Negative Cooperativity in NAD(P)H Quinone Oxidoreductase 1 (NQO1).

NAD(P)H quinone oxidoreductase-1 (NQO1) is a homodimeric protein that acts as a detoxifying enzyme or as a chaperone protein. Dicourmarol interacts with NQO1 at the NAD(P)H binding site and can both inhibit enzyme activity and modulate the interaction of NQO1 with other proteins. We show that the binding of dicoumarol and related compounds to NQO1 generates negative cooperativity between the monomers. This does not occur in the presence of the reducing cofactor, NAD(P)H, alone. Alteration of Gly150 (but not Gly149 or Gly174) abolished the dicoumarol-induced negative cooperativity. Analysis of the dynamics of NQO1 with the Gaussian network model indicates a high degree of collective motion by monomers and domains within NQO1. Ligand binding is predicted to alter NQO1 dynamics both proximal to the ligand binding site and remotely, close to the second binding site. Thus, drug-induced modulation of protein motion might contribute to the biological effects of putative inhibitors of NQO1.

A far-red fluorescent probe for sensing laccase in fungi and its application in developing an effective biocatalyst for the biosynthesis of antituberculous dicoumarin.

A far-red fluorescent probe has been developed for sensing fungal laccase. The probe was used to determine that Rhizopus oryzae had a high level endogenous laccase amongst 24 fungal strains. The Rhizopus oryzae was then used as a biocatalyst for the preparation of dicoumarin resulting in significant inhibition of Mycobacterium tuberculosis H37Ra.

Human liver-kidney model elucidates the mechanisms of aristolochic acid nephrotoxicity.

Environmental exposures pose a significant threat to human health. However, it is often difficult to study toxicological mechanisms in human subjects due to ethical concerns. Plant-derived aristolochic acids are among the most potent nephrotoxins and carcinogens discovered to date, yet the mechanism of bioactivation in humans remains poorly understood. Microphysiological systems (organs-on-chips) provide an approach to examining the complex, species-specific toxicological effects of pharmaceutical and environmental chemicals using human cells. We microfluidically linked a kidney-on-a-chip with a liver-on-a-chip to determine the mechanisms of bioactivation and transport of aristolochic acid I (AA-I), an established nephrotoxin and human carcinogen. We demonstrate that human hepatocyte-specific metabolism of AA-I substantially increases its cytotoxicity toward human kidney proximal tubular epithelial cells, including formation of aristolactam adducts and release of kidney injury biomarkers. Hepatic biotransformation of AA-I to a nephrotoxic metabolite involves nitroreduction, followed by sulfate conjugation. Here, we identify, in a human tissue-based system, that the sulfate conjugate of the hepatic NQO1-generated aristolactam product of AA-I (AL-I-NOSO3) is the nephrotoxic form of AA-I. This conjugate can be transported out of liver via MRP membrane transporters and then actively transported into kidney tissue via one or more organic anionic membrane transporters. This integrated microphysiological system provides an ex vivo approach for investigating organ-organ interactions, whereby the metabolism of a drug or other xenobiotic by one tissue may influence its toxicity toward another, and represents an experimental approach for studying chemical toxicity related to environmental and other toxic exposures.

Development of pyridine dicoumarols as potent anti HIV-1 leads, targeting HIV-1 associated topoisomeraseIIß kinase.

AIM: A structural study of a series of pyridine dicoumarol derivatives with potential activity against a novel Topoisomerase IIß kinase which was identified in the HIV-1 viral lysate, compounds were designed and synthesized based on a 3D-QSAR study. MATERIALS & METHODS: Based on QSAR model we have designed and synthesized a series of pyridine dicoumarol derivatives and characterized by spectral studies, all the molecules are biologically evaluated by kinase assay, cytotoxicity assay, ELISA and PCR method. RESULT: We demonstrated the achievement of water soluble disodium pyridine dicoumarate derivatives showing high anti-HIV-1 activity (IC50 <25 nM) which provides a crucial point for further development of pyridine dicoumarol series as HIV-1-associated topoisomerase IIß kinase inhibitors for clinical application against AIDS. CONCLUSION: A new class of anti-HIV-1 lead compounds have been designed and tested. Further studies would result in development of  novel and potential drugs.

