Biomedical subjects

D F Covey

Publications and source records attributed to D F Covey.

At least 55 records · Page 3Linked to original sources

PubMed1998

Structure-activity studies of fluoroalkyl-substituted gamma-butyrolactone and gamma-thiobutyrolactone modulators of GABA(A) receptor function.

Dihydro-2(3H)-furanones (gamma-butyrolactones) and dihydro-2(3H)-thiophenones (gamma-thiobutyrolactones) containing fluoroalkyl groups at positions C-3, C-4, and C-5 of the heterocyclic rings were prepared. The anticonvulsant/convulsant activities of the compounds were evaluated in mice. Brain concentrations of the compounds were determined and the effects of the compounds on [35S]-tert-butylbicyclophosphorothionate ([35S]TBPS) binding to the picrotoxin site on GABAA receptors were investigated. The effects of the compounds on GABAA receptor function were studied using electrophysiological methods and cultured rat hippocampal neurons. Fluorination at C-3 results in either subtle or pronounced effects on the pharmacological activity of the compounds. When hydrogens are replaced with fluorines at the methylene carbon of an ethyl group, as in 3-(1,1-difluoroethyl)dihydro-3-methyl-2(3H)-furanone (1), the anticonvulsant actions of the compound are not much changed from those found for the corresponding alkyl-substituted analogue. In marked contrast, fluorination at the methyl carbon of the ethyl group, as in dihydro-3-methyl-3-(2,2,2-trifluoroethyl)-2(3H)-furanone (3), produces a compound having convulsant activity. This convulsant activity seems to be due to an increased affinity of the compound for the picrotoxin site on GABAA receptors caused by an interaction that involves the trifluoromethyl group. Results obtained with gamma-butyrolactones containing either a 3-(1-trifluoromethyl)ethyl or a 3-(1-methyl-1-trifluoromethyl)ethyl substituent indicate that the interactions of the trifluoromethyl group with the picrotoxin binding site are subject to both stereochemical and steric constraints. Sulfur for oxygen heteroatom substitution, as in the corresponding gamma-thiobutyrolactones, affects the type (competitive, non-competitive, etc.) of binding interactions that these compounds have with the picrotoxin site in a complex manner. Fluorination of alkyl groups at the C-4 and C-5 positions of gamma-butyrolactones having convulsant activity increases convulsant potency.

4-Butyrolactone↗

PubMed1998

The anesthetic steroid (+)-3alpha-hydroxy-5alpha-androstane-17beta-carbonitrile blocks N-, Q-, and R-type, but not L- and P-type, high voltage-activated Ca2+ current in hippocampal and dorsal root ganglion neurons of the rat.

High voltage-activated (HVA) Ca2+ current (ICa) was recorded from neonatal rat hippocampal and adult rat dorsal root ganglion neurons. In both cell types, (+)-3alpha-hydroxy-5alpha-androstane-17beta-carbonitrile [(+)-ACN], a neuroactive steroid, had no effect on nifedipine- (L-type) or omega-agatoxin IVA- (P-type) sensitive ICa. Selective blockade of N-type current with omega-conotoxin GVIA and of Q-type current with omega-conotoxin MVIIC indicated that (+)-ACN inhibits both N- and Q-type current components in both cell types. Current persisting after blockade of all other current components (R-type) was also sensitive to (+)-ACN. Half-blockade of (+)-ACN-sensitive HVA current occurred in the range of 3-25 microM, with N-type current somewhat more sensitive than Q- or R-type. The (+)-ACN enantiomer, (-)-ACN, and pregnanolone were somewhat less effective at inhibiting total HVA current than (+)-ACN, whereas several steroid analogs, including alfaxalone, were relatively ineffective at inhibiting total HVA current. Neither guanosine-5'-O-(2-thio)diphosphate nor guanosine-5'-O-(3-thio)triphosphate altered the ability of (+)-ACN to inhibit HVA current in dorsal root ganglion neurons, indicating that (+)-ACN acts directly on Ca2+ channels. The partial selectivity exhibited by (+)-ACN among different HVA current components suggests that manipulations of steroid analogues may be a useful strategy in the generation of more selective, more potent, small-molecular-weight HVA channel blockers.

Androstanols↗

PubMed1998

Enantioselective blockade of T-type Ca2+ current in adult rat sensory neurons by a steroid that lacks gamma-aminobutyric acid-modulatory activity.

