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D F Covey

Publications and source records attributed to D F Covey.

At least 91 records · Page 5Linked to original sources
PubMed1990

Convulsant and anticonvulsant cyclopentanones and cyclohexanones.

The convulsant and/or anticonvulsant activity of unsubstituted and mono-alkyl-substituted cyclopentanones and cyclohexanones were examined by testing the ability of these compounds to produce seizures or to inhibit seizures induced by pentylenetetrazol and maximal electroshock in CF-1 mice. In addition, these compounds were tested for their ability to bind to the picrotoxin receptor. The unsubstituted compounds, cyclopentanone and cyclohexanone, prevented both pentylnetetrazol- and maximal electroshock-induced seizures. Cyclopentanones and cyclohexanones with small (less than 3 carbon atoms) alkyl substituents in the 2-position were also anticonvulsant; all of these compounds, except 2-ethylcyclohexanone, blocked both pentylenetrazol- and maximal electroshock-induced seizures. 2-Ethylcyclohexanone was very effective against pentylenetetrazol seizures but did not prevent maximal electroshock seizures. Cyclohexanones with larger alkyl substituents in the 2-position, 2-propylcyclohexanone and 2-t-butylcyclohexanone, caused clonic seizures following injection into mice. Of the cyclopentanones and cyclohexanones with alkyl substitutions in the 3-position that were studied, one was an anticonvulsant (3-methylcyclopentanone), one was a mixed convulsant/anticonvulsant (3-ethylcyclohexanone), and the other two (3-ethylcyclopentanone and 3-t-butylcyclohexanone) were convulsants. Finally, two cyclohexanones with alkyl substituents in the 4-position were studied. Both 4-ethylcyclohexanone and 4-t-butylcyclohexanone produced convulsions when injected into mice. All the neuroactive cyclopentanones and cyclohexanones competitively displaced [35S]t-butylbicyclophosphorothionate, a ligand specific for the picrotoxin receptor, from rat brain membranes. The convulsant compounds were generally more potent than the anticonvulsants. The cyclohexanones were more potent than their corresponding cyclopentanones and the binding potency of both increased as the size of the alkyl substituent increased. These results suggest that cyclopentanone, cyclohexanone, and their alkyl-substituted derivatives act at the picrotoxin receptor to increase or decrease neuronal activity. Thus, they appear to have sites and mechanisms of action similar to those of the neuroactive gamma-butyrolactones and gamma-thiobutyrolactones.

Animals↗
PubMed1990

Binding interactions of convulsant and anticonvulsant gamma-butyrolactones and gamma-thiobutyrolactones with the picrotoxin receptor.

Alkyl-substituted gamma-butyrolactones (GBLs) and gamma-thiobutyrolactones (TBLs) are neuroactive chemicals. beta-Substituted compounds are convulsant, whereas alpha-alkyl substituted GBLs and TBLs are anticonvulsant. The structural similarities between beta-alkyl GBLs and the convulsant picrotoxinin suggested that alkyl substituted GBLs and TBLs act at the picrotoxin receptor. To test this hypothesis we examined the interactions of convulsant and anticonvulsant GBLs and TBLs with the picrotoxin, benzodiazepine and gamma-aminobutyric acid (GABA) binding sites of the GABA receptor complex. All of these convulsants and anticonvulsants studied competitively displaced 35S-t-butylbicyclophosphorothionate (35S-TBPS), a ligand that binds to the picrotoxin receptor. This inhibition of 35S-TBPS binding was not blocked by the GABA antagonist bicuculline methobromide. The convulsant GBLs and TBLs also partially inhibited [3H]muscimol binding to the GABA site and [3H]flunitrazepam binding to the benzodiazepine site, but they did so at concentrations substantially greater than those that inhibited 35S-TBPS binding. The anticonvulsant GBLs and TBLs had no effect on either [3H]muscimol or [3H]flunitrazepam binding. In contrast to the GBLs and TBLs, pentobarbital inhibited TBPS binding in a manner that was blocked by bicuculline methobromide, and it enhanced both [3H]flunitrazepam and [3H]muscimol binding. Both ethosuximide and tetramethylsuccinimide, neuroactive compounds structurally similar to GBLs, competitively displaced 35S-TBPS from the picrotoxin receptor and both compounds were weak inhibitors of [3H] muscimol binding. In addition, ethosuximide also partially diminished [3H]flunitrazepam binding. These data demonstrate that the site of action of alkyl-substituted GBLs and TBLs is different from that of GABA, barbiturates and benzodiazepines. We suggest that the GBLs and TBLs act at the picrotoxin receptor.