Nuevos anticoagulantes frente a anticoagulantes clásicos: ventajas e inconvenientes / New versus classical anticoagulants: advantages and disadvantages

El descubrimiento de los anticoagulantes se inició en 1916 con la aparición de la heparina, en 1941 se aisló y caracterizo el 3,3’-metilenobis-(4-hidroxicumarina), que más tarde se conocería como dicumarol o bishidroxicumarina. El desarrollo de los nuevos anticoagulantes orales ha sido lento hasta la reciente introducción de dabigatrán, un inhibidor oral de trombina, y rivaroxabán, inhibidor oral del factor Xa. La heparina presenta una serie de inconvenientes, como la necesidad de administración parenteral, complicaciones hemorrágicas al inducir trombocitopenia, osteoporosis, reacciones cutáneas, reacciones de hipersensibilidad y elevación de las transaminasas. Los antagonistas de la vitamina K tienen un mecanismo de acción indirecto, presentan múltiples interacciones con fármacos y alimentos, dificultad de dosificación y precisan la monitorización de su efecto. Las limitaciones de los actuales fármacos antitrombóticos ha impulsado la búsqueda de nuevos agentes, que para superar las limitaciones de los AVK deben reunir, entre otras, las siguientes características: administración oral, bajo riesgo de hemorragia, una cinética predecible, que no requieran vigilancia de la coagulación, que no sea necesario ajustar la dosis y tener una baja interacción con fármacos y alimentos (AU)

The discovery of anticoagulants started in 1916 with the finding of heparin; in 1941 3,3’-metilenobis-(4-hidroxicumarin) was isolated and characterized, and would later become known as dicumarol or bishydroxycoumarin. The development of the new oral anticoagulants was slow until the recent introduction of dabigatran, a novel oral thrombin inhibitor, and rivaroxaban, an oral factor Xa (FXa) inhibitor-Heparin has a series of disadvantages, such as the need for parenteral administration, hemorrhagic complications if thrombocytopenia develops, osteoporosis, cutaneous reactions, hypersensitivity reactions, and transaminase elevation. In addition, monitoring is required. Vitamin K antagonists also require monitoring and have an indirect mechanism of action and multiple food and drug interactions. Dosing is also problematic. The limitations of currently available antithrombotic agents have prompted the search for new drugs. To overcome the limitations of VKA, these drugs should include the following characteristics, among others; oral administration, low bleeding risk, predictable kinetics, few food and drug interactions and no requirement for dose adjustments. (AU)

Identification of novel species-selective agonists of the G-protein-coupled receptor GPR35 that promote recruitment of ß-arrestin-2 and activate Gα13.

The poorly characterized G-protein-coupled receptor GPR35 has been suggested as a potential exploratory target for the treatment of both metabolic disorders and hypertension. It has also been indicated to play an important role in immune modulation. A major impediment to validation of these concepts and further study of the role of this receptor has been a paucity of pharmacological tools that interact with GPR35. Using a receptor-ß-arrestin-2 interaction assay with both human and rat orthologues of GPR35, we identified a number of compounds possessing agonist activity. These included the previously described ligand zaprinast. Although a number of active compounds, including cromolyn disodium and dicumarol, displayed similar potency at both orthologues of GPR35, a number of ligands, including pamoate and niflumic acid, had detectable activity only at human GPR35 whereas others, including zaprinast and luteolin, were markedly selective for the rat orthologue. Previous studies have demonstrated activation of Gα13 by GPR35. A Saccharomyces cerevisiae-based assay employing a chimaeric Gpa1-Gα13 G-protein confirmed that all of the compounds active at human GPR35 in the ß-arrestin-2 interaction assay were also able to promote cell growth via Gα13. Each of these ligands also promoted binding of [35S]GTP[S] (guanosine 5'-[γ-[35S]thio]triphosphate) to an epitope-tagged form of Gα13 in a GPR35-dependent manner. The ligands identified in these studies will be useful in interrogating the biological actions of GPR35, but appreciation of the species selectivity of ligands at this receptor will be vital to correctly attribute function.

Evaluation of selective competitive binding of basic drugs to alpha1-acid glycoprotein variants.

