Uncompetitive Manner Research Articles

Exploration of morpholine-thiophene hybrid thiosemicarbazones for the treatment of ureolytic bacterial infections via targeting urease enzyme: Synthesis, biochemical screening and computational analysis

An important component of the pathogenicity of potentially pathogenic bacteria in humans is the urease enzyme. In order to avoid the detrimental impact of ureolytic bacterial infections, the inhibition of urease enzyme appears to be an appealing approach. Therefore, in the current study, morpholine-thiophene hybrid thiosemicarbazone derivatives (5a-i) were designed, synthesized and characterized through FTIR, 1H NMR, 13C NMR spectroscopy and mass spectrometry. A range of substituents including electron-rich, electron-deficient and inductively electron-withdrawing groups on the thiophene ring was successfully tolerated. The synthesized derivatives were evaluated in vitro for their potential to inhibit urease enzyme using the indophenol method. The majority of compounds were noticeably more potent than the conventional inhibitor, thiourea. The lead inhibitor, 2-(1-(5-chlorothiophen-2-yl)ethylidene)-N-(2-morpholinoethyl)hydrazinecarbothioamide (5g) inhibited the urease in an uncompetitive manner with an IC50 value of 3.80 ± 1.9 µM. The findings of the docking studies demonstrated that compound 5g has a strong affinity for the urease active site. Significant docking scores and efficient binding free energies were displayed by the lead inhibitor. Finally, the ADME properties of lead inhibitor (5g) suggested the druglikeness behavior with zero violation.

The critical role of a conserved lysine residue in periplasmic nitrate reductase catalyzed reactions.

Periplasmic nitrate reductase NapA from Campylobacter jejuni (C. jejuni) contains a molybdenum cofactor (Moco) and a 4Fe-4S cluster and catalyzes the reduction of nitrate to nitrite. The reducing equivalent required for the catalysis is transferred from NapC → NapB → NapA. The electron transfer from NapB to NapA occurs through the 4Fe-4S cluster in NapA. C. jejuni NapA has a conserved lysine (K79) between the Mo-cofactor and the 4Fe-4S cluster. K79 forms H-bonding interactions with the 4Fe-4S cluster and connects the latter with the Moco via an H-bonding network. Thus, it is conceivable that K79 could play an important role in the intramolecular electron transfer and the catalytic activity of NapA. In the present study, we show that the mutation of K79 to Ala leads to an almost complete loss of activity, suggesting its role in catalytic activity. The inhibition of C. jejuni NapA by cyanide, thiocyanate, and azide has also been investigated. The inhibition studies indicate that cyanide inhibits NapA in a non-competitive manner, while thiocyanate and azide inhibit NapA in an uncompetitive manner. Neither inhibition mechanism involves direct binding of the inhibitor to the Mo-center. These results have been discussed in the context of the loss of catalytic activity of NapA K79A variant and a possible anion binding site in NapA has been proposed.

Inhibitory activity of luteolin on α‐glucosidase and pancreatic lipase in vitro and in vivo

SummaryObesity is becoming a top global health problem. Inhibition of digestive enzymes is an effective approach in weight management due to the reduction of energy absorption. In this study, the inhibitory effects of luteolin on α‐glucosidase and pancreatic lipase were investigated in vitro and in vivo via enzyme kinetics, fluorescence quenching, molecular docking and rat experiments. The results showed that luteolin inhibited α‐glucosidase and pancreatic lipase in a mixed‐type and uncompetitive manner with IC50 values of 29.31 and 42.74 μg mL−1 respectively. The binding of luteolin with the two enzymes caused the quenching of intrinsic fluorescence of protein. Molecular docking simulation revealed that luteolin could be located in the active pocket of α‐glucosidase and interact with the key amino acid residues. Moreover, a hydrophobic interaction and a van der Waals force were formed between luteolin and the key amino acid residues of pancreatic lipase (His264 and Ser153). In vivo studies further confirmed that luteolin could significantly decrease the absorption of glucose and triacylglyceride in rats by inhibiting α‐glucosidase and pancreatic lipase.