A number of steroids seem to have anesthetic effects resulting primarily from their ability to potentiate currents gated by gamma-aminobutyric acidA (GABAA) receptor activation. One such compound is (3alpha,5alpha, 17beta)-3-hydroxyandrostane-17-carbonitrile [(+)-ACN]. We were interested in whether carbonitrile substitution at other ring positions might result in other pharmacological consequences. Here we examine effects of (3beta,5alpha, 17beta)-17-hydroxyestrane-3-carbonitrile [(+)-ECN] on GABAA receptors and Ca2+ channels. In contrast to (+)-ACN, (+)-ECN does not potentiate GABAA-receptor activated currents, nor does it directly gate GABAA-receptor mediated currents. However, both steroids produce an enantioselective reduction of T-type current. (+)-ECN blocked T current with an IC50 value of 0.3 microM with a maximal block of 41%. (+)-ACN produced a partial block of T current (44% maximal block) with an IC50 value of 0.4 microM. Block of T current showed mild use- and voltage-dependence. The (-)-ECN enantiomer was about 33 times less potent than (+)-ECN, with an IC50 value of 10 microM and an amount of maximal block comparable to (+)-ECN. (+)-ECN was less effective at blocking high-voltage-activated Ca2+ current in DRG neurons (IC50 value of 9. 3 microM with maximal block of about 27%) and hippocampal neurons. (+)-ECN (10 microM) had minimal effects on voltage-gated sodium and potassium currents in rat chromaffin cells. The results identify a steroid with no effects on GABAA receptors that produces a partial inhibition of T-type Ca2+ current with reasonably high affinity and selectivity. Further study of steroid actions on T currents may lead to even more selective and potent agents.

Anesthetics↗

PubMed1998

Inhibition of voltage-dependent sodium channels by the anticonvulsant gamma-aminobutyric acid type A receptor modulator, 3-benzyl-3-ethyl-2-piperidinone.

3-Benzyl-3-ethyl-2-piperidinone (3-BEP) belongs to a family of compounds that includes alpha- substituted gamma-butyrolactones, gamma-thiobutyrolactones, 2-pyrrolidinones and hexahydro-2H-azepin-2-ones. Many of these drugs exhibit potent in vivo anticonvulsant activity in mice. Previous electrophysiological studies demonstrated that they potentiate gamma-aminobutyric acid- (GABA) mediated chloride currents. This GABAA receptor modulation was thought to be the main mechanism of anticonvulsant activity. We report that 3-BEP also modulates sodium channels. It decreased sodium currents in cultured rat hippocampal neurons in a voltage- and concentration-dependent manner. The drug's apparent affinity increased as neurons were depolarized. At a holding potential of -60 mV, the apparent IC50 was 487 microM. This concentration is comparable to its EC50 for GABAA modulation (575 microM). Current blockade occurred over all activation voltages tested. The steady state inactivation curve was shifted by 600 microM 3-BEP from V50 = -65.3 mV to -72.0 mV, and recovery from inactivation was slowed from tau = 4.9 to 12.8 msec. Sodium current inhibition was not observed for three related compounds, suggesting a degree of chemical specificity for this activity. We conclude that in addition to its known effects on GABAA receptors, 3-BEP modulates sodium channels. Therefore this compound may prevent seizures by both enhancing inhibition and diminishing neuronal excitability.

Animals↗

PubMed1997

Synthesis and anticonvulsant activities of 3,3-dialkyl- and 3-alkyl-3-benzyl-2-piperidinones (delta-valerolactams) and hexahydro-2H-azepin-2-ones (epsilon-caprolactams).

A series of 3-substituted 2-piperidinone (delta-valerolactam) and hexahydro-2H-azepin-2-one (epsilon-caprolactam) derivatives were prepared and evaluated as anticonvulsants in mice. In the 2-piperidinone series, 3,3-diethyl compound 7b is the most effective anticonvulsant against pentylenetetrazole-induced seizures (ED50, 37 mg/kg; PI (TD50/ED50), 4.46), and 3-benzyl compound 4c (ED50, 41 mg/kg; PI, 7.05) is the most effective anticonvulsant against seizures induced by maximal electroshock. By contrast, none of the epsilon-caprolactams tested had anticonvulsant effects below doses causing rotorod toxicity. log P values were correlated with neurotoxicity and [35S]TBPS displacement, but not with anticonvulsant activity. Electrophysiological evaluations of selected compounds from each series indicated that both the delta-valero-lactams and epsilon-caprolactams potentiated GABA-mediated chloride currents in rat hippocampal neurons.

Animals↗

PubMed1997

Lactone modulation of the gamma-aminobutyric acid A receptor: evidence for a positive modulatory site.