We examined the binding of various basic drugs to the F(1)S and A genetic variants of alpha(1)-acid glycoprotein (AGP), which were isolated from native human commercial AGP (total AGP) by chromatography on an immobilized copper(II) affinity adsorbent. The values of the dissociation constant (K(d)) of some basic drugs with the F(1)S variant in equilibrium dialysis differed characteristically from those with the A variant. The selective binding to these variants was evaluated by measuring the displacement ratio of dicumarol bound to the F(1)S variant or that of acridine orange bound to the A variant, using circular dichroism spectroscopy. There was reasonably good agreement between the K(d) values and displacement ratios. There was a characteristic difference between the values of inhibition constant (K(i)) of basic drugs towards dipyridamole binding to F(1)S and towards disopyramide binding to A in total AGP. We found that the K(i) values for dipyridamole binding were well correlated with the K(d) values for the F(1)S variant, whereas those for disopyramide binding were well correlated with the K(d) values for the A variant. In conclusion, the higher the affinity of basic drugs for AGP, the more they inhibit the binding of other basic drugs, and further, the inhibitory potency depends on the selectivity of binding to the AGP variants.

The crystal structure of NAD(P)H quinone oxidoreductase 1 in complex with its potent inhibitor dicoumarol.

NAD(P)H quinone oxidoreductase 1 (NQO1) is a ubiquitous flavoenzyme that catalyzes two-electron reduction of quinones to hydroquinones utilizing NAD(P)H as an electron donor. NQO1 binds and stabilizes several short-lived proteins including the tumor suppressors p53 and p73 and the enzyme ornithine decarboxylase (ODC). Dicoumarol is a widely used potent competitive inhibitor of NQO1 enzymatic activity, which competes with NAD(P)H for binding to NQO1. Dicoumarol also disrupts the binding of NQO1 to p53, p73, and ODC and induces their ubiquitin-independent proteasomal degradation. We report here the crystal structure of human NQO1 in complex with dicoumarol at 2.75 A resolution. We have identified the interactions of dicoumarol with the different residues of NQO1 and the conformational changes imposed upon dicoumarol binding. The most prominent conformational changes that occur in the presence of dicoumarol involve Tyr 128 and Phe 232 that are present on the surface of the NQO1 catalytic pocket. On the basis of the comparison of the NQO1 structure in complex with different NQO1 inhibitors and our previous analysis of NQO1 mutants, we propose that the specific conformation of Tyr 128 and Phe 232 is important for NQO1 interaction with p53 and other client proteins.

Aerobic 2- and 4-nitroreduction of CB 1954 by human liver.

5-(Aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) is an anti-tumour prodrug which recently entered clinical trials in combination with Escherichia coli nitroreductase in a gene-directed enzyme prodrug therapy (GDEPT) context. A Phase I trial of the prodrug, however, revealed dose-limiting hepatotoxicity (transaminitis). The aim of this study was to find out whether the prodrug undergoes reductive metabolism in human liver to cytotoxic metabolites which may contribute to this clinical toxicity. CB 1954 (2.5-250 microM) was incubated with human liver preparations (2-8 mg/mL of S9, cytosolic or microsomal proteins) in the presence of NAD(P)H (1 mM). The NADH- and NADPH-dependent formation of both 2- and 4-nitroreduction products was demonstrated, with NADPH being the preferred cofactor, by HPLC and mass spectrometry. The major metabolite formed in all three human liver preparations was the 4-hydroxylamine, a potent DNA cross-linking cytotoxin. The 2-hydroxylamine and 2-amine metabolites were also detected, both of which have also been demonstrated to be highly cytotoxic. 2-Nitroreduction was far greater in S9 compared with cytosol and was not detected in microsomal preparations. Although 2- and 4-nitroreduction of CB 1954 was inhibited under hyperoxic conditions, substantial metabolism was observed under atmospheric oxygen levels. These studies demonstrate that human liver is capable of aerobic reductive bioactivation of CB 1954 to cytotoxic metabolites in vitro, possibly involving multiple enzymes, which may account for the clinical hepatotoxicity observed.

AMP-activated protein kinase can induce apoptosis of insulin-producing MIN6 cells through stimulation of c-Jun-N-terminal kinase.