Mosloflavone from Fissistigma petelotii ameliorates oncogenic multidrug resistance by STAT3 signaling modulation and P-glycoprotein blockade

BackgroundOncogenic multidrug resistance (MDR) is a tough question in cancer therapy. However, no effective medications targeting oncogenic MDR are currently available. Studies have demonstrated that mosloflavone exerts anti-inflammatory effects, yet, its potential to ameliorate MDR remains unclear. PurposeThis study aimed to access the capability and elucidate molecular mechanisms of mosloflavone as a MDR resensitizing candidate. MethodsWe investigated the ability of mosloflavone to reverse oncogenic MDR and investigated its underlying mechanisms through cytotoxicity assay, cell cycle assay, apoptosis assay, and zebrafish xenograft model. The modulatory interplay between mosloflavone and P-gp was investigated through analysis of calcein-AM uptake, substrate efflux, ATPase assays, and molecular docking simulation. ResultsMosloflavone inhibited P-gp efflux function in an uncompetitive manner without altering ABCB1 gene expression. In addition, it stimulated P-gp ATPase activity by binding to an active site distinct from that of verapamil. Regarding MDR reversal potential, mosloflavone resensitized MDR cancer cells to chemotherapies by arresting cell cycle and triggering apoptosis, possibly by enhancing reactive oxygen species accumulation and reducing phospho-STAT3. Moreover, in the zebrafish xenograft model, mosloflavone significantly potentiated the antitumor effect of paclitaxel. ConclusionOur findings underscore the potential of mosloflavone as a novel dual modulator of STAT3 and P-gp, indicating it is a promising candidate for overcoming MDR in cancer treatment.

Inhibition of human glutathione transferase by catechin and gossypol: comparative structural analysis by kinetic properties, molecular docking and their efficacy on the viability of human MCF-7 cells.

Glutathione transferase Pi (GSTP1) expression is increased in many cancer types and is associated with multidrug resistance and apoptosis inhibition. Inhibitors of GSTP1-1 have the potential to overcome drug resistance and improve chemotherapy efficacy as adjuvant agents. This study investigated the effects of catechin and gossypol on human glutathione transferase Pi (GSTP1-1) activity and their cytotoxic effects on breast cancer cells (MCF-7) individually and in combination with tamoxifen (TAM). Gossypol effectively inhibited the enzyme with an IC50 value of 40μM, compared to 200μM for catechin. Gossypol showed stronger inhibition of GSTP1-1 activity (Ki = 63.3 ± 17.5μM) compared to catechin (Ki = 220 ± 44μM). Molecular docking analysis revealed their binding conformations to GSTP1-1, with gossypol binding at the subunit interface in an un-competitive manner and catechin showing mixed non-competitive inhibition. Gossypol had severe cytotoxic effects on both MCF-7 cells and normal BJ1 cells, while catechin had a weak cytotoxic effect on MCF-7 cells only. Combination therapy with TAM resulted in cytotoxicity of 27.3% and 35.2% when combined with catechin and gossypol, respectively. Gossypol showed higher toxicity to MCF-7 cells, but its strong effects on normal cells raised concerns about selectivity and potential side effects.

