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PubMed2000

Behavioural outcome of school-age children after prenatal exposure to coumarins.

In utero exposure to coumarin derivatives may affect the development of the central nervous system of the child, irrespective of the period of exposure in pregnancy. Little is known about effects on development in the long term. The aim of the present study was to determine whether prenatal exposure to coumarins affects behavioural outcome in children at school age. Behavioural outcome was assessed in a cohort of 305 exposed children, aged 7-15 years. Findings were compared with those in a cohort of 263 non-exposed controls, matched for sex, age, and demographic region. Behaviour was rated by parents and teachers using standardized questionnaires: the Groningen Behaviour Checklist Family situation (GBF) and the Groningen Behaviour Checklist School situation (GBS), respectively. The findings of the GBF were supported by the results of the GBS, filled in by teachers who were blind for the exposure status of the child. In comparison to the non-exposed children, the coumarin-exposed children scored lower on the cluster 'positive task orientation' (GBF P<0.05, GBS P<0.01), they scored higher on 'emotional instability' (GBF P<0.001, GBS P<0.05), and they had more problems on the social clusters (P<0.01). Based on the results of both questionnaires, we conclude that behavioural development may be negatively influenced in school-age children after in utero exposure to coumarins, leading to less favourable task-oriented and social-emotional behaviour. However, the frequency of clinically relevant 'problem behaviour' (GBF) was not increased in relation to coumarin exposure, the odds ratio was 1.2 (CI(95) 0.7-1.8).

Adolescent↗
PubMed2003

Diversity of selective environmental substrates for human cytochrome P450 2A6: alkoxyethers, nicotine, coumarin, N-nitrosodiethylamine, and N-nitrosobenzylmethylamine.

Cytochrome P450 2A6 constitutes 5-10% of the total microsomal CYPs of human liver. Although CYP2A6 is the major coumarin 7-hydroxylase, other known substrates of CYP2A6 include many toxicants and precarcinogens. The chemical structure diversity of these substrates raises the question of their selectivity. Thus, kinetic parameters were determined for the hydroxylation of five substrates of diverse chemical structures known to be selective for cytochrome P450 2A6: methyl tert-butyl ether (MTBE), nicotine, coumarin, N-nitrosobenzylmethylamine (NBzMA), and N-nitrosodiethylamine (NDEA). Sources of enzymes were either human liver microsomes or heterologously expressed CYPs. Coumarin was shown to be the substrate with the highest affinity, followed by NDEA, nicotine, NBzMA, and MTBE. Variability of CYP2A6 catalytic activities in human liver was between 24-fold for MTBE to sevenfold for coumarin, while CYP2A6 content varied 68-fold in human liver microsomes. These five catalytic activities were highly significantly correlated between them and with hepatic CYP2A6 content. The most selective chemical inhibitor of these five substrates was shown to be 8-methoxypsoralen. Based upon chemical inhibition of the enzymatic activities of pure recombinant human CYPs, it cannot be totally excluded that P450s other than CYP2A6, especially CYP2E1, are involved, although to a lesser extent, in NDEA and NBzMA metabolism. In conclusion, the prototype probes for CYP2A6 phenotyping are coumarin and nicotine.

Adult↗
PubMed2001

Coumarin formation in novobiocin biosynthesis: beta-hydroxylation of the aminoacyl enzyme tyrosyl-S-NovH by a cytochrome P450 NovI.