We have recently shown that conditions known to activate AMP-activated protein kinase (AMPK) in primary beta-cells can trigger their apoptosis. The present study demonstrates that this is also the case in the MIN6 beta-cell line, which was used to investigate the underlying mechanism. Sustained activation of AMPK was induced by culture with the adenosine analogue AICA-riboside or at low glucose concentrations. Both conditions induced a sequential activation of AMPK, c-Jun-N-terminal kinase (JNK) and caspase-3. The effects of AMPK on JNK activation and apoptosis were demonstrated by adenoviral expression of constitutively active AMPK, a condition which reproduced the earlier-described AMPK-dependent effects on pyruvate kinase and acetyl-coA-carboxylase. The effects of JNK activation on apoptosis were demonstrated by the observations that (i). its inhibition by dicumarol prevented caspase-3 activation and apoptosis, (ii). adenoviral expression of the JNK-interacting scaffold protein JIP-1/IB-1 increased AICA-riboside-induced JNK activation and apoptosis. In primary beta-cells, AMPK activation was also found to activate JNK, involving primarily the JNK 2 (p54) isoform. It is concluded that prolonged stimulation of AMPK can induce apoptosis of insulin-producing cells through an activation pathway that involves JNK, and subsequently, caspase-3.

Dicoumarol: a unique microtubule stabilizing natural product that is synergistic with Taxol.

In studies on the antiproliferative actions of coumarin compounds, we discovered that dicoumarol (a coumarin anticoagulant; 3,3'-methylenebis[4-hydroxycoumarin]) inhibits the first cleavage of Strongylocentrotus purpuratus (sea urchin) embryos in a concentration-dependent manner with 50% inhibition occurring at a concentration of 10 microM. Because first cleavage in sea urchin embryos is highly selective for microtubule-targeted agents, we thought that the active compounds might inhibit cell division by interacting with tubulin or microtubules. We found that dicoumarol binds to bovine brain tubulin with a K(d) of 22 microM and that 0.1 microM dicoumarol strongly stabilizes the growing and shortening dynamics at the plus ends of the microtubules in vitro. Dicoumarol reduces the rate and extent of shortening, it increases the percentage of time the microtubules spend in an attenuated (paused) state, and it reduces the overall dynamicity of the microtubules. The antimitotic effects of the widely used cancer chemotherapeutic agent Taxol (paclitaxel) are also mediated by suppressing microtubule dynamics. We demonstrate that exposure to combinations of Taxol and dicoumarol results in a synergistic inhibition of cell division of sea urchin embryos. The results suggest that the antiproliferative mechanism of action of dicoumarol and possibly related pharmacophores may be mediated by tubulin binding and the stabilization of spindle microtubule dynamics. Because of its low toxicity and simple chemical structure, there is potential interest to explore combinations of antimitotic coumarins with other chemotherapeutic agents to improve efficacy and lower toxicity.

Concerted action of DT-diaphorase and superoxide dismutase in preventing redox cycling of naphthoquinones: an evaluation.

It has been suggested that the enzymes DT-diaphorase and superoxide dismutase act in concert to prevent redox cycling of naphthoquinones and thus protect against the toxic effects of such substances. Little is known, however, about the scope of this process or the conditions necessary for its operation. In the presence of low levels of DT-diaphorase, 2-methyl-1,4-naphthoquinone was found to undergo redox cycling. This was very effectively inhibited by SOD, and in the presence of both enzymes the hydroquinone was maintained in the reduced form. The inhibitory effect of the enzyme combination was overcome, however, at high concentrations of the quinone, or by small increases in pH. Furthermore, redox cycling was re-established by addition of haemoproteins such as cytochrome c and methaemoglobin. DT-diaphorase and SOD strongly inhibited redox cycling by 2,3-dimethyl- and 2,3-dimethoxy-1,4-naphthoquinone, but not that of 2-hydroxy-, 5-hydroxy- or 2-amino-1,4-naphthoquinone. Inhibition of redox cycling by a combination of DT-diaphorase and SOD is therefore not applicable to all naphthoquinone derivatives, and when it does occur, it may be overwhelmed at high quinone concentrations, and it may not operate under slightly alkaline conditions or in the presence of tissue components capable of initiating hydroquinone autoxidation.

Measurement of dicumarol-sensitive NADPH: (menadione-cytochrome c) oxidoreductase activity results in an artifactual assay of DT-diaphorase in cell sonicates.