4-Butyrolactone↗
PubMed1990

Physiological regulation of the picrotoxin receptor by gamma-butyrolactones and gamma-thiobutyrolactones in cultured hippocampal neurons.

We examined the effects of alkyl-substituted gamma-butyrolactones (GBLs), and gamma-thiobutyrolactones (TBLs) on GABA currents in cultured, voltage-clamped rat hippocampal neurons. Convulsant GBLs and TBLs reversiby diminished GABA responses in a concentration-dependent manner. beta-Ethyl-beta-methyl GBL (beta-EMGBL) completely abolished GABA responses at 3 mM (IC(50)390 microM), while TBL and beta-ethyl-beta-methyl TBL (beta-EMTBL)-induced inhibition of GABA currents was incomplete, saturating at about 50% of control at 300 microM and 10 mM for beta-EMTBL and TBL, respectively. beta-EMGBL and beta-EMTBL both increased the rate of decay of inhibitory post-synaptic currents (IPSCs) and beta-EMGBL also decreased IPSC peak amplitude. In contrast, the anticonvulsant alpha-ethyl-alpha-methyl TBL (alpha-EMTBL) potentiated GABA currents at all GABA concentrations tested; maximal potentiation was 190% of control at 1 mM alpha-EMTBL (EC50 102 microM). Another anticonvulsant alpha-ethyl-alpha-methyl GBL (alpha-EMGBL), potentiated responses to low (0.5 microM) but not high (greater than or equal to 10 microM) GABA. It also blocked the inhibitory effects of picrotoxin and beta-EMGBL and the facilitative effect of alpha-EMTBL on responses to 30 microM GABA. alpha-EMGBL did not interfere with other agents which augment GABA currents. Both alpha-EMTBL and alpha-EMGBL decreased the rate of IPSC decay without altering IPSC peak amplitude. None of these compounds had any direct membrane effects. We propose that beta-alkyl GBLs diminish GABA currents, and therefore, we hypothesize that these compounds are picrotoxin receptor agonists. beta-Alkyl TBLs partially diminish GABA currents and may be partial agonists.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Butyrolactone↗
PubMed1989

Convulsant gamma-butyrolactones block GABA currents in cultured chick spinal cord neurons.

The gamma-aminobutyric acid (GABA) inhibiting properties of the beta-substituted gamma-butyrolactone convulsant, beta-isopropyl-gamma-butyrolactone (beta IPGBL), were studied using gigaseal recording techniques in cultured chick spinal cord neurons. beta IPGBL produced a dose-dependent inhibition of GABA currents with half maximal effect at 92 microM. The effects of beta IPGBL were immediate and completely reversible within minutes after exposure. The inhibition by beta IPGBL showed mixed competitive and non-competitive features with little voltage-dependence. beta IPGBL did not alter the GABA reversal potential nor the degree of GABA desensitization. At a single-channel level, beta IPGBL markedly diminished the opening of GABA channels and decreased the mean channel open time by 30-40% without affecting the amplitude of the single-channel current.

4-Butyrolactone↗
PubMed1989

Effects of steroid D-ring modification on suicide inactivation and competitive inhibition of aromatase by analogues of androsta-1,4-diene-3,17-dione.

Analogues of androsta-1,4-diene-3,17-dione (3a) in which the D ring is modified were prepared and tested as suicide inactivators and competitive inhibitors of human placental aromatase. As long as the five-membered ring is intact, modifications of the D ring such as reduction or removal of the carbonyl group or conversion to a gamma-butyrolactone cause a less than 6-fold decrease in affinity for and rate of inactivation of aromatase, compared to 3a. Thus, an oxygen atom at C-17 is not required for binding of these inhibitors to aromatase, suggesting that hydrogen bonding to the D-ring oxygen does not play a major role in binding. Opening the D ring converts the cyclopentane ring to an alkyl chain and causes a greater than 300-fold decrease in affinity; this can be partially reversed by shortening the chain length. These results are consistent with a model in which the free chain of the opened D ring adopts conformations that sterically interfere with binding of the inhibitor to the enzyme. These findings may have practical applications in drug design, by allowing the preparation of 17-deoxo analogues that have high affinity for aromatase but that are not subject to reduction of the 17-carbonyl group, which is a major mode of metabolism of 3a.

Androstadienes↗
PubMed1988

[19-14C]androstenedione: a new substrate for assaying aromatase and studying its reaction mechanism.