Inhibition mechanisms of four ellagitannins from terminalia chebula fruits on acetylcholinesterase by inhibition kinetics, spectroscopy and molecular docking analyses

Acetylcholinesterase (AChE) is an important therapeutic target for the treatment of Alzheimer's disease (AD), and the development of natural AChE inhibitors as candidates has played a significant role in drug discovery. In this study, the inhibition mechanisms of four ellagitannins, punicalagin, chebulinic acid, geraniin and corilagin, from Terminalia chebula fruits on AChE were investigated systematically by a combination of inhibition kinetics, multi-spectroscopic methods and molecular docking. The kinetic results showed that punicalagin, chebulinic acid and geraniin exhibited strong reversible inhibitory effects on AChE in an uncompetitive manner with the IC50 values of 0.43, 0.50, and 0.51 mM, respectively, while corilagin inhibited AChE activity in a mixed type with the IC50 value of 0.72 mM. The results of fluorescence and UV–vis spectra and fluorescence resonance energy transfer (FRET) revealed that four ellagitannins could significantly quenched the intrinsic fluorescence of AChE though a static quenching along with non-radiative energy transfer. Thermodynamic analyses showed that values of ΔG, ΔH and ΔS were negative, indicating that all binding processes were spontaneous, and the hydrogen bonding and Van der Waals forces might make a great contribution to the formation of inhibitor-AChE complexes. The synchronous fluorescence, three-dimensional (3D) fluorescence, UV–vis, and FT-IR spectra studies suggested that four ellagitannins could lead to alterations in the micro-environment and secondary structure of AChE, and thus the conformational change of AChE. Moreover, molecular docking demonstrated that four ellagitannins could interacted with main amino acid residues of AChE with affinity energies ranging from −9.9 to −8.7 kJ/mol, and further confirmed the above experimental results. This study provided valuable findings for the potential application of four ellagitannins as promising candidates in the exploration of natural AChE inhibitors for the treatment of AD.

Mechanism of interaction between urolithin A and α-glucosidase: Analysis by inhibition kinetics, fluorescence spectroscopy, isothermal titration calorimetry and molecular docking

In this study, the mechanism of interaction between urolithin A, a gut metabolite of ellagitannins, and α-glucosidase was characterized by inhibition kinetics, fluorescence spectroscopy, isothermal titration calorimetry, and molecular docking. Urolithin A exhibited potential reversible inhibitory activity for α-glucosidase in an uncompetitive manner with an IC50 value of (28.03 ± 0.59) μM. The results of fluorescence titration and isothermal titration calorimetry analysis showed that urolithin A statically quenched the endogenous fluorescence of α-glucosidase, which was a spontaneous exothermic process, mainly driven by hydrogen bond and/or van der Waals force. Synchronous fluorescence spectroscopy revealed that urolithin A increased the polarity of tryptophan microenvironment and the hydrophobicity of tyrosine microenvironment. The combination of urolithin A with acarbose, a competitive α-glucosidase inhibitor, showed an additive inhibitory effect. Furthermore, molecular docking showed that urolithin A formed hydrogen bonds with key residues Arg269, Thr273, and His258, which are the sites outside the active center of α-glucosidase. These findings could highlight the value of ellagitannin rich diet as an antihyperglycemic food in the prevention of postprandial hyperglycemia, and indicate that urolithin A may be a promising lead compound of α-glucosidase inhibitor.

Kitul, a food plant with antidiabetic-like effects: Reduction of intracellular reactive species in glucose-stimulated RIN-5F pancreatic β-cells and mitigation of pro-inflammatory mediators in activated RAW 264.7 macrophages

Kitul (Caryota urens L.) inflorescences are broadly used for sweet sap production in Asian countries and Kitul food products are known as being suitable for diabetic patients. Considering the strong ability to inhibit α-glucosidase, we hypothesize that kitul antidiabetic properties might also involve the modulation of inflammatory pathways and hyperglycaemia-induced oxidative damage. Hence, the effects of an inflorescence’s methanol extract were investigated in glucose-stimulated pancreatic cells (RIN-5F) and LPS-stimulated RAW 264.7 macrophages. The extract reduced the overproduction of intracellular reactive species in pancreatic cells and also NO, L-citrulline and IL-6 levels in LPS-stimulated RAW 264.7 macrophages. Inhibition of 5-lipoxygenase (IC50 = 166.1 µg/mL) through an uncompetitive manner was also recorded upon treatment with C. urens inflorescences extract. The phenolic profile of the inflorescences was characterized by HPLC-DAD, six hydroxycinnamic acids being identified and quantified. Overall, our data provide additional evidence on the pleiotropic mechanisms of Kitul inflorescences as an antidiabetic agent.