Purified DT-diaphorase can be assayed as either dicumarol-inhibitable NAD(P)H:menadione oxidoreductase or dicumarol-inhibitable NAD(P)H:dichlorophenolindophenol reductase. Both of these methods have been utilized to assay DT-diaphorase activity in tissue and cell homogenates. When DT-diaphorase activity was measured as dicumarol-inhibitable NADPH:dichlorophenolindophenol reductase in sonicates of two cell lines previously shown to not have any measurable activity of this enzyme, no enzymatic activity was detected. However, when the water-soluble bisulfite addition product of menadione was used as the electron acceptor, an artifactual activity for DT-diaphorase was detected in these cell lines. When another cell line was assayed utilizing menadione bisulfite, an apparent activity of about three times that found with dichlorophenolindophenol was measured, and thus, may overestimate DT-diaphorase activity in cells having activity. When menadione was used in place of menadione bisulfite, an artifactual DT-diaphorase activity was also detected, but was about one-half that obtained with menadione bisulfite. Polarographic determinations of the midpoint potentials for menadione and menadione bisulfite indicated that the latter compound was easier to reduce and may account for the greater apparent DT-diaphorase activity measured with this compound.

Active site studies of DT-diaphorase employing artificial flavins.

NAD(P)H:quinone oxidoreductase (EC 1.6.99.2) (DT-diaphorase) is an FAD-containing enzyme that catalyzes the 2-electron reduction of quinones to hydroquinones using either NADH or NADPH as the electron donor. In this study, FAD was removed by dialyzing the holoprotein against 2 M KBr, and synthetic analogs of FAD were substituted in the flavin binding site as structural probes. Spectral analysis indicates that the benzoquinoid forms of 8-mercapto-FAD and 6-mercapto-FAD are stabilized on binding to the enzyme. This is consistent with the fact that the native flavoprotein forms the anion flavin radical upon photoreduction and suggests the presence of a positive charge near the N(1)C(2)O position of the isoalloxazine ring. Reactivity studies using 8-chloro- and 8-mercapto-flavins suggest that the 8 position of the FAD is accessible to the solvent. However, the rates of the reactions were dramatically decreased in the presence of the competitive inhibitor, dicumarol. 6-Mercapto-, 6-thiocyanato-, 6-azido-, and 6-amino-flavins were also used as structural probes. The results indicate that the 6 position is accessible to solvent. Dicumarol binding increases the pK alpha of the enzyme-bound 6-mercapto-flavin from below pH 5.0 to higher than pH 9.0. The results suggest that DT-diaphorase shows the same properties as the C-C transhydrogenases, and the binding of dicumarol elicits a conformational change or an adjustment in the polarity of the FAD pocket. The enzyme reconstituted with oxidized 5-deaza-FAD has significant catalytic activity, confirming that DT-diaphorase is an obligatory 2-electron transfer enzyme and plays a role in the detoxification of quinones and quinoid compounds by reducing them to the relatively stable hydroquinones.

A comparative study of liver mixed function oxidases in camels (Camelus dromedarius), guinea pigs (Cavia porcellus) and rats (Rattus norvegicus).

1. The activities of the drug-metabolizing enzymes, benzphetamine N-demethylase, 7-ethoxycoumarin O-deethylase and dicoumarol oxidation have been measured in vitro in the liver of camels, guinea pigs and rats. 2. In these species, levels of hepatic microsomal parameters namely microsomal protein, cytochrome P450, cytochrome b5 and NADPH-cytochrome c reductase have also been determined. 3. In general, camels seemed to have the lowest enzyme activity when compared to rats and guinea pigs. 4. Some sex differences were observed in the levels of enzymes studied. In rats and guinea pigs, males had higher benzphetamine N-demethylase than females. However, in camels and guinea pigs, females had higher 7-ethoxycoumarin O-deethylase when compared to males.

Identification of a glycine-rich sequence as an NAD(P)H-binding site and tyrosine 128 as a dicumarol-binding site in rat liver NAD(P)H:quinone oxidoreductase by site-directed mutagenesis.

