Effects of bepridil on early cardiac development of zebrafish.

Bepridil is a commonly used medication for arrhythmia and heart failure. It primarily exerts hemodynamic effects by inhibiting Na+/K+ movement and regulating the Na+/Ca2+ exchange. In comparison to other Ca2+ inhibitors, bepridil has a long half-life and a complex pharmacology. Additionally, it is widely used in antiviral research and the treatment of various diseases. However, the toxicity of this compound and its other possible effects on embryonic development are unknown. In this study, we investigated the toxicity of bepridil on rat myocardial H9c2 cells. After treatment with bepridil, the cells became overloaded with Ca2+ and entered a state of cytoplasmic vacuolization and nuclear abnormality. Bepridil treatment resulted in several morphological abnormalities in zebrafish embryo models, including pericardium enlargement, yolk sac swelling, and growth stunting. The hemodynamic effects on fetal development resulted in abnormal cardiovascular circulation and myocardial weakness. After inhibiting the Ca2+ transmembrane, the liver of zebrafish larvae also displayed an ectopic and deficient spatial location. Additionally, the results of the RNA-seq analysis revealed the detailed gene expression profiles and metabolic responses to bepridil treatment in zebrafish embryonic development. Taken together, our study provides an important evaluation of antiarrhythmic agents for clinical use in prenatal heart patients.

Electropharmacological profile of an atrial-selective sodium channel blocker acehytisine assessed in the isoflurane-anesthetized guinea-pig model.

Experimental evidence regarding the risk of proarrhythmic potential of acehytisine is limited. We assessed its electropharmacological effect together with proarrhythmic potential at intravenous doses of 4 and 10 mg/kg (n = 6) using isoflurane-anesthetized guinea pigs in comparison with that of bepridil at 1 and 3 mg/kg, intravenously (n = 6). Acehytisine at therapeutic dose (4 mg/kg) decreased the heart rate, prolonged P wave duration, QRS width, QT interval, QTc, MAP90(sinus), MAP90(CL300) and MAP90(CL250). At supratherapeutic dose (10 mg/kg), it prolonged the PR interval besides enhancing the changes induced by the therapeutic dose. Quantitative assessment showed that peak changes in P wave duration by acehytisine at 10 mg/kg were 1.7 times longer than bepridil, and in MAP90(sinus), MAP90(CL300) and MAP90(CL250) by acehytisine were 1.9, 1.5 and 1.5 times shorter than bepridil, respectively. Importantly, qualitative assessment indicated that bepridil increased beat-to-beat variability and J-Tpeakc in a dose-related manner, confirming a higher proarrhythmic risk, whereas such dose-related responses were not observed in acehytisine, suggesting a lower proarrhythmic risk. These results suggest that acehytisine exhibits favorable pharmacological characters, i.e. potent atrial inhibition and lower proarrhythmic toxicity compared with bepridil, being a promising candidate for the treatment of paroxysmal supraventricular tachycardia.

Surface plasmon resonance and cytotoxicity assays of drug efficacies predicted computationally to inhibit p53/MDM2 interaction.

Docking on the p53-binding site of murine double minute 2 (MDM2) by small molecules restores p53's tumor-suppressor function. We previously assessed 3244 FDA-approved drugs via "computational conformer selection" for inhibiting MDM2 and p53 interaction. Here, we developed a surface plasmon resonance method to experimentally confirm the inhibitory effects of the known MDM2 inhibitor, nutlin-3a, and two drug candidates predicted by our computational method. This p53/MDM2 interaction displayed a dosage-dependent weakening when MDM2 is pre-mixed with drug candidates. The inhibition efficiency order is nutlin-3a (IC50 = 97 nM) > bepridil (206 nM) > azelastine (307 nM). Furthermore, we verified their anti-proliferation effects on SJSA-1 (wild-type p53 and overexpressed MDM2), SW480 (mutated p53), and SaOs-2 (deleted p53) cancer cell lines. The inhibitory order towards SJSA-1 cell line is nutlin-3a (IC50 = 0.8 µM) > bepridil (23 µM) > azelastine (25 µM). Our experimental results are in line with the computational prediction, and the higher IC50 values from the cell-based assays are due to the requirement of higher drug concentrations to penetrate cell membranes. The anti-proliferation effects of bepridil and azelastine on the cell lines with mutated and deleted p53 implied some p53-independent anti-proliferation effects.

