated 3T3-L1 adipocytes. In conclusion, our studies suggest that
teneligliptin reduces uric acid levels by suppressing Xdh expression in epididymal adipose tissue of obese subjects.
Dipeptidyl peptidase-4 (DPP-4), glycyl-prolyl-naphthylamidase, is a serine protease that catalyses the hydrolysis of various proline-containing polypeptides. It is involved in the inactivation of glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), having in this way a profound influence on glucose metabolism. During organ damage, stromal and endothelial cells produce a chemokine known as stromal cell-derived factor-1 (SDF-1), a powerful chemoattractant of stem/progenitor cells. SDF-1 binds to a specific α-chemokine receptor (CXCR4) and can be degraded by proteases, including matrix DPP-4/CD26, presented in the circulation, or activated in injured tissues. DPP-4 inhibition has received considerable attention because of its significant therapeutic benefits in the regulation of insulin secretion and tissue insulin sensitivity, the regulation of tumour growth and metastasis, angiogenesis, tissue repair, especially after myocardial infarction, and regulation of endocrine function. Inhibition of circulating proteases appears to maintain the optimal endogenous SDF-1 concentration and may enhance homing of endothelial progenitor cells. In the present article, we present an overview of some basic facts about therole of DPP-4 in glucose homeostasis, the mechanism of its inhibition, and a brief summary of available DPP-4 inhibitors. Furthermore, since protection against the overactivity of proteases is important for restorating cardiac function and repair after myocardial damage, necrosis and apoptosis, we propose that administration of a DPP-4 inhibitor may also be beneficial following myocardial infarction by the prevention of cleavage of stem cell chemoattractant cytokine SDF-1.
Two groups of benzimidazole derivatives were synthesized using as precursors 5(6)-substituted 2-mercapto-benzimidazol-thiols and their antioxidant activity was investigated using TBA-MDA test. In the group of 1,3-disubstituted-benzimidazol-2-imines the highest lipid peroxidation inhibition effect 74.04% (IC50=141.89μg/mL) revealed ethyl [3-(2-ethoxy-2-oxoethyl)-2-imino-5-benzoyl-2,3-dihydro-1H-benzimdazol-1-yl]acetate 12 while in the group of 2-substituted-1,3-thiazolo[3,2-a]benzimidazolones the highest inhibition effect showed 2-(4-fluorobenzylidene)-7-(phenylcarbonyl)[1,3]thiazolo[3,2-a]benzimidazol-3(2H)-one 17 90.76% (IC50=53.70μg/mL). In order to estimate the capability of the studied benzimidazoles to act as radical scavengers the structure of the most active derivative within the both subseries was optimized at B3LYP/6-311++G(∗∗) level and the respective bond dissociation enthalpies were calculated. The appropriate models for the HAT and SET-mechanism of the antioxidant activity were proposed. The antibacterial activity of the compounds was evaluated against two Gram-positive bacteria (Bacillus subtilis ATCC 6633 and Staphylococcus aureus ATCC 6538) and three Gram-negative bacteria (Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 9027 and Salmonella abony NCTC 6017). 1,3-Diphenylpropyl-5-methyl-1,3-dihydro-2H-benzimidazol-2-imine 14 exhibited significant activity against B. subtilis, S. aureus, S. abony and E. coli (with MIC values of 0.125, 0.016, 0.50 and 0.50mg/mL, respectively). The group of thiazolobenzimidazolones did not reveal antibacterial activity against the tested strains.
In humans, uric acid is the final oxidation product of purine catabolism. The serum uric acid level is based on the balance between the absorption, production and excretion of purine. Uric acid is similarly produced in the liver, adipose tissue and muscle and is primarily excreted through the urinary tract. Several factors, including a high-fructose diet and the use of xenobiotics and alcohol, contribute to hyperuricaemia. Hyperuricaemia belongs to a cluster of metabolic and haemodynamic abnormalities, called metabolic syndrome, characterised by abdominal obesity, glucose intolerance, insulin resistance, dyslipidaemia and hypertension. Hyperuricaemia reduction in the Pound mouse or fructose-fed rats, as well as hyperuricaemia induction by uricase inhibition in rodents and studies using cell culture have suggested that uric acid plays an important role in the development of metabolic syndrome. These studies have shown that high uric acid levels regulate the oxidative stress, inflammation and enzymes associated with glucose and lipid metabolism, suggesting a mechanism for the impairment of metabolic homeostasis. Humans lacking uricase, the enzyme responsible for uric acid degradation, are susceptible to these effects. In this review, we summarise the current knowledge of the effects of uric acid on the regulation of metabolism, primarily focusing on liver, adipose tissue and skeletal muscle.
Copyright © 2015. Published by Elsevier B.V.
We have previously shown that hyperuricemia plays an important role in the vascular complications of insulin resistance (IR). Here we investigated the effect of xanthine oxidase (XO) inhibition on the cardiac complications of IR.
IR was induced in rats by a high fructose high fat diet for 12 weeks. Allopurinol, a standard XO inhibitor, was administered in the last 4 weeks before cardiac hemodynamics and electrocardiography, serum glucose, insulin, tumor necrosis factor alpha (TNFα), 8-isoprostane, uric acid, lactate dehydrogenase (LDH) and XO activity were measured. Expression of cardiac angiotensin II (AngII) and angiotensin receptor 1 (AT1) were assessed by immunofluorescence.
IR animals had significant hyperuricemia which was inhibited by allopurinol administration. IR was associated with impaired ventricular relaxation (reflected by a decreased diastolic pressure increment and prolonged diastolic duration) and XO inhibition greatly attenuated impaired relaxation. IR was accompanied by cardiac ischemia (reflected by increased QTc and T peak trend intervals) while XO inhibition alleviated the ECG abnormalities. When subjected to isoproterenol-induced ischemia, IR hearts were less resistant (reflected by larger ST height depression and higher LDH level) while XO inhibition alleviated the accompanying ischemia. In addition, XO inhibition prevented the elevation of serum 8-isoprostane and TNFα, and blocked elevated AngII and AT1 receptor expression in the heart tissue of IR animals. However, XO inhibition did not affect the developed hyperinsulinemia or dyslipidemia.