Site-directed mutagenesis was utilized to identify binding sites for NAD(P)H and dicumarol in rat liver NAD(P)H:quinone oxidoreductase (NQOR, EC 1.6.99.2). The mutant cDNA clones were generated by a procedure based on the polymerase chain reaction and were expressed in Escherichia coli. The mutant enzymes were purified to apparent homogeneity as judged by SDS-polyacrylamide gel electrophoresis and were found to contain 2 FADs/enzyme molecule identical with that of the wild-type NQOR. Purified mutant enzymes Y128D, G150F, G150V, S151F, and Y155D showed dramatic decreases in activities in the reduction of dichlorophenolindophenol in comparison with the activities of the wild-type enzyme, whereas the activities of F124L, T127V, T127E, Y128V, Y128F, S151A, and Y155V were similar to those of NQOR. Enzyme kinetic analysis revealed that the Km values of T127E, Y128D, G150F, G150V, S151F, and Y155D were, respectively, 4-, 2-, 13-, 5-, 26-, and 19-fold higher than the Km of NQOR for NADPH, and were, respectively, 2-, 3-, 7-, 3-, 20-, and 11-fold higher than that of NQOR for NADH. The kcat values of Y128D, G150F, and G150V were also much lower than those of NQOR, but the kcat values of other mutants were similar to those of the wild-type enzyme. The Km values of the mutants for dichlorophenolindophenol were the same or slightly higher than that of NQOR. The apparent inhibition constants (Ki) for dicumarol on Y128V and F124L were elevated 12 and 8 times, respectively. Similar, but smaller, changes on Ki for 4-hydroxycoumarin were also observed. This study demonstrated that residues Gly150, Ser151, and Tyr155 in the glycine-rich region of NQOR are essential for NADPH and NADH binding and Tyr128 is important for dicumarol binding. Based on the results of the study, it is proposed that the glycine-rich region of the enzyme, along with other residues around the region, forms a beta sheet-turn-alpha helix structure important for the binding of the pyrophosphate group of NADPH and NADH.

Species differences and interindividual variation in liver microsomal cytochrome P450 2A enzymes: effects on coumarin, dicumarol, and testosterone oxidation.

Antibody against purified CYP2A1 recognizes two rat liver microsomal P450 enzymes, CYP2A1 and CYP2A2, that catalyze the 7 alpha- and 15 alpha-hydroxylation of testosterone, respectively. In human liver microsomes, this antibody recognizes a single protein, namely CYP2A6, which catalyzes the 7-hydroxylation of coumarin. To examine species differences in CYP2A function, liver microsomes from nine mammalian species (rat, mouse, hamster, rabbit, guinea pig, cat, dog, cynomolgus monkey, and human) were tested for their ability to catalyze the 7 alpha- and 15 alpha-hydroxylation of testosterone and the 7-hydroxylation of coumarin. Antibody against rat CYP2A1 recognized one or more proteins in liver microsomes from all mammalian species examined. However, liver microsomes from cat, dog, cynomolgus monkey, and human catalyzed negligible rates of testosterone 7 alpha- and/or 15 alpha-hydroxylation, whereas rat and cat liver microsomes catalyzed negligible rates of coumarin 7-hydroxylation. Formation of 7-hydroxycoumarin accounted for a different proportion of the coumarin metabolites formed by liver microsomes from each of the various species examined. 7-Hydroxycoumarin was the major metabolite (greater than 70%) in human and monkey, but only a minor metabolite (less than 1%) in rat. The 7-hydroxylation of coumarin by human liver microsomes was catalyzed by a single, high-affinity enzyme (Km 0.2-0.6 microM), which was markedly inhibited (greater than 95%) by antibody against rat CYP2A1. The rate of coumarin 7-hydroxylation varied approximately 17-fold among liver microsomes from 22 human subjects. This variation was highly correlated (r2 = 0.956) with interindividual differences in the levels of CYP2A6, as determined by immunoblotting. These results indicate that CYP2A6 is largely or entirely responsible for catalyzing the 7-hydroxylation of coumarin in human liver microsomes. Treatment of monkeys with phenobarbital or dexamethasone increased coumarin 7-hydroxylase activity, whereas treatment with beta-naphthoflavone caused a slight decrease. These results suggest that environmental factors can increase or decrease CYP2A expression in cynomolgus monkeys, which implies that environmental factors may be responsible for the large variation in CYP2A6 levels in humans, although genetic factors may also be important. In contrast to rats and mice, the expression of CYP2A enzymes in cynomolgus monkeys and humans was not sexually differentiated. Despite their structural similarity to coumarin, the anticoagulants dicumarol and warfarin do not appear to be substrates for CYP2A6. The overall rate of dicumarol metabolism varied approximately 5-fold among the human liver microsomal samples, but this variation correlated poorly (r2 = 0.126) with the variation observed in CYP2A6 levels and coumarin 7-hydroxylase activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Reversal of signs of induced cotton effects of dicumarol-alpha 1-acid glycoprotein systems by phenothiazine neuroleptics through ternary complexation.