Fluorescence Based Characterization of Calcium Sensitizer Action on the Troponin Complex.

Calcium sensitizers enhance the transduction of the Ca(2+) signal into force within the heart and have found use in treating heart failure. However the mechanisms of action for most Ca(2+) sensitizers remain unclear. To address this issue an efficient fluorescence based approach to Ca(2+) sensitizer screening was developed which monitors cardiac troponin C's (cTnC's) hydrophobic cleft. This approach was tested on four common Ca(2+) -sensitizers, EMD 57033, levosimendan, bepridil and pimobendan with the aim of elucidating the mechanisms of action for each as well as proving the efficacy of the new screening method. Ca(2+) -titration experiments were employed to determine the effect on Ca(2+) sensitivity and cooperativity of cTnC opening, while stopped flow experiments were used to investigate the impact on cTnC relaxation kinetics. Bepridil was shown to increase the sensitivity of cTnC for Ca(2+) under all reconstitution conditions, sensitization by the other drugs was context dependent. Levosimendan and pimobendan reduced the rate of cTnC closing consistent with a stabilization of cTnC's open conformation while bepridil increased the rate of relaxation. Experiments were also run on samples containing cTnT(T204E), a known Ca(2+) -desensitizing phosphorylation mimic. Levosimendan, bepridil, and pimobendan were found to elevate the Ca(2+) -sensitivity of cTnT(T204E) containing samples in this context.

Versatile cardiac troponin chimera for muscle protein structural biology and drug discovery.

Investigation of the molecular interactions within and between subunits of the heterotrimeric troponin complex, and with other proteins in the sarcomere, has revealed salient structural elements involved in regulation of muscle contraction. The discovery of new cardiotonic drugs and structural studies utilizing intact troponin, or regulatory complexes formed between the key regions identified in troponin C and troponin I, face intrinsic and technical difficulties associated with weak protein-protein interactions and with solubility, aggregation, stability of the overall architecture, isotope labeling, and size, respectively. We have designed and characterized a chimeric troponin C-troponin I hybrid protein with a cleavable linker that is useful for producing isotopically labeled troponin peptides, stabilizes their interaction, and has proven to be a faithful representation of the original complex in the systolic state, but lacking its disadvantages, making it particularly suitable for drug screening and structural studies.

Role of the activation gate in determining the extracellular potassium dependency of block of HERG by trapped drugs.

Drug induced long QT syndrome (diLQTS) results primarily from block of the cardiac potassium channel HERG (human-ether-a-go-go related gene). In some cases long QT syndrome can result in the lethal arrhythmia torsade de pointes, an arrhythmia characterized by a rapid heart rate and severely compromised cardiac output. Many patients requiring medication present with serum potassium abnormalities due to a variety of conditions including gastrointestinal dysfunction, renal and endocrine disorders, diuretic use, and aging. Extracellular potassium influences HERG channel inactivation and can alter block of HERG by some drugs. However, block of HERG by a number of drugs is not sensitive to extracellular potassium. In this study, we show that block of WT HERG by bepridil and terfenadine, two drugs previously shown to be trapped inside the HERG channel after the channel closes, is insensitive to extracellular potassium over the range of 0 mM to 20 mM. We also show that bepridil block of the HERG mutant D540K, a mutant channel that is unable to trap drugs, is dependent on extracellular potassium, correlates with the permeant ion, and is independent of HERG inactivation. These results suggest that the lack of extracellular potassium dependency of block of HERG by some drugs may in part be related to the ability of these drugs to be trapped inside the channel after the channel closes.

A computational and experimental approach to investigate bepridil binding with cardiac troponin.