XO inhibition alleviates cardiac ischemia and impaired relaxation in IR through the inhibition of low grade inflammation and the angiotensin system.
Thirty 2-amino-5-alkylidene-thiazol-4-ones were assayed for inhibitory activity against commercial enzyme xanthine oxidase (XO) in vitro and XO in rat liver homogenate as well as for anti-inflammatory response on human peripheral blood mononuclear cells (PBMCs). 4-((2-Benzylamino-4-oxothiazol-5(4H)-ylidene)-methyl)benzonitrile showed the most potent inhibitory effect against commercial XO (IC50=17.16μg/mL) as well as against rat liver XO (IC50=24.50μg/mL). All compounds containing the 4-cyanobenzylidene group or (indol-3-yl)methylene group at the position 5 of thiazol-4-one moiety were moderately potent inhibitors of commercial XO. The assayed compounds were docked into the crystal structures of XO enzyme complexes with three diverse inhibitors (PDB codes: 1FIQ, 1VDV, and 1V97) using OEDocking software. Our results strongly point to a correlation between the data on inhibitory activity against commercial XO and data on antioxidant activity of studied compounds, screened using a lipid peroxidation (LP) method. 2-(Benzylamino)-5-((thiophen-2-yl)methylene)thiazol-4(5H)-one showed the highest anti-inflammatory response on PBMCs, exerted most probably through the NF-κB inhibition. Studied 2-amino-5-alkylidene-thiazol-4-ones obey the "Rule of five" and meet all criteria for good solubility and permeability.
Copyright © 2015. Published by Elsevier Ireland Ltd.
We investigated whether sitagliptin, a dipeptidyl peptidase-4 (DPP-4) inhibitor, attenuates arrhythmias through inhibiting nerve growth factor (NGF) expression in post-infarcted normoglycemic rats, focusing on adenosine and reactive oxygen species production. DPP-4 bound adenosine deaminase has been shown to catalyse extracellular adenosine to inosine. DPP-4 inhibitors increased adenosine levels by inhibiting the complex formation. Normoglycemic male Wistar rats were subjected to coronary ligation and then randomized to either saline or sitagliptin in in vivo and ex vivo studies. Post-infarction was associated with increased oxidative stress, as measured by myocardial superoxide, nitrotyrosine and dihydroethidium fluorescent staining. Measurement of myocardial norepinephrine levels revealed a significant elevation in vehicle-treated infarcted rats compared with sham. Compared with vehicle, infarcted rats treated with sitagliptin significantly increased interstitial adenosine levels and attenuated oxidative stress. Sympathetic hyperinnervation was blunted after administering sitagliptin, as assessed by immunofluorescent analysis and western blotting and real-time quantitative RT-PCR of NGF. Arrhythmic scores in the sitagliptin-treated infarcted rats were significantly lower than those in vehicle. Ex vivo studies showed a similar effect of erythro-9-(2-hydroxy-3-nonyl) adenine (an adenosine deaminase inhibitor) to sitagliptin on attenuated levels of superoxide and NGF. Furthermore, the beneficial effects of sitagliptin on superoxide anion production and NGF levels can be reversed by 8-cyclopentyl-1,3-dipropulxanthine (adenosine A1 receptor antagonist) and exogenous hypoxanthine. Sitagliptin protects ventricular arrhythmias by attenuating sympathetic innervation via adenosine A1 receptor and xanthine oxidase-dependent pathways, which converge through the attenuated formation of superoxide in the non-diabetic infarcted rats.
Endothelial xanthine oxidoreductase (XOR) together with NAD(P)H oxidase and nitric oxide (NO) synthase plays a physiologic role in inflammatory signalling, the regulation of NO production and vascular function. The oxidative stress generated by these enzymes may induce endothelial dysfunction, leading to atherosclerosis, cardiovascular diseases and metabolic syndrome. XOR activity creates both oxidant and anti-oxidant products that are implicated in the development of hypertension, smoking vascular injury, dyslipidemia and diabetes, which are the main risk factors of atherosclerosis. In particular, uric acid may have a protective as well as a detrimental role in vascular alterations, thus justifying the multi-directional effects of XOR inhibition. Moreover, XOR products are associated with cell differentiation, leading to adipogenesis and foam cell formation, as well as to the production of monocyte chemoattractant protein-1 from arterial smooth muscle cells, after proliferation and migration. The role of XOR in adipogenesis is also connected with insulin resistance and obesity, two main features of type 2 diabetes.
Copyright © 2014 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.
Cell lines are the invitro models used for the drug permeability studies in the preclinical and clinical phases of the drug discovery. Cell line models are simple and quick to use and avoids the usage of animal models for pharmacological and toxicological studies and hence cost effective, produce reliable and reproducible results for understanding and evaluating the permeability characteristics of the potential lead drug candidates. Different cell line models used in the drug permeability studies, their characteristics has been summarized emphasizing on CACO-2. By virtue of its merits, CACO-2 cell line development, transport experiments, automated assays, optimization of experimental conditions and mechanistic uses of CACO-2 cell lines dealt comprehensively in the following context.
The present study demonstrates and validates the discovery of two novel hydrazine derivatives as selective dipeptidyl peptidase-IV (DPP-IV) inhibitors. Virtual screening (VS) of publicly available databases was performed using virtual screening workflow (VSW) of Schrödinger software against DPP-IV and the most promising hits were selected. Selectivity was further assessed by docking the hits against homology modeled structures of DPP8 and DPP9. Two novel hydrazine derivatives were selected for further studies based on their selectivity threshold. To assess their correct binding modes and stability of their complexes with enzyme, molecular dynamic (MD) simulation studies were performed against the DPP-IV protein and the results revealed that they had a better binding affinity towards DPP-IV as compared to DPP 8 and DPP 9. The binding poses were further validated by docking these ligands with different softwares (Glide and Gold). The proposed binding modes of hydrazines were found to be similar to sitagliptine and alogliptine. Thus, the study reveals the potential of hydrazine derivatives as highly selective DPP-IV inhibitors.