Citrus peel waste essential oil: Chemical composition along with anti‐amylase and anti‐glucosidase potential

SummaryThe most common waste in citrus processing is their skin, which contains high amounts of essential oil. The antioxidant, anti‐glucosidase, and anti‐amylase properties of essential oils extracted from fresh bitter orange, lime, lemon, pomelo, grapefruit, mandarin, and sweet orange peels were examined. Linalool (18.26%–29.08%), limonene (17.08%–22.44%), linalool acetate (17.17%–30.47%), α‐terpineol (6.08%–11.06%), geraniol (2.05%–6.30%), β‐ocimene (1.52%–5.02%), nerolidol (2.93%–4.00%), β‐pinene (2.71%–3.29%), farnesol (2.08%–2.97%), and geranyl acetate (1.89%–2.80%) were found to be the primary components of citrus peel essential oil. Citrus peel essential oil displayed a good antioxidant capacity ranging from 375 to 643 mg Trolox equivalent/g. Citrus peel essential oils strongly inhibit amylase (520 to 738 mg acarbose equivalent/g) and glucosidase (475 to 780 mg acarbose equivalent/g). According to kinetic analysis, fluorescence quenching, and ultraviolet absorption, citrus peel essential oils inhibit amylase and glucosidase with a mixed competitive, non‐competitive, and uncompetitive manner. Some citrus varieties have long been used to treat diabetes and obesity. While citrus fruits are consumed worldwide, the citrus peel is typically discarded; hence, using the citrus peel in the culinary and medical industries might have a positive economic impact on managing diabetes.

Phloretin exhibits potential food-drug interactions by inhibiting human UDP-glucuronosyltransferases in vitro

Phloretin is a well-known apple polyphenol possessing a wide variety of biological effects and has been widely used in many fields. However, it's unclear whether phloretin has an effect on the activity of human UGT enzymes. Our study indicated that phloretin inhibited human UGTs on a broad spectrum. Further kinetic analysis revealed that phloretin inhibited UGT1A1, 1A6, 1A9, 2B7, and 2B15 in a noncompetitive manner, with calculated Ki of 8.34 μM, 16.69 μM, 10.58 μM, 17.74 μM and 2.46μΜ, respectively, whereas phloretin inhibited UGT1A7 in an un-competitive manner, with calculated Ki of 5.70 μM. According to the quantitative risk prediction, co-administration of phloretin with drugs primarily metabolized by UGT1A7 and/or UGT2B15 may result in potential food-drug interactions. To sum up, when phloretin or phloretin-rich food is administered with medications metabolized by UGT1A7 and/or UGT2B15, concern should be exercised.

Abstract 2135: Targeting N-Myc in neuroblastoma cells with selective Aurora kinase A degraders