[19-14C]Androstenedione has been prepared and utilized as a substrate for assaying microsomal human placental aromatase. Enzyme activity is determined by measuring the rate at which [14C]formate is produced by aromatization of this 14C-labeled steroid. Isotope ratio experiments using [19-14C]androstenedione and [1 beta-3H]androstenedione demonstrate that an apparent kinetic hydrogen isotope effect exists for the aromatization of the tritiated steroid with kH/kT approximately 1.09. Metabolic switching occurs to a minor extent (approximately 3%) during aromatization of [1 beta-3H]androstenedione, but not during the aromatization of [19-14C]androstenedione.

Androstenedione↗
PubMed1988

Solid-state NMR observation of cysteine and lysine Michael adducts of inactivated estradiol dehydrogenase.

The inactivation of estradiol dehydrogenase by enzyme-generated 3-hydroxy-14,15-secoestra-1,3,5(10)-trien-15-yn-17-one is accompanied by the formation of a lysine enaminone. The experiments leading to this conclusion involved degradation of the inactivated enzyme with Pronase and subsequent analysis by solution-state 13C NMR. The present paper reports solid-state 13C NMR experiments on lyophilized intact inactivated enzyme which are free from problems due to Pronase digestion. These experiments combine conventional cross-polarization and magic-angle spinning with selective irradiation of resonances arising from a 13C double label in the steroid. Magnetization transfer between neighboring 13C nuclei is used to simplify the spectra and to identify peaks due to label. The formation of cysteine and lysine Michael adducts of the enzyme is established by comparisons with chemical shifts of solid model adducts.

17-Hydroxysteroid Dehydrogenases↗
PubMed1988

Alpha-substituted thiobutyrolactones potentiate GABA currents in voltage-clamped chick spinal cord neurons.

The effect of the experimental anticonvulsant, alpha-ethyl,methyl thiobutyrolactone (alpha-EMTBL), on gamma-aminobutyric acid (GABA) currents was examined in cultured chick spinal cord neurons using gigaseal recording techniques. alpha-EMTBL potentiated GABA responses in a dose-dependent fashion with half-maximal effect at 7 microM. alpha-EMTBL also augmented the response to maximal doses of GABA. In competition experiments, alpha-EMTBL relieved the block of GABA currents produced by saturating concentrations of picrotoxinin whereas diazepam and phenobarbital did not. At a single channel level, alpha-EMTBL increased the probability of opening GABA channels without significantly altering the mean channel open time or the single channel conductance. These results indicate that alpha-EMTBL potentiates GABA responses and has properties which distinguish it from benzodiazepines or barbiturates.

4-Butyrolactone↗
PubMed1988

Characterization of pregnant mare's serum gonadotropin-stimulated rat ovarian aromatase and its inhibition by 10-propargylestr-4-ene-3,17-dione.

Aromatase, the important regulatory enzyme that converts androgens to estrogens, is found in relatively high levels in the human placenta. However, since the ovary is the major source of the estrogens in females, we undertook studies to compare the rodent ovarian enzyme with that from human placenta. Pregnant mare's serum gonadotropin (PMSG) markedly increases aromatase activity in the ovaries of immature rats, and this model was used in order to reproducibly obtain high enzyme levels. An injection of PMSG resulted in a specific stimulation of aromatase activity 12 times the increase in ovarian weight in 48 h. Kinetic studies demonstrated that, although the PMSG-stimulated ovarian microsomes had one-tenth the specific activity of the human placenta, the Km values were similar (about 33 and 44 nM, respectively). The potent inhibitor of placenta aromatase, 10-propargylestr-4-ene-3,17-dione, was used to further characterize the enzyme. It inhibited the rat aromatase with an I50 of 36 nM and exhibited time-dependent inhibition with a half-life of inactivation of 16 min and a Ki of 15 nM. These values are similar to those we obtained with the human enzyme (10 nM, 12 min, and 5 nM, respectively). The enzyme parameters in the presence and absence of the inhibitor suggest that the enzymes from the two sources are kinetically quite similar.

Androstenedione↗
PubMed1987

Metabolism of 19-methyl-substituted steroids by human placental aromatase.