Figure
Structures of sitagliptine and the novel hydrazine identified by screening
Xanthine oxidoreductase is a ubiquitous cytoplasmic protein that catalyzes the final two steps in purine catabolism. We have
previously investigated the catalytic mechanism of the enzyme by rapid reaction kinetics and x-ray crystallography using the
poor substrate 2-hydroxy-6-methylpurine, focusing our attention on the orientation of substrate in the active site and the
role of Arg-880 in catalysis. Here we report additional crystal structures of as-isolated, functional xanthine oxidase in
the course of reaction with the pterin substrate lumazine at 2.2 Å resolution and of the nonfunctional desulfo form of the
enzyme in complex with xanthine at 2.6 Å resolution. In both cases the orientation of substrate is such that the pyrimidine
subnucleus is oriented opposite to that seen with the slow substrate 2-hydroxy-6-methylpurine. The mechanistic implications
as to how the ensemble of active site functional groups in the active site work to accelerate reaction rate are discussed.
While the free radical-generating enzyme xanthine oxidase is a central mechanism of injury in postischemic tissues, questions remain regarding how xanthine oxidase-mediated radical generation is triggered during ischemia and reperfusion. There is controversy regarding whether radical generation is caused by enzyme formation of that of its substrates xanthine and hypoxanthine. Therefore, studies were performed in isolated rat hearts correlating the magnitude and time course of radical generation with alteration in xanthine oxidase and its substrates. Radical generation was measured by electron paramagnetic resonance spectroscopy and correlated with spectrophotometric assays of tissue xanthine oxidase activity and chromatographic measurements of tissue and effluent concentrations of xanthine oxidase substrates and products. Xanthine oxidase was present in preischemic hearts and slightly increased during 30-min global ischemia. Hypoxanthine and xanthine were not present prior to ischemia but accumulated greatly during ischemia due to ATP degradation. These substrate concentrations rapidly declined over the first 5 min of reperfusion matching the observed time course of radical generation, whereas xanthine oxidase activity was largely unchanged. Both substrates were also observed in the coronary effluent during the first 5 min of reflow along with the product uric acid. Thus, the burst of xanthine oxidase-mediated free radical generation upon reperfusion is triggered and its time course controlled by a large increase in substrate formation that occurs secondary to the degradation of ATP during ischemia.
In the presence of its substrates hypoxanthine and xanthine, xanthine oxidase generates oxygen free radicals that cause postischemic injury. Recently, it has been demonstrated that the burst of xanthine oxidase-mediated free radical generation in the reperfused heart is triggered by a large increase in substrate formation, which occurs secondary to the degradation of adenine nucleotides during ischemia. It is not known, however, whether blocking this substrate formation is sufficient to prevent radical generation and functional injury. Therefore, studies were performed in isolated rat hearts in which xanthine oxidase substrate formation was blocked with the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), and measurements of contractile function and free radical generation were performed. Chromatographic measurements of the intracellular adenine nucleotide pool showed that preischemic administration of EHNA blocked postischemic hypoxanthine, xanthine, and inosine formation. Electron paramagnetic resonance spin trapping measurements of free radical generation showed that inhibition of adenosine deaminase with EHNA blocked free radical generation and that it also increased the recovery of contractile function by more than 2-fold. Exogenous infusion of hypoxanthine and xanthine totally reversed the protective effects of EHNA. These results demonstrate that blockade of xanthine oxidase substrate formation by adenosine deaminase inhibition can prevent free radical generation and contractile dysfunction in the postischemic heart.
Therapeutic strategies against free radicals have mostly focused on the augmentation of antioxidant defenses (eg, vitamins C and E). A novel approach is to prevent free radical generation by the enzyme system xanthine oxidase. We examined whether the inhibition of xanthine oxidase with allopurinol can improve endothelial function in subjects with type 2 diabetes and coexisting mild hypertension compared with control subjects of a similar age. We examined 23 subjects (11 patients with type 2 diabetes and 12 healthy age-matched control subjects) in 2 parallel groups. The subjects were administered 300 mg allopurinol in a randomized, placebo-controlled study in which both therapies were administered for 1 month. Endothelial function was assessed with bilateral venous occlusion plethysmography, in which the forearm blood flow responses to intra-arterial infusions of endothelium-dependent and -independent vasodilators were measured. Allopurinol significantly increased the mean forearm blood flow response to acetylcholine by 30% (3.16+/-1.21 versus 2.54+/-0.76 mL. 100 mL(-1). min(-1) allopurinol versus placebo; P=0.012, 95% CI 0.14, 1.30) but did not affect the nitroprusside response in patients with type 2 diabetes. There was no significant impact on either endothelium-dependent or -independent vascular responses in age-matched control subjects. Allopurinol improved endothelial function to near-normal levels. Regarding markers of free radical activity, the level of malondialdehyde was significantly reduced (0.30+/-0.04 versus 0. 34+/-0.05 micromol/L for allopurinol versus placebo, P=0.03) in patients with type 2 diabetes but not in control subjects. The xanthine oxidase inhibitor allopurinol improves endothelial dysfunction in patients with type 2 diabetes with mild hypertension but not in matched control subjects. In the former group, allopurinol restored endothelial function to near-normal levels.
The aim of this work was to study the mechanism of free radical formation in type 1 diabetes and its possible prevention. We have found oxidation of blood glutathione and an increase in plasma lipoperoxide levels in both human type 1 diabetes and experimental diabetes. Peroxide production by mitochondria does not increase in diabetes. On the contrary, the activity of xanthine oxidase, a superoxide-generating enzyme, increases in liver and plasma of diabetic animals. The increase in plasma xanthine oxidase activity may be explained by the increase in the hepatic release of this enzyme, which is not due to nonspecific membrane damage: release of other hepatic enzymes, such as the amino transferases, does not increase in diabetes. Superoxide formation by aortic rings of rabbits increases significantly in diabetes. This is completely inhibited by allopurinol, an inhibitor of xanthine oxidase. Heparin, which releases xanthine oxidase from the vessel wall, also decreases superoxide formation by aortic rings of diabetic animals. Treatment with allopurinol decreases oxidative stress in type 1 diabetic patients: hemoglobin glycation, glutathione oxidation, and the increase in lipid peroxidation are prevented. These results may have clinical significance in the prevention of late-onset vascular complications of diabetes.