BACKGROUND: Coumarin group antibiotics, such as novobiocin, coumermycin A1 and clorobiocin, are potent inhibitors of DNA gyrase. These antibiotics have been isolated from various Streptomyces species and all possess a 3-amino-4-hydroxy-coumarin moiety as their structural core. Prior labeling experiments on novobiocin established that the coumarin moiety was derived from L-tyrosine, probably via a beta-hydroxy-tyrosine (beta-OH-Tyr) intermediate. Recently the novobiocin gene cluster from Streptomyces spheroides was cloned and sequenced and allows analysis of the biosynthesis of the coumarin at the biochemical level using overexpressed and purified proteins. RESULTS: Two open reading frames (ORFs), NovH and NovI, from the novobiocin producer S. spheroides have been overexpressed in Escherichia coli, purified and characterized for tyrosine activation and oxygenation which are the initial steps in coumarin formation. The 65 kDa NovH has two predicted domains, an adenylation (A) and a peptidyl carrier protein (PCP), reminiscent of non-ribosomal peptide synthetases. Purified NovH catalyzes L-tyrosyl-AMP formation by its A domain, can be posttranslationally phosphopantetheinylated on the PCP domain, and accumulates the covalent L-tyrosyl-S-enzyme intermediate on the holo PCP domain. The second enzyme in the pathway, NovI, is a 45 kDa heme protein that functions as a cytochrome P450-type monooxygenase with specificity for the tyrosyl-S-NovH acyl enzyme. The product beta-OH-tyrosyl-S-NovH was detected by alkaline release and high performance liquid chromatography analysis of radioactive [3H]beta-OH-Tyr and by mass spectrometry. Also detected was 4-OH-benzaldehyde, a retro aldol breakdown product of beta-OH-Tyr. The amino acid released was (3R,2S)-3-OH-Tyr by comparison with authentic standards. CONCLUSIONS: This work establishes that NovH and NovI are responsible for the formation of a beta-OH-Tyr intermediate that is covalently tethered to NovH in novobiocin biosynthesis. Comparable A-PCP/P450 pairs for amino acid beta-hydroxylation are found in various biosynthetic gene clusters, such as ORF19/ORF20 in the chloroeremomycin cluster for tyrosine, CumC/CumD in the coumermycin A1 cluster for tyrosine, and NikP1/NikQ in the nikkomycin cluster for histidine. This phenomenon of covalent docking of the amino acid in a kinetically stable thioester linkage prior to chemical modification by downstream tailoring enzymes, could represent a common strategy for controlling the partitioning of the amino acid for incorporation into secondary metabolites.

Acylation↗
PubMed2003

Liquid chromatographic determination of the glutathione conjugate and ring-opened metabolites formed from coumarin epoxidation.

Species differences in the biotransformation of coumarin are thought to play an important role in its toxicity. Since the putative toxic metabolite is coumarin 3,4-epoxide (CE), methods to measure the metabolites of CE were developed. The glutathione (GSH) conjugate of CE (CE-SG) at the 3-position was purified by reversed-phase (RP)-high performance liquid chromatography (HPLC), and characterized by mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR). An RP-HPLC method was developed to quantify CE-SG in hepatic microsomal mixtures and a separate RP-HPLC method was also developed to quantify the three ring-opened coumarin metabolites; o-hydroxyphenylacetic acid (o-HPAA), o-hydroxyphenylethanol (o-HPE) and o-hydroxyphenylacetaldehyde (o-HPA) in hepatic microsomal mixtures. Detection limits for all four products of coumarin epoxidation exceeded 3.5 ng/ml and recovery from hepatic microsomal mixtures was essentially quantitative with RSD values less than 8%. Species differences in o-HPA detoxification were consistent with sensitivity to coumarin, thereby demonstrating that these methods have utility in addressing the fate of CE and its contribution to toxicity.

Animals↗
PubMed2002

DNA gyrase interaction with coumarin-based inhibitors: the role of the hydroxybenzoate isopentenyl moiety and the 5'-methyl group of the noviose.

DNA gyrase is a major bacterial protein that is involved in replication and transcription and catalyzes the negative supercoiling of bacterial circular DNA. DNA gyrase is a known target for antibacterial agents since its blocking induces bacterial death. Quinolones, coumarins, and cyclothialidines have been designed to inhibit gyrase. Significant improvements can still be envisioned for a better coumarin-gyrase interaction. In this work, we obtained the crystal costructures of the natural coumarin clorobiocin and a synthetic analogue with the 24 kDa gyrase fragment. We used isothermal titration microcalorimetry and differential scanning calorimetry to obtain the thermodynamic parameters representative of the molecular interactions occurring during the binding process between coumarins and the 24 kDa gyrase fragment. We provide the first experimental evidence that clorobiocin binds gyrase with a stronger affinity than novobiocin. We also demonstrate the crucial role of both the hydroxybenzoate isopentenyl moiety and the 5'-alkyl group on the noviose of the coumarins in the binding affinity for gyrase.

Anti-Infective Agents↗
PubMed1999

Probing the binding of coumarins and cyclothialidines to DNA gyrase.