The interaction of dicumarol and phenothiazine neuroleptics binding to alpha 1-acid glycoprotein (AGP) was investigated by circular dichroism (CD) and equilibrium dialysis. The induced CD spectra of the dicumarol-AGP complex were affected differently by the different substituents of the phenothiazine molecule. The sign of the induced Cotton effect of dicumarol bound to AGP was reversibly changed with the introduction of the propyldimethylamine substituent at position 10 or chloride group at position 2 of the phenothiazine molecule. Chlorpromazine, which contains both of these substituents reversed the sign of the induced Cotton effect with the highest intensity. The addition of trifluoperazine, fluphenazine, and promethazine containing neither of the two substituents generated a new negative CD band. However, the addition of opromazine, which contains sulfoxide at position 5, decreased the CD intensity of the dicumarol-AGP complex without changing the shape of the CD spectra. Equilibrium dialysis studies revealed that the interaction of dicumarol-AGP with phenothiazine derivatives occurred simultaneously, and the interaction followed a cooperative and anticooperative binding model. Further, among the six phenothiazine derivatives that reversed the signs of the induced Cotton effects of the dicumarol-AGP complex, a linear relationship was observed between coupling constants and the difference in the induced optical ellipticity. The opromazine and dicumarol interaction was competitive for a common binding site on the AGP molecule. Removal of sialic acid did not have any effect on this interaction. These data support the hypothesis that the acidic and the basic drug binding sites overlap each other.

Interaction mode of dicumarol and its derivatives with human serum albumin, alpha 1-acid glycoprotein and asialo alpha 1-acid glycoprotein.

The interaction of dicumarol and its seven other derivatives with human serum albumin (HSA), alpha 1-acid glycoprotein (AGP) and desialylatedAGP (asialoAGP) has been investigated by circular dichroism (CD) and fluorescence. The binding parameters of dicumarol and its derivatives obtained from fluorescence almost agreed with those obtained from CD. The binding data indicated that the total binding affinities (nK) to HSA were higher than the binding affinities to AGP and asialoAGP. Hydrophobic interaction was the driving force for the binding to all the three proteins and nK values in the binding process were found to be increased with the increase of hydrophobicity of the compound. This was evidenced by the attempts taken to correlate binding affinities with partition coefficients. Both electrostatic and van der Waals interactions were not found to play any significant role in the binding of these compounds to any of these three proteins. However, in case of the AGP and asialoAGP binding, apart from the hydrophobic interaction, some other forces may be involved as evidenced from the experimental data. Binding was exothermic, entropy driven and spontaneous. The change of enthalpy (delta H degree) was compensated for by the change in entropy (delta S degree). Relative contribution of hydrophobic interactions in the binding of these compounds to HSA was higher than to AGP or asialoAGP. Sialic acid was not found to impart any significant role in the binding of these compounds to AGP.

Effects of tricyclic drug on induced circular dichroism spectra of dicumarol bound to alpha 1-acid glycoprotein.

Effects of both tricyclic and non-tricyclic drugs on the extrinsic Cotton effects of dicumarol bound to human alpha 1-acid glycoprotein (AGP) have been investigated. Basic tricyclic drugs caused the reversal of the signs of the induced Cotton effects of the circular dichroism (CD) spectra of the dicumarol-AGP system while the basic drugs not possessing tricyclic rings and acidic drugs decreased the observed ellipticities without changing the signs of its CD spectra. There was no reversal of the CD signs of the drugs not containing two hydroxycoumarin rings bound to AGP by basic tricyclic drugs. Raising of pH and temperature, and the addition of guanidine hydrochloride decreased the observed ellipticities of the CD spectra of the dicumarol-AGP system without showing any change in the signs of the Cotton effects. The mutual displacement data showed that protriptyline increased its own binding and that of dicumarol with AGP. The results of CD titration and equilibrium dialysis experiments suggest that dicumarol-AGP and dicumarol-AGP-protriptyline form a 1:1 binary complex and a 1:1:1 ternary complex, respectively.