Molybdenum is widely distributed in biology and is usually found as a mononuclear metal center in the active sites of many enzymes catalyzing oxygen atom transfer. The molybdenum hydroxylases are distinct from other biological systems catalyzing hydroxylation reactions in that the oxygen atom incorporated into the product is derived from water rather than molecular oxygen. Here, we present the crystal structure of the key intermediate in the hydroxylation reaction of xanthine oxidoreductase with a slow substrate, in which the carbon-oxygen bond of the product is formed, yet the product remains complexed to the molybdenum. This intermediate displays a stable broad charge-transfer band at approximately 640 nm. The crystal structure of the complex indicates that the catalytically labile Mo-OH oxygen has formed a bond with a carbon atom of the substrate. In addition, the MoS group of the oxidized enzyme has become protonated to afford Mo-SH on reduction of the molybdenum center. In contrast to previous assignments, we find this last ligand at an equatorial position in the square-pyramidal metal coordination sphere, not the apical position. A water molecule usually seen in the active site of the enzyme is absent in the present structure, which probably accounts for the stability of this intermediate toward ligand displacement by hydroxide.
Adenosine deaminase (ADA), a protein whose deficit leads to severe combined immunodeficiency, binds to the cell surface by means of either CD26, A1 adenosine receptors, or A2B adenosine receptors. The physiological role of these interactions is not well understood. Our results show that by a 3-fold reduction in the EC50 for the antigen, ADA potentiated T cell proliferation in autologous cocultures with antigen-pulsed immature or mature dendritic cells. Costimulation was not due to the enzymatic activity but to the interaction of ADA–CD26 complexes in T cells with an ADA-anchoring protein in dendritic cells. From colocalization studies, it is deduced that ADA colocalizing with adenosine receptors on dendritic cells interact with CD26 expressed on lymphocytes. This costimulatory signal in the immunological synapse leads to a marked increase (3- to 34-fold) in the production of the T helper 1 and proimmflamatory cytokines IFN-γ, TNF-α, and IL-6.
• adenosine deaminase
• costimulation
• immunosynapse
Multi-targeting compounds comprising activity on more than a single biological target have gained remarkable relevance in drug discovery owing to the complexity of multifactorial diseases such as cancer, inflammation or the metabolic syndrome. Polypharmacological drug profiles can produce additive or synergistic effects while reducing side effects and significantly contribute to the high therapeutic success of indispensable drugs such as aspirin. While their identification has long been the result of serendipity, medicinal chemistry now tends to design polypharmacology. Modern in vitro pharmacological methods and chemical probes allow a systematic search for rational target combinations and recent innovations in computational technologies, crystallography, or fragment-based design equip multi-target compound development with valuable tools. In this perspective, we analyze the relevance of multiple ligands in drug discovery and the versatile toolbox to design polypharmacology. We conclude that despite some characteristic challenges remaining unresolved, designed polypharmacology holds enormous potential to secure future therapeutic innovation.
The objective of this study was to develop a nanodelivery system containing a mucoadhesive polymer hyaluronic acid (HA) for oral delivery. Metformin was used as a model drug. Blank and drug-loaded HA nanostructures were prepared by precipitation method and characterized for particle size (PS), zeta potential (ZP), physical stability (over 65 days), surface morphology, moisture content, and physical state of the drug in the nanostructures. The cytotoxicity and hemolysis potential of the delivery system was assessed in Caco-2 cells and whole human blood, respectively. The in vitro release of metformin and its uptake in Caco-2 cells was evaluated using high-performance liquid chromatography. Ex vivo permeability of metformin was measured through goat intestinal membrane. The nanoparticles were physically stable and neutrally charged with an average PS of 114.53 ± 12.01 nm. This nanodelivery system existed as nanofibers containing metformin in a crystalline state. This delivery system released the drug rapidly with > 50% of metformin released within 1 h. Cellular uptake studies on Caco-2 cells indicated higher uptake of metformin from nanoparticle as compared to metformin in solution, up to first 45 min. Ex vivo permeability studies on the other hand showed a higher metformin permeability from solution relative to that from nanoparticles through the goat intestinal membrane. Metformin nanoparticles were non-toxic at therapeutic concentrations in Caco-2 cells and showed no hemolytic effect to RBCs. This study indicates the preparation, characterization, as well as the potential use of HA nanostructures for oral delivery.
Gout is a common inflammatory arthritis caused by the deposition of urate crystals within joints. It is increasingly in prevalence during the past few decades as shown by the epidemiological survey results. Xanthine oxidase (XO) is a key enzyme to transfer hypoxanthine and xanthine to uric acid, whose overproduction leads to gout. Therefore, inhibiting the activity of xanthine oxidase is an important way to reduce the production of urate. In the study, in order to identify the potential natural products targeting XO, pharmacophore modeling was employed to filter databases. Here, two methods, pharmacophore based on ligand and pharmacophore based on receptor-ligand, were constructed by Discovery Studio. Then GOLD was used to refine the potential compounds with higher fitness scores. Finally, molecular docking and dynamics simulations were employed to analyze the interactions between compounds and protein. The best hypothesis was set as a 3D query to screen database, returning 785 and 297 compounds respectively. A merged set of the above 1082 molecules was subjected to molecular docking, which returned 144 hits with high-fitness scores. These molecules were clustered in four main kinds depending on different backbones. What is more, molecular docking showed that the representative compounds established key interactions with the amino acid residues in the protein, and the RMSD and RMSF of molecular dynamics results showed that these compounds can stabilize the protein. The information represented in the study confirmed previous reports. And it may assist to discover and design new backbones as potential XO inhibitors based on natural products.