Abstract Overexpression of the MYCN proto-oncogene is one of the strongest biomarkers for poor prognosis in neuroblastoma patients. N-Myc, encoded by MYCN, is a highly attractive yet “undruggable” target in neuroblastoma drug discovery. N-Myc is a short-lived protein in normal neuronal progenitor cells; however, in MYCN-amplified neuroblastoma cells, overexpressed N-Myc is stabilized by high levels of Aurora kinase A (Aurora-A). Several Aurora-A allosteric modulators have been developed to disassociate N-Myc binding and promote its degradation. These compounds function through a competitive mechanism against N-Myc, and clinical trial results suggest limited efficacy in high-risk neuroblastoma patients where N-Myc is highly expressed. To develop Aurora-A modulators with greater effects in destabilizing N-Myc, targeted degradation of Aurora-A was investigated in this study. First, we developed a highly selective Aurora-A-binding ligand by chemically modifying ribociclib, which targets CDK4/6. Next, a series of potent Aurora-A/N-Myc degraders were developed using proteolysis targeting chimaera (PROTAC) technology. Our lead compound, HLB-0532259, was found to substantially decrease N-Myc levels in neuroblastoma cells following the degradation of Aurora-A with nanomolar potency. Mechanistic studies suggest that HLB-0532259 binds to Aurora-A in an uncompetitive manner with respect to N-Myc, which contributes to its high potency for degrading the Aurora-A/N-Myc pool. Mass spectrometry analysis demonstrates that HLB-0532259 possesses remarkable proteome-wide selectivity for Aurora-A and N-Myc degradation. Further studies have revealed that HLB-0532259 exhibits nanomolar cytotoxicities against a panel of MYCN-amplified neuroblastoma cells, good pharmacokinetic properties, and favorable therapeutic outcomes in a xenograft neuroblastoma mouse model. Taken together, our data strongly support HLB-0532259 as a promising lead compound for neuroblastoma therapy. Citation Format: Jian Tang, Ramkumar Moorthy, Ozlem Demir, Zachary D. Baker, Jordan A. Naumann, Katherine F. Jones, Michael J. Grillo, Ella Haefner, Ke Shi, Michaella J. Levy, Hideki Aihara, Reuben S. Harris, Rommie E. Amaro, Nicholas M. Levinson, Daniel A. Harki. Targeting N-Myc in neuroblastoma cells with selective Aurora kinase A degraders [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2135.

Inhibitory mechanism of angiotensin-converting enzyme inhibitory peptides from black tea.

The aim of this work is to discover the inhibitory mechanism of tea peptides and to analyse the affinities between the peptides and the angiotensin-converting enzyme (ACE) as well as the stability of the complexes using in vitro and in silico methods. Four peptide sequences identified from tea, namely peptides I, II, III, and IV, were used to examine ACE inhibition and kinetics. The half maximal inhibitory concentration (IC50) values of the four peptides were (210.03±18.29), (178.91±5.18), (196.31±2.87), and (121.11±3.38) μmol/L, respectively. The results of Lineweaver-Burk plots showed that peptides I, II, and IV inhibited ACE activity in an uncompetitive manner, which requires the presence of substrate. Peptide III inhibited ACE in a non-competitive manner, for which the presence of substrate is not necessary. The docking simulations showed that the four peptides did not bind to the active sites of ACE, indicating that the four peptides are allosteric inhibitors. The binding free energies calculated from molecular dynamic (MD) simulation were -72.47, -42.20, -52.10, and -67.14 kcal/mol (1 kcal=4.186 kJ), respectively. The lower IC50 value of peptide IV may be attributed to its stability when docking with ACE and changes in the flexibility and unfolding of ACE. These four bioactive peptides with ACE inhibitory ability can be incorporated into novel functional ingredients of black tea.

Effect of Vernonia amygdalina on phospholipase activity from Naja Mossambica (Cobra)

Background: Phospholipases are one of the numerous enzymes found in the Naja mossambica venom. They play a major role in snakebite envenomation, and also responsible for the hydrolysis of a phospholipid, disrupting the membrane integrity. In this study, we evaluated the effect of Vernonia amygdalina on Phospholipase activity from Naja mossambica (Cobra) Results: Partially purified phospholipase had maximal velocity (Vmax) and Michaelis Menten constant (Km) of 7.6 × 10-5 mol/min and 1.7mg/ml, while the crude phospholipase had Vmax and Km of 9.4 × 10-5mol/min and 2.5mg/ml respectively. Inhibition study of aqueous extracts of Vernonia amygdalina leaf shows that the extract is a potent inhibitor of crude phospholipase in a dose-dependent pattern. The different doses of extract 15 %, 10 % and 5% produced percentage inhibition of 74.04 %, 78.6 % and 86.63% respectively. The kinetic binding constant (Ki) values of crude phospholipase for different concentrations of extracts 5%, 10% and 15% were 0.21mg/ml, 0.29mg/ml and 0.39mg/ml, while the partially purified phospholipase for different concentrations of extracts 5%, 10% and 15% were were 0.48mg/ml, 0.42mg/ml and 0.41mg/ml respectively. It can be deduced from the results that the extract inhibits the phospholipase activity in an uncompetitive manner. Conclusion: Aqueous extract of Vernonia amygdalina leaves may contain some bioactive agents that could serve as potent inhibitors of phospholipase from Naja mossambica venom.