DNA gyrase is the target of a number of antibacterial agents, including the coumarins and the cyclothialidines. To extend our understanding of the mechanism of action of these compounds, we have examined the previously published crystal structures of the complexes between the 24 kDa fragment of GyrB and coumarin and cyclothialidine drugs and made mutations by site-directed mutagenesis. We used proteolysis as a probe of drug binding to wild-type and mutant proteins. Limited proteolysis of gyrase revealed that binding of these antibiotics is associated with a characteristic proteolytic fingerprint, suggesting a drug-induced conformational change. The ability of the mutants to bind the drugs was studied by testing their ability to induce the coumarin-associated proteolytic signature and to bind to a novobiocin-affinity column. To analyze further the interaction of the drugs with gyrase, we studied the binding using surface plasmon resonance. Mutation of Asn46 to Asp has only a modest effect on the binding of coumarins, while an Asn46 to Leu mutation results in a 10-fold decrease in the affinity. Mutation of Asp73 to Asn completely abolishes binding to both coumarins and cyclothialidines. Mutations at these residues also abolish ATP hydrolysis, explaining the inability of such mutations to occur spontaneously.

Adenosine Triphosphate↗
PubMed1999

Identification of coumarins from the fruit of Citrus hystrix DC as inhibitors of nitric oxide generation in mouse macrophage RAW 264.7 cells.

Three known coumarins have been isolated from Citrus hystrix DC as inhibitors of both lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma)-induced nitric oxide (NO) generation in RAW 264.7 cells. The inhibitory activity of bergamottin (IC(50) = 14 microM) was comparable to that of N-(iminoethyl)-L-ornithine (L-NIO) (IC(50) = 7. 9 microM), whereas oxypeucedanin and 5-[(6',7'-dihydroxy-3', 7'-dimethyl-2-octenyl)oxy]psoralen, structurally different from bergamottin only in their side-chain moieties, were notably less active. Using 21 coumarins, we structurally classified various types of coumarins into groups A-C: (A) bearing an isoprenyl (IP) or a geranyl (GR) group, highly active; (B) bearing an IP group cyclized to a coumarin ring, moderately active; (C) bearing an IP group modified with hydroxyl group(s) and/or having other functional groups except for the IP, completely inactive. Cellular uptake studies suggested that coumarins in group C are inactive because of poor permeability to the cell membrane.

Animals↗
PubMed2005

Efficient synthesis of tyrosine-derived marine sponge metabolites via acylation of amines with a coumarin.

Condensation of N-acetylglycine with aldehyde 15 in acetic anhydride gave acetamido coumarin 16. Hydrolysis to the enol coumarin 17 and reaction with hydroxylamine gave the oximino coumarin 18. Reaction of the oximino coumarin 18 with a range of nucleophiles gave the phenolic oximes in excellent yield. The rates of acylation of histamine with the oximino coumarin 18 and methyl ester 9 were compared. Oxidative spirocyclization of three representative phenolic oximes with polymer-supported (diacetoxyiodo) benzene gave the spiroisoxazolines.

Acylation↗
PubMed2005

Study of energy transfer from 7-amino coumarin donors to the rhodamine 6G acceptor in lecithin vesicles and sodium taurocholate-lecithin mixed aggregates.

The energy transfer using 7-amino coumarin dyes as the donor and rhodamine 590 (Rh6G) as the acceptor was investigated in lecithin vesicles and sodium taurocholate (NaTC)-lecithin mixed aggregates using steady-state and time-resolved fluorescence spectroscopy. All energy transfer parameters were calculated. The coumarin 153-Rh6G pair is the most efficient donor-acceptor pair as reflected by the value of k(ET). With addition of NaTC in lecithin, in the case of the coumarin 153-Rh6G pair, the energy transfer rate or efficiency does not change very much, whereas in the case of the coumarin 151-Rh6G pair, the energy transfer rate decreases 2-fold upon going from lecithin vesicles to NaTC-lecithin mixed aggregates where the molar ratio is 2.5. It is mainly due to the deeper location of coumarin 153 in the lipid bilayer or in mixed aggregates. Rotational relaxation data also support this idea.

Coumarins↗
PubMed1998

A comparison of the bioconversion rates and the Caco-2 cell permeation characteristics of coumarin-based cyclic prodrugs and methylester-based linear prodrugs of RGD peptidomimetics.