The gamma-aminobutyric acid-A (GABA(A)) receptor complex is allosterically modulated by a variety of substances, some of clinical importance. Barbiturates and neurosteroids augment GABA-currents and also directly gate the channel. A variety of gamma-butyrolactone analogues also modulate GABA-induced currents, with some potentiating and others inhibiting. Because several gamma-thiobutyrolactone analogues have biphasic effects on GABA currents, experiments with wild-type and picrotoxinin-insensitive GABA(A) receptors were performed to analyze whether some gamma-thiobutyrolactones interact with two distinguishable sites on the GABA(A) receptor. beta-Ethyl-beta-methyl-gamma-thiobutyrolactone inhibited GABA-induced currents at low concentrations (0.001-1 mM), but potentiated GABA-induced currents at higher concentrations (3-10 mM) in wild-type alpha1beta2gamma2-subunit containing ionophores. The related alpha-ethyl-alpha-methyl-gamma-thiobutyrolactone potentiated submaximal GABA currents in wild-type receptors at both low and high concentrations (0.1-10 mM). Mutations in the second transmembrane domain of alpha1, beta2, or gamma2 conferred picrotoxinin-insensitivity onto GABA(A) receptor complexes. When these mutated alpha1, beta2, or gamma2 subunits were incorporated into the receptor complex, beta-ethyl-beta-methyl-gamma-thiobutyrolactone potentiated GABA currents over the entire concentration range (0.1-10 mM). Neither the potentiating activity nor the EC50 of alpha-ethyl-alpha-methyl-gamma-thiobutyrolactone changed in the mutant receptors. Further studies demonstrated that the mutations did not affect the EC50 of chlordiazepoxide or phenobarbital. These and our earlier results identify a modulatory site on the GABA(A) receptor distinct from that interacting with barbiturates, benzodiazepines, and steroids. Additionally, they show that the gamma-butyrolactones probably interact at two different sites on the ionophore to produce opposite effects on GABA-mediated current.

4-Butyrolactone↗

PubMed1997

Voltage-dependent calcium channels as targets for convulsant and anticonvulsant alkyl-substituted thiobutyrolactones.

Alkyl-substituted thiobutyrolactones increase or decrease gamma-aminobutyric acidA responses at or near the picrotoxin site, but they are structurally similar to ethosuximide, which prompted us to determine the actions of thiobutyrolactones on voltage-dependent Ca++ currents. We measured Ca++ currents in cultured neonatal rat dorsal root ganglion neurons in the absence and presence of the anticonvulsant alpha-ethyl,alpha-methyl-gamma-thiobutyrolactone (alpha-EMTBL) and the convulsant beta-ethyl,beta-methyl-gamma-thiobutyrolactone (beta-EMTBL). Low-voltage-activated (T-type) currents were reduced in a concentration-dependent manner, with a maximal reduction of 26% and 30% by alpha-EMTBL and beta-EMTBL, respectively. alpha-EMTBL reduced high-voltage-activated currents in a concentration- and voltage-dependent manner: maximal responses were 7% when evoked from -80 mV, with more rapid current inactivation; 29% when evoked from -40 mV, with little effect on current inactivation. beta-EMTBL increased high-voltage-activated currents < or = 20% at 10 to 300 microM, but reduced currents at higher concentrations; the latter action was similar to that of alpha-EMTBL in its magnitude and voltage dependence. Block of N-type channels with omega-conotoxin GVIA (10 microM) reduced the effect of alpha-EMTBL and eliminated its voltage dependence. The L-type current component was also reduced by alpha-EMTBL, with little effect on P- or Q-type current components. The related compound, alpha-ethyl,alpha-methyl-gamma-butyrolactone, had no effect on Ca++ currents. We conclude that thiobutyrolactones affect voltage-dependent Ca++ currents in a concentration- and voltage-dependent manner, with greater potency on low-voltage. activated channels. Both the ring structure and the position of its alkyl substitutions determine the identity of the targeted Ca++ channel subtypes and the manner of regulation.

4-Butyrolactone↗

PubMed1996

Neurosteroid analogues. 4. The effect of methyl substitution at the C-5 and C-10 positions of neurosteroids on electrophysiological activity at GABAA receptors.

A series of analogues of the neuroactive steroids 3 alpha-hydroxy-5 alpha-pregnan-20-one and 3 alpha-hydroxy-5 beta-pregnan-20-one were studied to elucidate the mode of binding of 5 alpha-and 5 beta-reduced steroids to steroid binding sites on GABAA receptors. Analogues which were either 3 alpha-hydroxy-20-ketosteroids or 3 alpha-hydroxysteroid-17 beta-carbonitriles and which contained various methyl group substitution patterns at C-5 and C-10 were prepared. Evaluations utilized whole-cell patch clamp electrophysiological methods carried out on cultured rat hippocampal neurons, and the results obtained with the rigid 17 beta-carbonitrile analogs were analyzed using molecular modeling methods. The molecular modeling results provide a rationale for the observation that the configuration of the hydroxyl group at C-3 is a greater determinant of anesthetic potency than the configuration of the A,B ring fusion at C-5. The electrophysiological results identify steric restrictions for the space that can be occupied in 5 alpha- and 5 beta-reduced steroid modulators of GABAA receptors in the regions of space proximate to the steroid C-5, C-10, and possibly C-4 positions. This information is useful for the development of nonsteroidal analogues that can modulate GABAA receptors via interactions at steroid binding sites.