Mechanistic inhibition of non-enzymatic glycation and aldose reductase activity by naringenin: Binding, enzyme kinetics and molecular docking analysis

The aldose reductase (AR) enzyme is considered a potential target for the management of diabetic complications. In this study, we describe the binding and enzyme kinetics of AR by naringenin, a bioflavonoid present in many dietary sources. Naringenin showed an inhibitory effect on the activity of AR with an IC50 value of 2.6 μM in an uncompetitive manner. Binding studies confirmed that the naringenin-AR complex has high spontaneous affinity (Ka = 1.94–7.88 × 104) with negative ΔG° value (−5.78 kcal mol−1). The interaction was enthalpy driven and the microenvironment of aromatic residues of AR was also altered. Various stages of protein oxidation and glycation were also measured. Naringenin inhibited fructosamine content by approximately 31.6% at 10 μM, and at the same concentration, >93% inhibition of fluorescent advanced glycation end-products (AGEs) was achieved. There was a significant recovery in free thiol groups and carbonyl content of bovine serum albumin (BSA). Furthermore, molecular docking of naringenin with AR revealed that naringenin formed two hydrogen bonds (Asn160 and Ile260), and three Pi-Pi interactions (two with Trp20 and one with His110). This study provides molecular insight of naringenin-AR interaction and mechanism of antiglycation which may be useful in the development of inhibitors for AGEs formation.

Beta-estradiol protects against copper-ascorbate induced oxidative damage in goat liver mitochondria in vitro by binding with ascorbic acid

Aimsβ-Estradiol (β-E), one of the chemical forms of female gonad hormone exhibited antioxidant efficacy in biochemical system, in vitro. The aim of the study was to investigate whether any other mechanism of protection by β-E to hepatic mitochondria in presence of stressor agent i.e.,a combination of Cu2+ and ascorbic acid is involved. Main methodsFreshly prepared goat liver mitochondria was incubated with stressors and 1 μM β-E and post incubated with the same concentration at 37 °C at pH 7.4. Mitochondrial viability, biomarkers of oxidative stress, activities of Krebs cycle enzymes, mitochondrial membrane potential, Ca2+ permeability were measured. Mitochondrial morphology and binding pattern of β-E with stressors were also studied. Key findingsUpon incubation of mitochondria with Cu, ascorbic acid and their combination there is a significant decline in activities of four of Krebs cycle enzymes in an uncompetitive manner with a concomitant increase in Ca2+ permeability and membrane potential of inner mitochondrial membrane, which is withdrawn during co-incubation with β-E, but was not reversed during post incubation with the β-E. The final studies on mitochondrial membrane morphology using scanning electron microscope also exhibited damage. Isothermal titration calorimetry data also showed the negative heat change in the mixture of β-E with ascorbic acid and also its combination with Cu2+. SignificanceOur results for the first time demonstrated that β-E protects againstCu2+-ascorbate induced oxidative stress by binding with ascorbic acid. The new mechanism of binding of β-E with stress agents may have a future therapeutic relevance.