PURPOSE: To compare the bioconversion rates in various biological media and the Caco-2 cell permeation characteristics of coumarin based cyclic prodrugs (3a, 3b) and methylester-based linear prodrugs (1b, 2b) of two RGD peptidomimetics (1a, 2a). METHODS: Bioconversion rates of the prodrugs to the RGD peptidomimetics were determined in Hank balances salt solution (HBSS), pH 7,4, at 37 degrees C and in various biological media (human blood plasma, rat liver homogenate, Caco-2 cell homogenate) known to have esterase activity. Transport rates of the prodrugs and the RGD peptidomimetics were determined using Caco-2 cell monolayers, an in vitro cell culture model of the intestinal mucosa. RESULTS. In HBSS, pH 7,4, the coumarin-based cyclic prodrugs 3a and 3b degraded slowly and quantitatively to the RGD peptidomimetics 1a and 2a, respectively (3a, t1/2 = 630+2-14 min; 3b, t1/2 = 301 +/-12 min). The methylester-based linear prodrugs 1b and 2b were more stable to chemical hydrolysis (1b and 2b, t1/2 > 2000 min). Both the coumarin-based cyclic prodrugs and the methylester-based linear prodrugs degraded more rapidly in biological media containing esterase activity (e.g., 90% human blood plasma: 1b, t1/2 < 5 min; 2b, t1/2 < 5 min; 3a, t1/2 < 91+/-1 min; 3b, 1/2 < 57+/-2 min). When the apical (AP)-to-basolateral (BL) permeation characteristics were determined using Caco-2 cell monolayers, it was found that the methylester prodrugs 1b and 2b underwent esterase bioconversion (>80%) to the RGD peptidomimetics 1a and 2a, respectively, In contrast, the cyclic prodrugs 3a and 3b permeated the cell monolayers intact. Considering the appearance of both the prodrug and the RGD peptidomimetic on the BL side, the methylester prodrugs 1b and 2b were approximately 12-fold more able to permeate than were the RGD peptidomimetics 1a and 2a. When similar analysis of the transport data for the coumarin prodrugs 3a and 3b was performed, they were shown to be approximately 6-fold and 5-fold more able to permeate than were the RGD peptidomimetics 1a and 12a, respectively. CONCLUSION: The coumarin-based cyclic prodrugs 3a and 3b were chemically less stable, but metabolically more stable, then the methylester based linear prodrugs. The esterase stability of the cyclic prodrugs 3a and 3b means that they are transported intact across the Caco-2 cell monolayer in contrast to the methylester prodrugs 1b and 2b, which undergo facile bioconversion during their transport to the RGD peptidomimetics. However, both prodrug systems successfully delivered more (5-12-fold) of the RGD peptidomimetic and/or the precursor (prodrug) than did the RGD peptidomimetics themselves.

Animals↗
PubMed1965

A spectrofluorimetric study of the 7-hydroxylation of coumarin by liver microsomes.

1. The fluorescence characteristics of 3- and 7-hydroxycoumarin, and 7-hydroxy-and 7-methoxy-4-methylcoumarin, have been determined. 7-Hydroxycoumarin shows excited-state ionization from pH1 to 9. 2. A sensitive and specific fluorimetric method for the determination of 7-hydroxycoumarin (umbelliferone), and its application to liver homogenates and other tissue preparations, are described. 3. The enzymic hydroxylation of coumarin to 7-hydroxycoumarin has been studied by this method and the optimum conditions have been determined for rabbit-liver preparations. The enzymic activity was found in the microsomal fraction and required NADPH(2) and oxygen. Activity with NADH(2) was one-third of that with NADPH(2). 4. Addition of NADP was necessary for full activity of 10000g supernatant preparations of liver. Nicotinamide added during preparation preserved coenzymic activity in tissue stored at -12 degrees . Glucose 6-phosphate had no effect on the activity of stored or fresh tissue. 5. Inhibition occurred with p-chloromercuribenzoate, and with the usual inhibitors of the microsomal drug-metabolizing enzymes, SKF acid, SKF 525A, and Lilly 7132, but not with 2,2'-bipyridyl. 6. Liver homogenates from rabbit, guinea pig, coypu, cat and pigeon showed activity, but preparations of rat or mouse liver, and of locust fat bodies, did not hydroxylate coumarin to umbelliferone. The enzyme system was absent from rat-liver homogenates and microsomal preparations. Moreover, rat liver also contained inhibitors of the rabbit-liver coumarin-7-hydroxylase system and of the further metabolism of umbelliferone by guinea-pig liver. Guinea-pig-liver preparations hydroxylated coumarin to umbelliferone and then converted this product into its glucuronide. 7. The coumarin-7-hydroxylase activity of female rabbit liver was two to three times that of male rabbit liver.