The 19-methyl analogues of androstenedione and its aromatization intermediates (19-hydroxyandrostenedione and 19-oxoandrostenedione) were evaluated as substrates of microsomal aromatase in order to determine the effect of a 19-alkyl substituent on the enzyme's regiospecificity. Neither the androstenedione analogue [10-ethylestr-4-ene-3,17-dione (1c)] nor the 19-oxoandrostenedione analogue [10-acetylestr-4-ene-3,17-dione (3c)] was converted to estrogens or oxygenated metabolites by placental microsomes. In contrast, both analogues of 19-hydroxyandrostenedione [10-[(1S)-1-hydroxyethyl]estr-4-ene-3,17-dione (2c) and 10-[(1R)-1-hydroxyethyl]estr-4-ene-3,17-dione (2e)] were converted to the intermediate analogue 3c in a process requiring O2 and either NADH or NADPH. No change in enzyme regiospecificity was detected. The absolute configuration of 2e was determined by X-ray crystallography. Experiments with 18O2 established that 3c generated from 2c retained little 18O (less than 3%), while 3c arising from 2e retained a significant amount of 18O (approximately equal to 70%). All four 19-methyl steroids elicited type I difference spectra from placental microsomes in addition to acting as competitive inhibitors of aromatase (KI = 81 nM, 11 microM, 9.9 microM, and 150 nM for 1c, 2c, 2e, and 3c, respectively). Pretreatment of microsomes with 4-hydroxyandrostenedione (a suicide inactivator of aromatase) abolished the metabolism of 2c and 2e to 3c, as well as the type I difference spectrum elicited by 2c and 2e.(ABSTRACT TRUNCATED AT 250 WORDS)

Aromatase↗
PubMed1987

Cytotoxic activity of a polyamine analogue, monoaziridinylputrescine, against the PC-3 human prostatic carcinoma cell line.

We have previously demonstrated that prostate and prostate-derived rodent tumors can be manipulated into increasing their accumulation of radiolabeled putrescine by alpha-difluoromethylornithine (DFMO)-induced depletion of intracellular putrescine and spermidine. As methods which increase intracellular accumulation of cytotoxic agents often increase the chemotherapeutic effectiveness of the agent, we examined whether an alkylating derivative of putrescine would be cytotoxic to tumor cells. We present here our findings on the cytotoxicity of the aziridinyl derivative of putrescine (AZP) against prostatic cancer cells. The apparent Km for putrescine was 2.5 microM with or without DFMO pretreatment and the apparent Ki for AZP was 1 microM with or without DFMO pretreatment. Intracellular polyamine depletion by DFMO pretreatment resulted in a 3.7-fold greater accumulation of AZP compared to non-DFMO-treated cells. The growth inhibitory activity of AZP was increased with prior polyamine depletion by DFMO with the 50% effective dose decreasing from 18 microM to 2.1 microM. Putrescine was able to block the cytotoxic effect of AZP. Putrescine was also able to rescue the AZP-treated PC-3 cells for up to 6 h following a 1-h exposure to AZP. It appears that aziridinylputrescine behaves like putrescine in that it competes with putrescine for uptake into the cell and, like putrescine, has its uptake into the cell increased by prior polyamine depletion. It differs from putrescine in that it expresses cytotoxic activity and inhibits the growth of the human prostate-derived PC-3 cell line. This cytotoxic activity is also increased by prior polyamine depletion. The cytotoxic behavior of AZP is dependent both on the concentration and duration of exposure. Putrescine can rescue the cells from the effect of AZP. AZP is a potentially useful cytotoxic analogue that utilizes the polyamine transport system for its uptake into the cell.

Cell Line↗
PubMed1986

Inactivation of Streptomyces hydrogenans 20 beta-hydroxysteroid dehydrogenase by an enzyme-generated ethoxyacetylenic ketone in the presence of a thiol.

Replacement of the 21-methyl group of 20 beta-hydroxypregn-4-en-3-one with an ethoxyacetylene group yields a compound that is an excellent substrate (pH 7.4, Km = 2.3 microM, Vmax = 4.6 nmol min-1 micrograms-1) for the Streptomyces hydrogenans NAD(H)-dependent 20 beta-hydroxysteroid dehydrogenase (EC 1.1.1.53). The enzyme-generated ethoxyacetylenic ketone product is a potent inactivator of the enzyme. Gel filtration chromatography of enzyme inactivated with radiolabeled steroid demonstrates that covalent modification of the enzyme has occurred. Both NAD and NADH retard the rate of inactivation, suggesting that only free enzyme is susceptible to covalent modification. Consequently, enzymatically formed ethoxyacetylenic ketone does not react with the enzyme while it is part of the ternary complex. Moreover, the kinetically preferred release of this reactive ketone prior to NADH release assures that enzyme inactivation occurs only when released ketone subsequently encounters free enzyme. Kinetic analysis of inactivations carried out with chemically prepared ethoxyacetylenic ketone and enzyme at pH 7.4 and 9.2 yields bimolecular rate constants for the inactivation process of 1.15 X 10(4) L mol-1 s-1 and 6.94 X 10(4) L mol-1 s-1, respectively. This bimolecular reaction is faster than the bimolecular reaction of the ethoxyacetylenic ketone with either glutathione, mercaptoethanol, or dithiothreitol. Thus, complete inactivation by ketone generated from 5 microM alcohol and 5 microM NAD occurs in 30 min at pH 7.4 in the presence of 1 mM glutathione.