The inhibition of dipeptidyl peptidase‐IV (DPP‐IV) has emerged over the last decade as one of the most effective treatments for type 2 diabetes mellitus, and consequently (a) 11 DPP‐IV inhibitors have been on the market since 2006 (three in 2015), and (b) 74 noncovalent complexes involving human DPP‐IV and drug‐like inhibitors are available at the Protein Data Bank (PDB). The present review aims to (a) explain the most important activity cliffs for DPP‐IV noncovalent inhibition according to the binding site structure of DPP‐IV, (b) explain the most important selectivity cliffs for DPP‐IV noncovalent inhibition in comparison with other related enzymes (i.e., DPP8 and DPP9), and (c) use the information deriving from this activity/selectivity cliff analysis to suggest how virtual screening protocols might be improved to favor the early identification of potent and selective DPP‐IV inhibitors in molecular databases (because they have not succeeded in identifying selective DPP‐IV inhibitors with IC50 ≤ 100 nM). All these goals are achieved with the help of available homology models for DPP8 and DPP9 and an analysis of the structure–activity studies used to develop the noncovalent inhibitors that form part of some of the complexes with human DPP‐IV available at the PDB.
Diabetes is a fast growing chronic metabolic disorder around the world. Dipeptidyl peptidase-4 (DPP-4) is a new promising target during type 2 diabetes glycemic control. Thus, a number of potent DPP-4 inhibitors were developed and play a rapidly evolving role in the management of type 2 diabetes in recent years. This article reviews the development of synthetic and natural DPP-4 inhibitors from 2012 to 2017 and provides their physico-chemical properties, biological activities against DPP-4 and selectivity over dipeptidyl peptidase-8/9. Moreover, the glucose-lowering mechanisms and the active site of DPP-4 are also discussed. We also discuss strategies and structure-activity relationships for identifying potent DPP-4 inhibitors, which will provide useful information for developing potent DPP-4 drugs as type 2 diabtes treatments.
Objective:
We aimed to evaluate whether xanthine oxidase (XO), a key enzyme in uric acid (UA) metabolism and a major source of reactive oxygen species, plays a causal and important role in the development of type 2 diabetes mellitus (T2DM) in a large prospective cohort study.
Research design and methods:
A total of 4,412 diabetes-free adults (2,071 women and 2,341 men) aged 30-65 years at baseline in 2008 were involved. Participants were followed for incident change of glucose metabolism during an average of 4.7 years. At baseline, serum XO and UA, serum lipids, and glucose homeostasis indexes including fasting blood glucose (FBG), 2-h blood glucose (PBG), glycosylated hemoglobin A1c (HbA1c), and fasting insulin were tested for analysis.
Results:
During an average follow-up period of 4.7 years, 249 women and 360 men developed new-onset T2DM. Serum XO activity was positively associated with UA concentration (all P values <0.001). When XO activity and UA concentration were considered in the same model of the sex-specific analysis, only XO activity was significantly associated with the incidence of T2DM, with the hazard ratios from the bottom to the top quartile of XO activity being 1.00, 1.67 (95% CI 1.00-2.79), 1.86 (1.11-3.13), and 2.36 (1.43-3.90) in women and 1.00, 1.01 (0.68-1.52), 1.41 (0.98-2.03), and 1.90 (1.30-2.78) in men.
Conclusions:
Elevated serum XO activity, but not UA concentration, was associated with an increased risk of developing T2DM in women and men with mutual adjustment for XO and UA. Further studies are needed to examine the underlying mechanisms.
This review summarizes recent published literature on the association between serum uric acid and cardiovascular disease, a relationship which is complex and not fully elucidated. Uric acid may be a marker for risk, a causative agent in cardiovascular disease, or both. Various biologic factors can influence serum uric acid levels, and serum uric acid level itself is closely related to conditions such as hypertension, dyslipidemia, obesity, and impaired glucose metabolism, that contribute to cardiovascular disease pathophysiology. Serum uric acid levels have been found to be associated with adverse outcomes, including mortality, in the general population. In addition, serum uric acid is associated with increased risk for incident coronary heart disease, heart failure, and atrial fibrillation. In the setting of established systolic heart failure, serum uric acid is positively associated with disease severity and mortality risk. Whether targeting treatment based on uric acid levels might affect clinical outcomes is still being studied.
Copyright © 2015. Published by Elsevier Ireland Ltd.
The role of hyperuricaemia as cardiovascular risk factor has exhaustingly been debated for decades. While the association of elevated uric acid (UA) levels with increased mortality risk as convincingly been shown, the question whether UA is independently predictive of just a related effect within a more complex risk factor profile (including metabolic, inflammatory and haemodynamic risk factors) is still a matter of dispute. In heart failure the independent prognostic and functional impact of elevated UA has not only been shown but also the pathophysiologic mechanism(s) and the potential of targeted therapeutic interventions have been investigated in some detail. The emerging picture suggests the increased activity of the enzyme xanthine oxidase (XO) with corresponding increased production of free oxygen radical (ROS) as a main underlying principle with the resulting increase in UA levels being mostly a marker of this up-regulated pathway. While this concept will not diminish the value of UA as a prognostic marker, it provides the basis for a novel metabolic treatment option and the means to identify those patients most eligible for this tailored therapy. This review will summarize the recent evidence on XO as a novel and promising therapeutic target in heart failure.
Copyright © 2015. Published by Elsevier Ireland Ltd.
Chrysin, a bioactive flavonoid, was investigated for its potential to inhibit the activity of xanthine oxidase (XO), a key enzyme catalyzing xanthine to uric acid and finally causing gout. The kinetic analysis showed that chrysin possessed a strong inhibition on XO ability in a reversible competitive manner with IC50 value of (1.26±0.04)×10(-6) mol L(-1). The results of fluorescence titrations indicated that chrysin bound to XO with high affinity, and the interaction was predominately driven by hydrogen bonds and van der Waals forces. Analysis of circular dichroism demonstrated that chrysin induced the conformational change of XO with increases in α-helix and β-sheet and reductions in β-turn and random coil structures. Molecular simulation revealed that chrysin interacted with the amino acid residues Leu648, Phe649, Glu802, Leu873, Ser876, Glu879, Arg880, Phe1009, Thr1010, Val1011 and Phe1013 located within the active cavity of XO. The mechanism of chrysin on XO activity may be the insertion of chrysin into the active site occupying the catalytic center of XO to avoid the entrance of xanthine and causing conformational changes in XO. Furthermore, the interaction assays indicated that chrysin and its structural analogue apigenin exhibited an additive effect on inhibition of XO.
Copyright © 2015. Published by Elsevier B.V.