Animals↗
PubMed2002

Overanticoagulation associated with combined use of antifungal agents and coumarin anticoagulants.

BACKGROUND: Several case reports have associated combined use of coumarins and antifungal agents with overanticoagulation. However, we are not aware of epidemiologic studies that quantify the risk of overanticoagulation caused by antifungal agents. We conducted a follow-up study in a large population-based cohort to investigate the antifungal agents that are associated with overanticoagulation during therapy with coumarins. METHODS: All patients in the Rotterdam Study who were treated with acenocoumarol or phenprocoumon in the study period from April 1, 1991, through Dec 31, 1998, and for whom international normalized ratio data were available were followed up until an INR > or =6.0, the end of their treatment, death, or the end of the study period. Proportional hazards regression analysis was used to estimate the risk of an INR > or =6.0 in relation to concomitant use of an oral anticoagulant and antifungal agents, after adjustment for several potentially confounding factors such as age, gender, hepatic dysfunction, malignancies, and heart failure. RESULTS: Of the 1124 patients in the cohort, 351 had an INR > or =6.0. The incidence rate was 6.9 per 10,000 treatment days. Oral miconazole most strongly increased the risk of overanticoagulation with an adjusted relative risk of 36.6 (95% confidence interval [CI], 12.4-108.0). CONCLUSIONS: In this study among outpatients of an anticoagulant clinic who were on a regimen of coumarins, oral miconazole was especially strongly associated with overanticoagulation. Awareness of these drug interactions and more frequent monitoring of INR values during the initial stages of treatment with some antifungal drugs in patients taking coumarins may minimize the risk of bleeding complications. The concurrent use of miconazole and coumarins should be discouraged.

Acenocoumarol↗
PubMed2003

Coumarin inhibits the growth of carrot (Daucus carota L. cv. Saint Valery) cells in suspension culture.

We used a carrot (Daucus carota L. cv. Saint Valery) cell suspension culture as a simplified model system to study the effects of the allelochemical compound coumarin (1,2 benzopyrone) on cell growth and utilisation of exogenous nitrate, ammonium and carbohydrates. Exposure to micromolar levels of coumarin caused severe inhibition of cell growth starting from the second day of culture onwards. At the same time, the presence of 50 mumol/L coumarin caused accumulation of free amino acids and of ammonium in the cultured cells, and stimulated their glutamine synthetase, glutamate dehydrogenase, glucose-6-phosphate dehydrogenase and phosphoenolpyruvate carboxylase activities. Malate dehydrogenase, on the other hand, was inhibited under the same conditions. These effects were interpreted in terms of the stimulation of protein catabolism and/or interference with protein biosynthesis induced by coumarin. This could have led to a series of compensatory changes in the activities of enzymes linking nitrogen and carbon metabolism. Because coumarin seemed to abolish the exponential phase and to accelerate the onset of the stationary phase of cell growth, we hypothesise that such allelochemical compounds may act in nature as an inhibitor of the cell cycle and/or as a senescence-promoting substance.

2,4-Dichlorophenoxyacetic Acid↗
PubMed2002

Triterpene and coumarins from Skimmia laureola.

In the course of our studies on the chemical constituents of the leaves of Skimmia laureola, a new triterpene O-methyl cyclolaudenol (1) and a new coumarin, (+)-7-methoxy-6-(2'R-methoxy-3'-hydroxy-3'-methyl butyl) coumarin (2) were isolated. In addition five known coumarins, isogospherol (3), heraclenol (4), 5,8-dimethoxy coumarin-2H-1-benzopyran-2-one (5), 7-methoxy-6[2'-oxo-3'-methyl butyl] coumarin (6), and (+)-ulopterol (7) were also isolated for the first time from this plant. The structures were identified by spectroscopic studies and the stereochemistry at C-2' in compounds 3 and 4 were established by Horeau's procedure.