Cardiac troponin is a Ca(2+)-dependent switch for the contraction in heart muscle and a potential target for drugs in the therapy of heart failure. Bepridil is a drug that binds to troponin and increases calcium sensitivity of muscle contraction. Because bepridil has been well studied, it is a good model for analysis by computational and experimental methods. Molecular dynamics (MD) simulations were performed on troponin complexes of different sizes in the presence and absence of bepridil bound within the hydrophobic pocket at the N-terminal domain of troponin C. About 100 ns of simulation trajectory data were generated, which were analyzed using cross-correlation analyses and MMPBSA and MMGBSA techniques. The results indicated that bepridil binding within the hydrophobic pocket of cardiac TnC decreases the interaction of TnC with TnI at both the N-domain of TnC and the C-domain of TnC, and decreases the correlations of motions among the segments of the troponin subunits. The estimated calcium-binding affinities using MMPBSA showed that bepridil has a sensitizing effect for the isolated system of TnC, but loses this effect for the complex. Our experimental measurements of calcium dissociation rates were consistent with that prediction. We also observed that while bepridil enhanced the troponin-tropomyosin-actin-activated ATPase activity of myosin S1 at low calcium concentrations it was slightly inhibitory at high calcium concentrations. Bepridil increases the ATPase activity and force generation in muscle fibers, but its effects appear to depend on the concentration of calcium.

Possible role of calcium ions, calcium channels and calmodulin in excystation and metacystic development of Entamoeba invadens.

The effect of calcium ions (Ca(2+)) and calmodulin (CaM) on the excystation and metacystic development of Entamoeba invadens was examined by transfer of cysts to a growth medium containing calcium antagonists and CaM inhibitors. Excystation, which was assessed by counting the number of metacystic amoebae after induction of excystation, was inhibited by the calcium chelators ethyleneglycol bis (beta-aminoethyl ether)- N,N'-tetraacetate (EGTA) and ethylene-diaminetetraacetate (EDTA), with EDTA being more potent than EGTA. The inhibitory effect of higher concentrations of these chelators on excystation was associated with reduced viability of cysts. Metacystic development, when determined by the number of nuclei in an amoeba, was delayed by EGTA, because the percentage of four-nucleate amoebae was higher than in controls at day 3 of incubation. EDTA made metacystic development unusual by producing a large number of metacystic amoebae with more than ten nuclei. The inhibition of excystation by these chelators was partially abrogated by their removal. A putative antagonist of intracellular calcium flux, 8-( N,N-diethylamino) octyl-3,4,5-trimethoxybenzoate (TMB-8) also inhibited the excystation and metacystic development, but had little effect on cyst viability. The slow Na(+)-Ca(2+) channel blocker bepridil but not verapamil inhibited the excystation and metacystic development, associating with reduced cyst viability at higher concentrations. The inhibitory effect of bepridil on excystation was abrogated by removal of the drug. The CaM inhibitor trifIuoperazine (TFP) but not W-7 [ N-(6-aminohexyl)-chloro-1-naphtalene sulphonamide] inhibited the excystation and metacystic development. The inhibitory effect of TFP on excystation was also abrogated by removal of the drug. These results indicate that extracellular calcium ions, amoebic intracellular calcium flux, calcium channels, and a CaM-dependent process contribute to the excystation and metacystic development of E. invadens.

Antioxidants from the bark of Burkea africana, an African medicinal plant.

The bark of the tree Burkea africana is used medicinally in large areas of sub-Saharan Africa. The constituents responsible for its putative activity are not well known. We have investigated the bark of B. africana for antioxidant and radical scavenging activity. A hydroethanol bark extract showed high activity, and most of this activity was located in semipolar fractions of the extract. From chromatographic purification and spectroscopical structure studies, we conclude that the active constituents are proanthocyanidins. Two major components appear to be fisetinidol-(4alpha- --> 8)-catechin 3-gallate and bis-fisetinidol-(4alpha- --> 6, 4alpha- --> 8)-catechin 3-gallate. The latter compound is a new natural product. Smaller amounts of monomeric flavan-3-ols (catechin, epicatechin and fisetinidol) were also found.

Antioxidant effects of 1,5-anhydro-D-fructose, a new natural sugar, in vitro.

The antioxidant effects of 1,5-anhydro-D-fructose (1,5-AF), a unique anhydrohexulose, were studied in 1,1-diphenyl-2-picrylhydrazyl (DPPH) solution, in human cells along with lipid peroxidation of low-density lipoprotein (LDL). We have confirmed that 1,5-AF scavenges DPPH radicals directly in solution and inhibits the formation of hydrogen peroxide and superoxide anion, typical reactive oxygen species (ROS), induced by phorbol myristate acetate (PMA) in a dose-dependent manner in THP-1 cells. We also observed the dose-dependent antioxidant effects of 1,5-AF on copper-mediated LDL oxidation. These findings suggest that 1,5-AF might play a role in reducing the risk of atherosclerosis and may help prevent coronary heart disease.