Effects of D-Limonene on aldose reductase and protein glycation in diabetic rats

Diabetes mellitus and hyperglycemia can lead to other health complications. The pathogenesis of diabetes mellitus can affect numerous tissues in the eye structure and cataracts are one of the major causes of visual impairment in diabetes mellitus patients. In this study, we aim to investigate the ability of D-limonene to inhibit aldose reductase hypoglycemic activity in lens organ culture and its anti-glycating properties in STZ-induced cataracts of rats with diabetes. D-limonene repressed aldose reductase in-vitro with IC50 ranges of ~13.9 ± 0.31 µg/mL in an uncompetitive manner. This compound also prevented hyperglycemic induced increases in aldose reductase activity, accumulation of sorbitol, altered crystalline proteins (α, β and γ), and opacification of the rat lens in ex-vivo lens organ culture. Supplementation of D-limonene in STZ-induced diabetic rats restored these changes and delayed the progression of cataracts. This study demonstrates that D-limonene is a pharmaceutically active component that inhibits rat-lens aldose reductase. Therefore, this study showed that D-limonene prevents advanced glycation end product formation contributing to the integrity of α-crystallin chaperone activity and delays cataract development. It has been concluded, D-limonene has a role in inhibiting aldose reductase activity and advanced glycation end products formation in vivo, and in delaying the development of diabetic cataracts. Further studies of D-limonene are needed to assess other complications in DM as they may provide additional evidence for establishing D-limonene as a potential pharmacological target therapy for diabetes mellitus complications.

Inhibitory effect of corosolic acid on α-glucosidase: kinetics, interaction mechanism, and molecular simulation.

The suppression of α-glucosidase activity to retard glucose absorption is an important therapy for type-2 diabetes. Corosolic acid (CRA) is a potential antidiabetic component in many plant-based foods and herbs. In this study, the interplay mechanism between α-glucosidase and corosolic acid was investigated by several methods, including three-dimensional fluorescence spectra, circular dichroism spectra, and molecular simulation. Corosolic acid significantly inhibited α-glucosidase reversibly in an uncompetitive manner and its IC50 value was 1.35 × 10-5 mol L-1 . A combination of CRA with myricetin exerted a weak synergy against α-glucosidase. The intrinsic fluorescence of α-glucosidase was quenched via a static quenching course and the binding constant was 3.47 × 103 L mol-1 at 298 K. The binding of CRA to α-glucosidase was mainly driven by hydrophobic forces and resulted in a partial extension of the protein polypeptide chain with a loss of α-helix content. The molecular simulation illustrated that CRA bound to the entrance part of the active center of α-glucosidase and interacted with the amino acid residues Ser157, Arg442, Phe303, Arg315, Tyr158, and Gln353, which could hinder the release of substrate and catalytic reaction product, eventually suppressing the catalytic activity of α-glucosidase. These results may suggest new insights into corosolic acid from food sources as a potential α-glucosidase inhibitor that could better control diabetes. © 2019 Society of Chemical Industry.

Dual actions of norathyriol as a new candidate hypouricaemic agent: uricosuric effects and xanthine oxidase inhibition

Hyperuricaemia, which results from the overproduction and underexcretion of uric acid, has been linked with chronic renal dysfunction, cardiovascular diseases, diabetes and metabolic syndrome. However, available clinical drugs cannot simultaneously target the production and excretion of uric acid. Norathyriol, a natural xanthone, was expected to have the dual actions. We previously reported that norathyriol possessed potent anti-hyperuricaemic activity related to the inhibition of uric acid production. Here, we investigated the uricosuric actions in hyperuricaemic animals and explored the possible molecular mechanisms associated with renal urate transporters and xanthine oxidase (XO). The results showed that norathyriol (0.5–4.0 mg/kg) dose- and time-dependently decreased serum urate levels in uric acid-induced hyperuricaemic mice and markedly increased the fractional excretion of urate in oxonate-induced hyperuricaemic rats, demonstrating a promotion of urate excretion in the kidney. Further evidence showed that norathyriol markedly increased renal mRNA and protein expression of the secretory organic anion transporter 1 (OAT1) in hyperuricaemic mice and strengthened its transport function in vitro. Moreover, norathyriol also upregulated the mRNA expression of the secretory transporters OAT3, ATP-binding cassette transporter G2 and multidrug resistance protein 4, but not their protein expression. Changes in the expression of the reabsorptive transporters were not observed. This is the first report that norathyriol has uricosuric effects by targeting OAT1. Moreover, norathyriol significantly inhibited XO activity in an uncompetitive manner. Taken together, these findings suggest that norathyriol has the potential to be developed as a new anti-hyperuricaemic agent with dual actions that activate OAT1 and inhibit XO activity.