Coumarins↗
PubMed1996

The structure of a coumarin derivative related to the carcinogen benz[a]anthracene.

The three-dimensional structure of 3-methyl-2H-anthra[1,2-b]pyran-2-one, an anticarcinogenic coumarin related to the carcinogen benz[a]anthracene, has been determined by X-ray diffraction techniques. The molecule, apart from hydrogen atoms in the methyl group, is flat, the maximum deviation from its least squares best plane being 0.13 angstroms. The carbonyl C=O bond length is normal [1.206(1) angstroms] and the bonding throughout the molecule indicates localization of double bonds within the coumarin ring, but some delocalization of electrons in the other rings. Molecules pack in planes parallel to each other, the coumarin ring oxygen atom lying between two aromatic rings of other coumarin molecules. The bulky methyl groups are not involved in such stacking, while the carbonyl groups attract C-H groups in neighboring molecules by way of C-H...O interactions. These are the types of interactions that such coumarins could make if they bound to hydrophobic areas in biological macromolecules.

Antineoplastic Agents↗
PubMed1997

Effect of naturally occurring coumarins on the formation of epidermal DNA adducts and skin tumors induced by benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene in SENCAR mice.

Several naturally occurring coumarins previously found to be potent inhibitors of mouse hepatic ethoxyresorufin-O-deethylase (EROD) and/or pentoxyresorufin-O-dealkylase (PROD) were examined for their effects on formation of benzo[a]pyrene (B[a]P) and 7,12-dimethylbenz[a]anthracene (DMBA) DNA adducts in mouse epidermis, as well as, their effects on skin tumor initiation by these polycyclic aromatic hydrocarbons (PAH). Bergamottin, a potent inhibitor of hepatic EROD, given topically 5 min prior to an initiating dose of B[a]P, significantly decreased total covalent binding of B[a]P to DNA in a dose-dependent manner 24 h after treatment. A dose of 400 nmol bergamottin reduced covalent binding of B[a]P by 72%. Coriandrin, at a dose of 400 nmol also significantly reduced total covalent binding of B[a]P by 59%. In addition, formation of the major (+)anti-B[a]P-diol epoxide-N2-dGuo adduct was selectively reduced by both of these coumarins. In contrast, bergamottin and coriandrin did not significantly decrease covalent binding of DMBA to epidermal DNA at doses of either 400 nmol or 800 nmol. Imperatorin and isopimpinellin, which are more potent inhibitors of hepatic PROD activity, significantly reduced overall binding of DMBA to epidermal DNA by 67% and 52%, respectively, when applied at doses of 400 nmol. These two coumarins also inhibited B[a]P-DNA adduct formation at similar doses but to a lesser extent. Imperatorin at a dose of 400 nmol dramatically decreased formation of covalent DNA adducts derived from both the anti and syn diol epoxides of DMBA. Bergamottin was a potent inhibitor of tumor initiation by B[a]P while coriandrin was less effective in this regard. Imperatorin was an effective inhibitor of skin tumor initiation by DMBA and also inhibited complete carcinogenesis by this PAH. At dose levels higher than those effective against DMBA, imperatorin also inhibited tumor initiation by B[a]P. The results demonstrate that several naturally occurring coumarins possess the ability to block DNA adduct formation and tumor initiation by PAHs such as B[a]P and DMBA. The mechanism for reduced DNA adduct formation and tumor initiation appears to involve inhibition of the P450s involved in the metabolic activation of these hydrocarbons. Finally, the differential effects of certain coumarins on B[a]P vs DMBA DNA adduct formation and tumor initiation may be useful for dissecting the role of specific cytochromes P450 in their metabolic activation.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2000

Novobiocin and related coumarins and depletion of heat shock protein 90-dependent signaling proteins.