Interest in adenosine deaminase (ADA) in the context of medicine has mainly focused on its enzymatic activity. This is justified by the importance of the reaction catalyzed by ADA not only for the intracellular purine metabolism, but also for the extracellular purine metabolism as well, because of its capacity as a regulator of the concentration of extracellular adenosine that is able to activate adenosine receptors (ARs). In recent years, other important roles have been described for ADA. One of these, with special relevance in immunology, is the capacity of ADA to act as a costimulator, promoting T-cell proliferation and differentiation mainly by interacting with the differentiation cluster CD26. Another role is the ability of ADA to act as an allosteric modulator of ARs. These receptors have very general physiological implications, particularly in the neurological system where they play an important role. Thus, ADA, being a single chain protein, performs more than one function, consistent with the definition of a moonlighting protein. Although ADA has never been associated with moonlighting proteins, here we consider ADA as an example of this family of multifunctional proteins. In this review, we discuss the different roles of ADA and their pathological implications. We propose a mechanism by which some of their moonlighting functions can be coordinated. We also suggest that drugs modulating ADA properties may act as modulators of the moonlighting functions of ADA, giving them additional potential medical interest.
The proline-specific dipeptidyl aminopeptidase IV (DPP IV, DPP-4, CD26), widely expressed in mammalians, releases X-Pro/Ala dipeptides from the N-terminus of peptides. DPP IV is responsible of the degradation of the incretin peptide hormones regulating blood glucose levels. Several families of DPP IV inhibitors have been synthesized and evaluated, Their positive effects on the degradation of the incretins and the control of blood glucose levels have been demonstrated in biological models and in clinical trials. Presently, several DPP IV inhibitors, the "gliptins", are approved for type 2 diabetes or are under clinical evaluation. However, the gliptins may also be of therapeutic interest for other diseases beyond the inhibition of incretin degradation. In this Perspective, the biological functions and potential substrates of DPP IV enzymes are reviewed and the characteristics of the DPP IV inhibitors are discussed, in view of type 2 diabetes and further therapeutic interest.
The vascular endothelium is a multifunctional organ and is critically involved in modulating vascular tone and structure. Endothelial cells produce a wide range of factors that also regulate cellular adhesion, thromboresistance, smooth muscle cell proliferation, and vessel wall inflammation. Thus, endothelial function is important for the homeostasis of the body and its dysfunction is associated with several pathophysiological conditions, including atherosclerosis, hypertension and diabetes. Patients with diabetes invariably show an impairment of endothelium-dependent vasodilation. Therefore, understanding and treating endothelial dysfunction is a major focus in the prevention of vascular complications associated with all forms of diabetes mellitus. The mechanisms of endothelial dysfunction in diabetes may point to new management strategies for the prevention of cardiovascular disease in diabetes. This review will focus on the mechanisms and therapeutics that specifically target endothelial dysfunction in the context of a diabetic setting. Mechanisms including altered glucose metabolism, impaired insulin signaling, low-grade inflammatory state, and increased reactive oxygen species generation will be discussed. The importance of developing new pharmacological approaches that upregulate endothelium-derived nitric oxide synthesis and target key vascular ROS-producing enzymes will be highlighted and new strategies that might prove clinically relevant in preventing the development and/or retarding the progression of diabetes associated vascular complications.
Xanthine oxidase (XO) and dipeptidyl peptidase IV (DPP-IV) inhibition by amino acids and dipeptides was studied. Trp and Trp-containing dipeptides (Arg-Trp, Trp-Val, Val-Trp, Lys-Trp and Ile-Trp) inhibited XO. Three amino acids (Met, Leu and Trp) and eight dipeptides (Phe-Leu, Trp-Val, His-Leu, Glu-Lys, Ala-Leu, Val-Ala, Ser-Leu and Gly-Leu) inhibited DPP-IV. Trp and Trp-Val were multifunctional inhibitors of XO and DPP-IV. Lineweaver and Burk analysis showed that Trp was a non-competitive inhibitor of XO and a competitive inhibitor of DPP-IV. Molecular docking with Autodock Vina was used to better understand the interaction of the peptides with the active site of the enzyme. Because of the non-competitive inhibition observed, docking of Trp-Val to the secondary binding sites of XO and DPP-IV is required. Trp-Val was predicted to be intestinally neutral (between 25% and 75% peptide remaining after 60min simulated intestinal digestion). These results are of significance for the reduction of reactive oxygen species (ROS) and the increase of the half-life of incretins by food-derived peptides.
2-aryl-1-arylmethyl-1H-benzimidazole derivatives having different side chains on the structure were examined in-vitro for their antioxidant abilities by DPPH (2,2-diphenyl-1-picryl hydrazine) radical scavenging activity, reducing ability, OH radical scavenging activity, inhibition of polyphenol oxidase (PPO) and xanthine oxidase (XO). Overall, with few exceptions, all the 2-aryl-1-arylmethyl-1H-benzimidazoles showed moderate biological activity with all parameters examined. The 2-aryl-1-arylmethyl-1H-benzimidazoles were found to be reactive towards DPPH radical and had considerable reducing ability, with significant XO inhibition. With few exceptions, all the compounds under study were found to possess moderate to poor OH radical scavenging activity and inhibited PPO significantly. These findings suggest that, these 2-aryl-1-arylmethyl-1H-benzimidazoles can be considered as potential antioxidant and XO inhibitory agents, those might be further, explored for the design of lead antioxidant and anti-gout drug candidates using in vivo trials. This article is protected by copyright. All rights reserved.
Serum uric acid may be an independent risk factor for cardiovascular disease. This review examines this association, potential mechanisms, and explores whether strategies to reduce uric acid will improve outcomes. The recent studies of xanthine oxidase inhibition are given particular focus. Epidemiological evidence supports the theory that uric acid is an independent risk factor for cardiovascular disease. Recent studies of losartan, atorvastatin and fenofibrate suggest that uric acid reduction contributes to the risk reduction offered by these therapies. Several small studies of xanthine oxidase inhibition have shown improvements in measures of cardiovascular function of a similar magnitude to that of other proven preventative treatments. These trial data and the convincing epidemiological evidence mandate that large clinical trials of uric acid-lowering strategies are performed in patients with or at high risk of cardiovascular disease. If such approaches are shown to be effective in reducing cardiovascular events, they would represent a novel and cost-effective preventative approach.