First Report of Plant-Derived β-Sitosterol with Antithrombotic, in Vivo Anticoagulant, and Thrombus-Preventing Activities in a Mouse Model.

Inhibitors of thrombin, a key enzyme in the blood coagulation cascade, are of great interest because of their selective specificity and effectiveness in anticoagulation therapy against cardiovascular disorders. The natural soybean phytosterol, β-sitosterol (BSS) demonstrated anticoagulant activity by dose-dependent inhibition of thrombin in an uncompetitive manner with a Ki value of 0.267 μM as well as by partial inhibition of thrombin-catalyzed platelet aggregation with a half-maximal inhibitory concentration (IC50) value of 10.45 ± 2.88 μM against platelet-rich plasma and 9.2 ± 1.2 μM against washed platelets. An in silico study indicated binding of BSS to thrombin, which was experimentally verified by spectrofluorometric and isothermal calorimetric analyses. Under in vitro conditions, BSS demonstrated thrombolytic activity by activating plasminogen, albeit it is devoid of protease (fibrinogenolytic) activity. BSS was noncytotoxic to mammalian cells, nonhemolytic, demonstrated its in vivo anticoagulant activity when administered orally, and inhibited k-carrageen-induced thrombus formation in the tails of mice. Our results suggest that dietary supplementation of BSS may help toprevent thrombosis-associated cardiovascular disorders.

NO-sGC-cGMP signaling influence the anxiolytic like effect of lithium in mice in light and dark box and elevated plus maze

Glutamate is an excitatory neurotransmitter implicated in the pathogenesis of psychiatric disorders. Glutamate results in the activation of an enzyme called glycogen synthase kinase-3 (GSK-3) acting through N-methyl-d-aspartate (NMDA) receptors. Impaired expression of GSK-3 affects behavior and neurochemicals level in the brain responsible for the pathogenesis of mood disorders. It has been reported that lithium acts as an inhibitor of GSK-3 and inhibit the enzyme GSK-3 in an uncompetitive manner. In the present study, anxiolytic like effect of lithium in mice is investigated through light and dark box (LDB) and elevated plus maze (EPM). Lithium (50, 100 and 200 mg/kg, i.p.) was administered to the mice to determine the anxiety related behavior. Results obtained suggests that the administration of lithium (100 mg/kg, i.p.) reversed the anxiety related behavior of mice and decreased the levels of glutamate and nitrite as compared to control. Glutamate acting through the NMDA receptor has been found to regulate the expression of enzyme neuronal nitric oxide synthase (nNOS), which is responsible for the release of nitric oxide (NO), suggesting a possible link between NO and GSK-3 also. Therefore, to determine the possible interaction with NO, sub-effective dose of lithium was administered in combination with NO donor i.e. l-Arginine (50 mg/kg, i.p.), NOS and soluble guanylate cyclase (sGC) inhibitor i.e. methylene blue (1 mg/kg, i.p.) and phosphodiesterase inhibitor i.e. sildenafil (1 mg/kg, i.p.). The results obtained demonstrated that the anxiolytic like effect of lithium was abolished by the pretreatment with NO donor and potentiated by the pretreatment with NOS inhibitor. Therefore, it is suggested that NO signaling pathway influence the anxiolytic like activity of lithium in mice, further suggesting the link between the GSK-3 and NO signaling in the regulation of anxiety related behavior.