BACKGROUND: Heat shock protein 90 (Hsp90) interacts with and stabilizes several oncogenic protein kinases (e.g., p185(erbB2), p60(v-src), and Raf-1) and is required for the stability and dominant-negative function of mutated p53 protein. Two unrelated antibiotics, geldanamycin and radicicol, bind specifically to an atypical nucleotide-binding pocket of Hsp90, a site that shares homology with the adenosine triphosphate (ATP)-binding domain of bacterial DNA gyrase B. This interaction leads to destabilization of proteins that interact with Hsp90. Since the nucleotide-binding site of gyrase B is targeted by coumarin antibiotics (e.g., novobiocin), we investigated whether these drugs can also interact with Hsp90 and affect its activity. METHODS: We used immobilized novobiocin, geldanamycin, or radicicol to isolate either endogenous Hsp90 from cell lysates or Hsp90 deletion fragments translated in vitro. Effects of the coumarin antibiotics novobiocin, chlorobiocin, and coumermycin A1 on several proteins interacting with Hsp90 were assessed in vitro and in vivo. RESULTS: Hsp90 binding to immobilized novobiocin was competed by soluble coumarins and ATP but not by geldanamycin or radicicol. A carboxy-terminal Hsp90 fragment bound immobilized novobiocin but not immobilized geldanamycin, while a geldanamycin-binding amino-terminal fragment did not bind novobiocin. All three coumarins markedly reduced cellular levels of p185(erbB2), p60(v-src), Raf-1, and mutated p53. Furthermore, novobiocin reduced Raf-1 levels in the spleens of mice treated with the drug. CONCLUSIONS: These coumarin antibiotics, particularly novobiocin, represent a first-generation alternative to other Hsp90-targeting drugs that are not as well tolerated. Novobiocin's unique interaction with Hsp90 identifies an additional site on this protein amenable to pharmacologic interference with small molecules.

Aminocoumarins↗
PubMed2004

Metabolic detoxification determines species differences in coumarin-induced hepatotoxicity.

Hepatotoxicity of coumarin is attributed to metabolic activation to an epoxide intermediate, coumarin 3,4-epoxide (CE). However, whereas rats are most susceptible to coumarin-induced hepatotoxicity, formation of CE is greatest in mouse liver microsomes, a species showing little evidence of hepatotoxicity. Therefore, the present work was designed to test the hypothesis that detoxification of CE is a major determinant of coumarin hepatotoxicity. CE can either rearrange spontaneously to o-hydroxyphenylacetaldehyde (o-HPA) or be conjugated with gluatathione (GSH). o-HPA is hepatotoxic and is further detoxified by oxidation to o-hydroxyphenylacetic acid (o-HPAA). In vitro experiments were conducted using mouse liver microsomes to generate a constant amount of CE, and cytosols from F344 rats, B6C3F1 mice, and human liver were used to characterize CE detoxification. All metabolites were quantified by HPLC methods with UV detection. In rats and mice, GSH conjugation occurred non-enzymatically and through glutathione-S-transferases (GSTs), and the kinetics of GSH conjugation were similar in rats and mice. In rat liver cytosol, oxidation of o-HPA to o-HPAA was characterized with a high affinity K(m) of approximately 12 microM, and a V(max) of approximately 1.5 nmol/min/mg protein. In contrast, the K(m) and V(max) for o-HPA oxidation in mouse liver cytosol were approximately 1.7 microM and 5 nmol/min/mg protein, respectively, yielding a total intrinsic clearance through oxidation to o-HPAA that was 20 times higher in mouse than in rats. Human cytosols (two separate pools) detoxified CE through o-HPA oxidation with an apparent K(m) of 0.84 microM and a V(max) of 5.7 nmol/min/mg protein, for a net intrinsic clearance that was more than 50 times higher than the rat. All species also reduced o-HPA to o-hydroxyphenylethanol (o-HPE), but this was only a major reaction in rats. In the presence of a metabolic reaction replete with all necessary cofactors, GSH conjugation accounted for nearly half of all CE metabolites in rat and mouse, whereas the GSH conjugate represented only 10% of the metabolites in human cytosol. In mouse, o-HPAA represented the major ring-opened metabolite, accounting for the remaining 50% of metabolites, and in human cytosol, o-HPAA was the major metabolite, representing nearly 90% of all CE metabolites. In contrast, no o-HPAA was detected in rats, whereas o-HPE represented a major metabolite. Collectively, these in vitro data implicate o-HPA detoxification through oxidation to o-HPAA as the major determinant of species differences in coumarin-induced hepatotoxicity.

Acetaldehyde↗