The design, synthesis, and structure-activity relationships of a new class of potent and orally active non-peptide dipeptidyl peptidase IV (DPP-4) inhibitors, 3-aminomethyl-1,2-dihydro-4-phenyl-1-isoquinolones, are described. We hypothesized that the 4-phenyl group of the isoquinolone occupies the S1 pocket of the enzyme, the 3-aminomethyl group forms an electrostatic interaction with the S2 pocket, and the introduction of a hydrogen bond donor onto the 6- or 7-substituent provides interaction with the hydrophilic region of the enzyme. Based on this hypothesis, intensive research focused on developing new non-peptide DPP-4 inhibitors has been carried out. Among the compounds designed in this study, we identified 2-[(3-aminomethyl-2-(2-methylpropyl)-1-oxo-4-phenyl-1,2-dihydro-6-isoquinolinyl)oxy]acetamide (35a) as a potent, selective, and orally bioavailable DPP-4 inhibitor, which exhibited in vivo efficacy in diabetic model rats. Finally, X-ray crystallography of 35a in a complex with the enzyme validated our hypothesized binding mode and identified Lys554 as a new target-binding site available for DPP-4 inhibitors.
In this study, we investigated the effect of the xanthine oxidase (XO) inhibitor, allopurinol (ALP), on cardiac dysfunction, oxidative-nitrosative stress, apoptosis, poly(ADP-ribose) polymerase (PARP) activity and fibrosis associated with diabetic cardiomyopathy in mice. Diabetes was induced in C57/BL6 mice by injection of streptozotocin. Control and diabetic animals were treated with ALP or placebo. Left ventricular systolic and diastolic functions were measured by pressure-volume system 10 weeks after established diabetes. Myocardial XO, p22(phox), p40(phox), p47(phox), gp91(phox), iNOS, eNOS mRNA and/or protein levels, ROS and nitrotyrosine (NT) formation, caspase3/7 and PARP activity, chromatin fragmentation and various markers of fibrosis (collagen-1, TGF-beta, CTGF, fibronectin) were measured using molecular biology and biochemistry methods or immunohistochemistry. Diabetes was characterized by increased myocardial, liver and serum XO activity (but not expression), increased myocardial ROS generation, p22(phox), p40(phox), p47(phox), p91(phox) mRNA expression, iNOS (but not eNOS) expression, NT generation, caspase 3/7 and PARP activity/expression, chromatin fragmentation and fibrosis (enhanced accumulation of collagen, TGF-beta, CTGF and fibronectin), and declined systolic and diastolic myocardial performance. ALP attenuated the diabetes-induced increased myocardial, liver and serum XO activity, myocardial ROS, NT generation, iNOS expression, apoptosis, PARP activity and fibrosis, which were accompanied by improved systolic (measured by the evaluation of both load-dependent and independent indices of myocardial contractility) and diastolic performance of the hearts of treated diabetic animals. Thus, XO inhibition with ALP improves type 1 diabetes-induced cardiac dysfunction by decreasing oxidative/nitrosative stress and fibrosis, which may have important clinical implications for the treatment and prevention of diabetic cardiomyopathy and vascular dysfunction.
Adenosine is released from the myocardium in response to a decrease in the oxygen supply/demand ratio, as is seen in myocardial ischemia; its protective role is manifested by coronary and collateral vessel vasodilation that increase oxygen supply and by multiple effects that act in concert to decrease myocardial oxygen demand (i.e., negative inotropism, chronotropism, and dromotropism). During periods of oxygen deprivation, adenosine enhances energy production via increased glycolytic flux and can act as a substrate for purine salvage to restore cellular energy charge during reperfusion. Adenosine limits the degree of vascular injury during ischemia and reperfusion by inhibition of oxygen radical release from activated neutrophils, thereby preventing endothelial cell damage, and by inhibition of platelet aggregation. These effects help to preserve endothelial cell function and microvascular perfusion. Long-term exposure to adenosine may also induce coronary angiogenesis.
The gastrointestinal tract remains the most popular and acceptable route of administration for drugs. It offers the great advantage of convenience and many compounds are well absorbed and thereby provide acceptable plasma concentration-time profiles. Currently there is considerable interest from the pharmaceutical industry in development of cell culture systems that would mimic the intestinal mucosa in order to evaluate strategies for investigating and/or enhancing drug absorption. The intestinal epithelial cells of primary interest, from the standpoint of drug absorption and metabolism, are the villus cells, which are fully differentiated cells. An in vitro cell culture system consisting of a monolayer of viable, polarized and fully differentiated villus cells, similar to that found in the small intestine, would be a valuable tool in the study of drug and nutrient transport and metabolism. The Caco-2 cell line, which exhibits a well-differentiated brush border on the apical surface and tight junctions, and expresses typical small-intestinal microvillus hydrolases and nutrient transporters, has proven to be the most popular in vitro model (a) to rapidly assess the cellular permeability of potential drug candidates, (b) to elucidate pathways of drug transport (e.g., passive versus carrier mediated), (c) to assess formulation strategies designed to enhance membrane permeability, (d) to determine the optimal physicochemical characteristics for passive diffusion of drugs, and (e) to assess potential toxic effects of drug candidates or formulation components on this biological barrier. Since differentiated Caco-2 cells express various cytochrome P450 isoforms and phase II enzymes such as UDP-glucuronosyltransferases, sulfotransferases and glutathione-S-transferases, this model could also allow the study of presystemic drug metabolism.