20-Hydroxysteroid Dehydrogenases↗
PubMed1986

Mechanism-based inactivation of 17 beta,20 alpha-hydroxysteroid dehydrogenase by an acetylenic secoestradiol.

14,15-Secoestra-1,3,5(10)-trien-15-yne-3,17 beta-diol (1) is a mechanism-based inactivator of human placental 17 beta,20 alpha-hydroxysteroid dehydrogenase (estradiol dehydrogenase, EC 1.1.1.62). Inactivation with alcohol 1 requires NAD-dependent enzymic oxidation and follows approximately pseudo-first-order kinetics with a limiting t1/2 of 82 min and a "Ki" of 2.0 microM at pH 9.2 and 25 degrees C. At saturating concentrations of NAD, the initial rate of inactivation is slower than in the presence of 5 microM NAD, suggesting that cofactor binding to free enzyme impedes the inactivation process. Glutathione completely protects the enzyme from inactivation at both cofactor concentrations. Inactivation with 45 microM tritiated alcohol 1 followed by dialysis and gel filtration demonstrates a covalent interaction and affords an estimated stoichiometry of 1.4 molecules of steroid per subunit (2.8 per dimer). Chemically prepared 3-hydroxy-14,15-secoestra-1,3,5(10)-trien-15-yn-17-one (2) rapidly inactivates estradiol dehydrogenase with biphasic kinetics. From the latter phase, a Ki of 2.8 microM and a limiting t1/2 of 12 min at pH 9.2 were determined. Estradiol, NADH, and NAD all retard this latter inactivation phase. We propose that enzymatically generated ketone 2 inactivates estradiol dehydrogenase after its release from and return to the active site of free enzyme.

20-Hydroxysteroid Dehydrogenases↗
PubMed1986

Studies of the inactivation of human placental aromatase by 17 alpha-ethynyl-substituted 10 beta-hydroperoxy and related 19-nor steroids.

The inactivation of human placental aromatase by 17 alpha-ethynyl-10 beta-hydroperoxy-17 beta-hydroxy-4-estren-3-one (SCH 10015) was investigated. In either the presence or absence of added NADPH, SCH 10015 (Ki = 41 microM) caused a time-dependent loss of aromatase activity (e.g. 50% loss after 20 min with 20 microM SCH 10015). Evidence for the oxidation of an active site sulfhydryl group as the molecular basis for SCH 10015 inactivation is presented. The contraceptive 17 alpha-ethynyl-substituted 19-nor steroids, norethisterone (Ki = 48 microM) and norethynodrel (Ki = 38 microM), were evaluated and found not to inactivate aromatase, suggesting that the potential conversion of either compound to SCH 10015 did not occur to a significant extent in these microsomal incubations. It is speculated that the previously observed potent contraceptive effects of SCH 10015 may have been the result of irreversible inhibition of estrogen biosynthesis.

Aromatase Inhibitors↗
PubMed1986

Alkyl-substituted thiolo-, thiono-, and dithio-gamma-butyrolactones: new classes of convulsant and anticonvulsant agents.

A series of sulfur-containing congeners have been prepared from alpha-ethyl-alpha-methyl-gamma-butyrolactone, beta-ethyl-beta-methyl-gamma-butyrolactone, and alpha,alpha,beta,beta-tetramethyl-gamma-butyrolactone as potential neuropharmacologic agents. The lactones were treated with benzyl mercaptide anion to form 4-(benzylthio)butyric acid, which, on treatment with trifluoroacetic acid, cyclized to yield thiololactones. The thiono- and dithiolactones were prepared by treating the corresponding lactones either with Lawesson's reagent or with phosphorus pentasulfide, respectively. As had been observed previously for the lactones, the beta-substituted and alpha,beta-substituted congeners were potent convulsants that caused generalized clonic and tonic seizures in mice. The alpha-substituted congeners were effective in inhibiting pentylenetetrazole-induced seizures in mice. alpha-Ethyl-alpha-methylthiolo-gamma-butyrolactone showed an increase in potency over the congeneric alpha-ethyl-alpha-methyl-gamma-butyrolactone and, additionally, was effective against maximal electroshock seizures. In no cases was a convulsant converted to an anticonvulsant or vice versa by sulfur-for-oxygen substitution.

4-Butyrolactone↗