Antioxidant capacities of ten edible North American plants.

The EtOAc extract obtained from ten edible North American plants, Acorus calamus, Clintonia borealis, Gaultheria shallon, Juniperus osteosperma, Opuntia polyacantha, Prunus americana, Prunus virginiana, Sambucus cerulea, Sorbus americana and Vaccinium parvifolium, were tested in the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical assay. High antioxidant activity was obtained from the extracts of three fruits, Gaultheria shallon, Sambucus cerulea and Prunus americana and one extracted rhizome, Acorus calamus. Catechin and epicatechin, potent polyphenolic antioxidants, were identified in the EtOAc extracts of Gaultheria shallon and Sambucus cerulea by reversed-phase thin-layer chromatography (TLC) and reversed-phase high-performance liquid chromatography (HPLC).

Antioxidant enzymes and DPPH-radical scavenging activity in chilled and heat-shocked rice (Oryza sativa L.) seedlings radicles.

Chilling whole rice seedlings at 5 degrees C significantly increased the time needed to recover linear growth and reduced the subsequent linear rate of radicle growth. Subjecting nonchilled seedlings to a 45 degrees C heat shock for up to 20 min did not alter subsequent growth, whereas a 3 min heat shock was optimal in reducing growth inhibition caused by 2 days of chilling. The activity of five antioxidant enzymes [superoxide dismutase (EC 1.15.1.1), catalase (CAT; EC 1.11.1.6), ascorbate peroxidase (APX; EC 1.11.1.11), glutathione reductase (GR; EC 1.6.4.2), and guaiacol peroxidase (GPX; EC 1.11.1.7)] and DPPH (1,1-diphenyl-2-picrylhydrazyl)-radical scavenging activity were measured in heat-shocked and/or chilled radicles. Heat shock slightly increased the activity of CAT, APX, and GR and suppressed the increase of GR and GPX activity during recovery from chilling. Increased CAT, APX, GR, and DPPH-radical scavenging activity and protection of CAT activity during chilling appear to be correlated with heat shock-induced chilling tolerance.

Interaction of bepridil with the cardiac troponin C/troponin I complex.

We have investigated the binding of bepridil to calcium-saturated cardiac troponin C in a cardiac troponin C/troponin I complex. Nuclear magnetic resonance spectroscopy and [(15)N,(2)H]cardiac troponin C permitted the mapping of bepridil-induced amide proton chemical shifts. A single bepridil-binding site in the regulatory domain was found with an affinity constant of approximately 140 microM(-1). In the presence of cardiac troponin I, bepridil binding to the C domain of cardiac troponin C was not detected. The pattern of bepridil-induced chemical shifts is consistent with stabilization of more open regulatory domain conformational states. A similar pattern of chemical shift perturbations was observed for interaction of the troponin I cardiac-specific amino-terminus with the cardiac troponin C regulatory domain. These results suggest that both bepridil and the cardiac-specific amino-terminus may mediate an increase in calcium affinity by interacting with and stabilizing open regulatory domain conformations. Chemical shift mapping suggests a possible role for inactive calcium-binding site I in the modulation of calcium affinity.

Assessment of antioxidant activity of some antileprotic drugs.

DPPH (alpha,alpha-diphenyl-beta-picryl hydrazyl) (CAS 217-591-8), a stable free radical can be used to determine the antioxidant activity (AOA) of some drugs. In the present study the DPPH method was used for the first time to test AOA of dapsone (CAS 80-08-0), clofazimine (CAS 2030-63-9) and rifampicin (CAS 13282-42-1) in vitro and deproteinated blood method. Ascorbic acid (CAS 50-81-7) was used as a control in the study, which showed concentration-dependent antioxidant activity. Rifampicin showed a per se effect but it showed concentration dependent decrease in the DPPH absorbance. Ascorbic acid, dapsone and rifampicin showed DPPH scavenging activity both in vitro and deproteinated blood method. Clofazimine did not have any influence on DPPH. This method may be extended to different drugs for testing their AOA in biological fluids.