TEI-6720 (2-(3-cyano-4-isobutoxyphenyl)-4-methyl-5-thiazolecarboxylic acid) is an extremely potent inhibitor of xanthine oxidoreductase. Steady state kinetics measurements exhibit mixed type inhibition with K(i) and K(i)' values of 1.2 +/- 0.05 x 10(-10) m and 9 +/- 0.05 x 10(-10) m, respectively. Fluorescence-monitored titration experiments showed that TEI-6720 bound very tightly to both the active and the inactive desulfo-form of the enzyme. The dissociation constant determined for the desulfo-form was 2 +/- 0.03 x 10(-9) m; for the active form, the corresponding number was too low to allow accurate measurements. The crystal structure of the active sulfo-form of milk xanthine dehydrogenase complexed with TEI-6720 and determined at 2.8-A resolution revealed the inhibitor molecule bound in a long, narrow channel leading to the molybdenum-pterin active site of the enzyme. It filled up most of the channel and the immediate environment of the cofactor, very effectively inhibiting the activity of the enzyme through the prevention of substrate binding. Although the inhibitor did not directly coordinate to the molybdenum ion, numerous hydrogen bonds as well as hydrophobic interactions with the protein matrix were observed, most of which are also used in substrate recognition.
There is substantial evidence that oxidative stress participates in the pathophysiology of cardiovascular disease. Biochemical, molecular and pharmacological studies further implicate xanthine oxidoreductase (XOR) as a source of reactive oxygen species in the cardiovascular system. XOR is a member of the molybdoenzyme family and is best known for its catalytic role in purine degradation, metabolizing hypoxanthine and xanthine to uric acid with concomitant generation of superoxide. Gene expression of XOR is regulated by oxygen tension, cytokines and glucocorticoids. XOR requires molybdopterin, iron-sulphur centres, and FAD as cofactors and has two interconvertible forms, xanthine oxidase and xanthine dehydrogenase, which transfer electrons from xanthine to oxygen and NAD(+), respectively, yielding superoxide, hydrogen peroxide and NADH. Additionally, XOR can generate superoxide via NADH oxidase activity and can produce nitric oxide via nitrate and nitrite reductase activities. While a role for XOR beyond purine metabolism was first suggested in ischaemia-reperfusion injury, there is growing awareness that it also participates in endothelial dysfunction, hypertension and heart failure. Importantly, the XOR inhibitors allopurinol and oxypurinol attenuate dysfunction caused by XOR in these disease states. Attention to the broader range of XOR bioactivity in the cardiovascular system has prompted initiation of several randomised clinical outcome trials, particularly for congestive heart failure. Here we review XOR gene structure and regulation, protein structure, enzymology, tissue distribution and pathophysiological role in cardiovascular disease with an emphasis on heart failure.
Hyperuricemia has been linked to cardiovascular and renal diseases, possibly through the generation of reactive oxygen species (ROS) and subsequent endothelial dysfunction. The enzymatic effect of xanthine oxidase is the production of ROS and uric acid. Studies have shown that inhibiting xanthine oxidase with allopurinol can reverse endothelial dysfunction. Furthermore, rat studies have shown that hyperuricemia-induced hypertension and vascular disease is at least partially reversed by the supplementation of the nitric oxide synthase (NOS) substrate, L-arginine. Therefore, we hypothesized that uric acid induces endothelial dysfunction by inhibiting nitric oxide production.
Hyperuricemia was induced in male Sprague-Dawley rats with an uricase inhibitor, oxonic acid, by gavage; control rats received vehicle. Allopurinol was placed in drinking water to block hyperuricemia. Rats were randomly divided into four groups: (1) control, (2) allopurinol only, (3) oxonic acid only, and (4) oxonic acid + allopurinol. Rats were sacrificed at 1 and 7 days, and their serum analyzed for serum uric acid and nitrites/nitrates concentrations. The effect of uric acid on nitric oxide production was also determined in bovine aortic endothelial cells.
Oxonic acid induced mild hyperuricemia at both 1 and 7 days (P < 0.05). Allopurinol reversed the hyperuricemia at 7 days (P < .001). Serum nitrites and nitrates (NO(X)) were reduced in hyperuricemic rats at both 1 and 7 days (P < .001). Allopurinol slightly reversed the decrease in NO(X) at 1 day and completely at 7 days (P < .001). There was a direct linear correlation between serum uric acid and NO(X) (R(2)= 0.56) and a trend toward higher systolic blood pressure in hyperuricemic rats (P= NS). Uric acid was also found to inhibit both basal and vascular endothelial growth factor (VEGF)-induced nitric oxide production in bovine aortic endothelial cells.
Hyperuricemic rats have a decrease in serum nitric oxide which is reversed by lowering uric acid levels. Soluble uric acid also impairs nitric oxide generation in cultured endothelial cells. Thus, hyperuricemia induces endothelial dysfunction; this may provide insight into a pathogenic mechanism by which uric acid may induce hypertension and vascular disease.
The prototypical xanthine oxidase (XO) inhibitor allopurinol, has been the cornerstone of the clinical management of gout and conditions associated with hyperuricemia for several decades. More recent data indicate that XO also plays an important role in various forms of ischemic and other types of tissue and vascular injuries, inflammatory diseases, and chronic heart failure. Allopurinol and its active metabolite oxypurinol showed considerable promise in the treatment of these conditions both in experimental animals and in small-scale human clinical trials. Although some of the beneficial effects of these compounds may be unrelated to the inhibition of the XO, the encouraging findings rekindled significant interest in the development of additional, novel series of XO inhibitors for various therapeutic indications. Here we present a critical overview of the effects of XO inhibitors in various pathophysiological conditions and also review the various emerging therapeutic strategies offered by this approach.
Structure-based drug design seeks to exploit the structure of protein-ligand or protein-protein binding sites, but the site is not always known at the outset. Even when the site is known, the researcher may wish to identify alternative prospective binding sites that may result in different biological effects or new class of compounds. It is also vital in lead optimization to clearly understand the degree to which known binders or docking hits satisfy or violate complementarity to the receptor. SiteMap is a new technique for identifying potential binding sites and for predicting their druggability in lead-discovery applications and for characterizing binding sites and critically assessing prospective ligands in lead-optimization applications. In large-scale validation tests, SiteMap correctly identifies the known binding site in > 96% of the cases, with best results (> 98%) coming for sites that bind ligands tightly. It also accurately distinguishes between sites that bind ligands and sites that don't. In binding-site analysis, SiteMap provides a wealth of quantitative and graphical information that can help guide efforts to modify ligand structure to enhance potency or improve physical properties. These attributes allow SiteMap to nicely complement techniques such as docking and computational lead optimization in structure-base drug design.