Antiradical and antilipoperoxidative effects of some plant extracts used by Sri Lankan traditional medical practitioners for cardioprotection.

Reactive oxygen species (ROS) are implicated in many pathogenic processes including the cardiovascular system. Detoxification of ROS by antioxidants (AO) therefore affords protection against such diseases. There is a growing body of evidence suggesting that antioxidants contribute to cardioprotection. Therefore, nine plants that are components of Ayurvedic formulations used for the therapy of cardiovascular diseases were investigated to determine whether antioxidant activity is one of the mechanisms by which these plants exert cardioprotection. Initially aqueous freeze dried extracts of the plants were prepared and the antioxidant activity was measured (a) in vitro, by DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging and deoxyribose damage protection assays, and (b) in vivo, by effects on lipid peroxidation. Terminalia arjuna showed significant DPPH radical scavenging activity with EC(50) 8.3 +/- 0.3 microg/mL (similar to L-ascorbic acid). The potency of this activity was much lower in Cassia fistula (EC(50) = 59.0 +/- 2.7 microg/mL). The other seven extracts demonstrated no such activity in the concentration range tested. In the deoxyribose damage protection assay, T. arjuna> demonstrated no significant effect in the concentration range 0-20 microg/mL, but above -20 microg/mL concentration (20-125 microg/mL), a pro-oxidant activity was observed (although markedly less than demonstrated by L-ascorbic acid). A similar trend was observed with Vitex negundo. In contrast, C. fistula afforded a 30% protection against such damage at 125 microg/mL concentration. Other plant extracts did not show any activity in this assay. At a dose of 90 mg/kg (single dose) T. arjuna, cardiac lipid peroxidation in male Wistar rats was reduced by 38.8% +/- 2.6% (p<0.05) whereas the reduction was only 11.6% +/- 3.5% in the case of C. fistula even at a dose of 120 mg/kg. Of all the plants tested, T. arjuna demonstrated the highest antioxidant activity. Overall results show that only some plants used in the therapy of cardiovascular disease exert their beneficial effects via antioxidant activity.

1,1-Diphenyl-2-picrylhydrazyl radical-scavenging active compounds from greater cardamom (Amomum subulatum Roxb.).

Constituents of the fruits of greater cardamom (Amomum subulatum) were fractionated into three fractions, the dichloromethane extract, and the ethyl acetate-soluble and water-soluble fractions of the 70% aqueous acetone extract. The ethyl acetate-soluble fraction showed a high radical-scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH). Four compounds were isolated from the ethyl acetate-soluble fraction, and their structures were ascribed to protocatechualdehyde (1), protocatechuic acid (2), 1,7-bis(3,4-dihydroxyphenyl)hepta-4E,6E-dien-3-one (3) and 2,3,7-trihydroxy-5-(3,4-dihydroxy-E-styryl)-6,7,8,9-tetrahydro-5H-benzocycloheptene (4) on the basis of spectroscopic evidence. This is the first isolation of these compounds from greater cardamom. In particular, 4 was a new type of cyclic diarylheptanoid. DPPH radical-scavenging activity of these compounds was measured by colorimetric analysis. Compounds 1 and 3 showed stronger activity than such natural antioxidants as alpha-tocopherol and L-ascorbic acid. Compounds 2 and 4 were comparable to alpha-tocopherol and L-ascorbic acid.

Antioxidant activity of 5-alkoxymethyl-6-chromanols.

The 5-alkoxymethyl-2,2,7,8-tetramethyl-6-chromanols (II) are excellent antioxidants against autoxidising safflower oil (ASO), although not as good as 2,2,5,7,8-pentamethyl-6-chromanol (I), the model compound of alpha-tocopherol. The aim of this work was to determine whether the rate of reaction of (II) with the radicals diphenylpicrylhydrazyl (DPP*) and galvinoxyl (ArO*) was directly proportional to their antioxidant activity against ASO. Compounds (II) reacted faster with DPP* than with ArO* but, in each case, slower than compound (I). The rates of reaction of I and II with both radicals followed the order I > II (R = H) > II (R = CH3) > II (R = other alkyls) and were directly proportional to their antioxidant activity against ASO.

Cyclooxygenase-inhibitory and antioxidant constituents of the aerial parts of Antirhea acutata.

Two new compounds, (6S)-hydroxy-29-nor-3,4-seco-cycloart-4(30),24-dien-3-oic acid (1) and 8-[1-(3,4-dihydroxyphenyl)-3-methoxy-3-oxopropyl]epicatechin (3), were isolated by bioassay-guided fractionation from the aerial parts of Antirhea acutata (DC.) Urb. (Rubiaceae). Compound 1 showed moderate inhibitory activities in cyclooxygenase-1 and -2 assays (IC(50) 43.7 and 4.7 microM, respectively), while compound 3 was active in 1,1-diphenyl-2-picrylhydrazyl free-radical and cytochrome c reduction antioxidant assays (IC(50) 29.1 and 16.3 microM, respectively). Additionally, one further new compound was isolated, (3S,24S)-25-trihydroxy-9,19-cycloartane-29-oic acid (2), but this was inactive in the bioassay systems used. Compound 1 is based on the unprecedented 29-nor-3,4-seco-cycloartane skeleton.

In vitro evaluation of the antioxidant activity and biomembrane interaction of the lazaroid U-74389G.

The aim of this paper was to clarify whether the interaction of the lazaroid U-74389G with phospholipid membranes might be relevant as to its antioxidant activity. Thus we evaluated the "in vitro" antioxidant activity of U-74389G in two experimental models: 1) bleaching of the stable 1,1-diphenyl-2-picrylhydrazyl radical; 2) peroxidation, induced by the water-soluble radical initiator 2,2'-azobis(2-amidino-propane) hydrochloride, on mixed dipalmitoyl-phosphatidylcholine/linoleic acid unilamellar vesicles. Moreover, given that biophysical techniques may help in explaining the role of a drug in its interaction with the microenvironment of the model lipid membranes, we used a classical approach to investigate the U-74389G/model membrane interaction: the differential scanning calorimetry technique on dimyristoylphosphatidylcholine multilamellar and unilamellar vesicles and the Langmuir-Blodgett technique on dimyristoylphosphatidylcholine monolayers. The results evidenced the strong antioxidant activity of U-74389G (especially in a membranous system) and its capability to interact with and be transported across model membranes. Thus one can speculate that U-74389G can act as scavenger of chain-propagating lipid peroxyl radicals within the membranes and may be able to protect not only cell membranes, but also intracellular components against peroxidative attack. Furthermore, also if there is no certain proof that the effect on the lipid packing order may play a key role in its antioxidant activity, the fluidifying effect on phospholipid bilayers of U-74389G favourably complements its free radical scavenging characteristics.

Broussochalcone A, a potent antioxidant and effective suppressor of inducible nitric oxide synthase in lipopolysaccharide-activated macrophages.

The antioxidant properties of broussochalcone A (BCA) and its effects on nitric oxide (NO) production in lipopolysaccharide (LPS)-activated macrophages were investigated in this study. BCA, isolated from Broussonetia papyrifera Vent., inhibited iron-induced lipid peroxidation in rat brain homogenate in a concentration-dependent manner with an IC(50) of 0.63 +/- 0.03 microM. It was as potent as butylated hydroxytoluene, a common antioxidant used for food preservation. In a diphenyl-2-picrylhydrazyl assay system, the radical-scavenging activity of BCA seemed to be more potent than that of alpha-tocopherol, its IC(0.200) being 7.6 +/- 0.8 microM. BCA could directly scavenge superoxide anion and hydroxyl radicals. These results indicated that BCA was a powerful antioxidant with versatile free radical-scavenging activity. On the other hand, we found that BCA suppressed NO production concentration-dependently, with an IC(50) of 11.3 microM in LPS-activated macrophages. This effect was not the consequence of a direct inhibitory action on the enzyme activity of inducible NO synthase (iNOS). Our results indicated that BCA exerts potent inhibitory effects on NO production, apparently mediated by its suppression of IkappaBalpha phosphorylation, IkappaBalpha degradation, nuclear factor-kappa B activation, and iNOS expression. Therefore, we conclude that the antioxidant activities of BCA and its inhibition of IkappaBalpha degradation and iNOS protein expression may have therapeutic potential, given that excessive free radicals and NO production have been associated with various inflammatory diseases.