Animals↗

PubMed1996

3,3-Dialkyl- and 3-alkyl-3-benzyl-substituted 2-pyrrolidinones: a new class of anticonvulsant agents.

A series of 3,3-dialkyl- and 3-alkyl-3-benzyl-substituted 2-pyrrolidinones (lactams) have been prepared and evaluated for their anticonvulsant activities. In the pentylenetetrazole mouse seizure model, 3,3-diethyl lactam 7c and 3-benzyl-3-ethyl lactam 7j are the most effective anticonvulsants (ED50 = 46 and 42 mg/kg, respectively) and have protective index (PI = TD50/ED50) values of 5.65 and 3.00, respectively. These protective index values compare favorably to those of the clinically used antiepileptic drugs ethosuximide (ED50 = 161 mg/kg), phenobarbital (ED50 = 22 mg/kg), and valproic acid (ED50 = 133 mg/kg), which have PI values of 2.35, 4.00, and 2.12, respectively. The benzyl compounds [3-substituents are Bn, H (7h); Bn, Me (7i); and Bn, Et (7j)] are also very effective anticonvulsants against seizures induced by maximal electroshock (ED50 = 41, 55, and 74 mg/kg, respectively) and have PI values of 3.51, 3.04, and 1.70, respectively. The corresponding PI values for phenobarbital and valproic acid are 1.37 and 5.18, respectively. As a class of anticonvulsants, the 3,3-disubstituted 2-pyrrolidinones have a broad spectrum of action and may be useful for the treatment of human epilepsies.

Animals↗

PubMed1996

Physiological comparison of alpha-ethyl-alpha-methyl-gamma-thiobutyrolactone with benzodiazepine and barbiturate modulators of GABAA receptors.

The GABAA receptor/chloride ionophore (GABAR) is allosterically modulated by several classes of anticonvulsant agents, including benzodiazepines and barbiturates, and some alkyl-substituted butyrolactones. To test the hypothesis that the anticonvulsant butyrolactones act at a distinct positive-modulatory site on the GABAR, we examined the physiological effects of a butyrolactone, a benzodiazepine and a barbiturate on GABA-mediated currents in voltage-clamped neurons and cells transfected with various subunit combinations. The butyrolactone, alpha-ethyl-alpha-methyl-gamma-thiobutyrolactone (alpha EMTBL), altered the EC50 for GABA and changed the apparent cooperativity of GABA responses. In contrast, the benzodiazepine chlordiazepoxide altered the EC50 for GABA with no effect on apparent cooperativity. The barbiturate phenobarbital altered both the EC50 and the amplitude of the maximal GABA response without altering apparent cooperativity. The GABA-mediated effect of the barbiturate, but not the benzodiazepine, added to the maximal effect of the butyrolactone, supporting the hypothesis that butyrolactones do not exert their effect at the barbiturate effector site. Both alpha EMTBL and phenobarbital potentiated GABA currents in transfected cells containing the alpha 1 beta 2 and alpha 1 gamma 2 subunit combinations, as well as alpha 1 subunits alone. Chlordiazepoxide had the minimum requirement of an alpha subunit and a gamma subunit. Specific GABARs lacking benzodiazepine or barbiturate modulation were tested for modulation by alpha EMTBL. The alpha 6 beta 2 gamma 2 combination was modulated by the butyrolactone but not chlordiazepoxide. However, GABARs comprising rho1 subunits were sensitive to both phenobarbital and alpha EMTBL. Although the molecular determinants for alpha EMTBL action appear similar to the barbiturates, our data support the conclusion that alpha EMTBL interacts with GABARs in a distinct manner from barbiturates and benzodiazepines.

4-Butyrolactone↗

PubMed1996

Effects of neurosteroid and benz[e]indene enantiomers on GABAA receptors in cultured hippocampal neurons and transfected HEK-293 cells.

The effects of the enantiomers of the neurosteroid, 3 alpha-hydroxy-5 alpha-pregnan-20-one (DHP), and the benz[e]indene, BI-1, on gamma-aminobutyric acid (GABA) responses were studied using whole-cell recording techniques in cultured rat hippocampal neurons and human embryonic kidney cells (HEK-293) transfected with either alpha 1 beta 2 gamma 2 or alpha 6 beta 2 gamma 2 GABAA receptor subunits. At 10 microM, the (+)-enantiomers enhanced currents gated by 2 microM GABA in all cells, whereas the (-)-enantiomers were significantly less effective. The enhancement of 2 microM GABA responses in HEK-293 cells transfected with alpha 6 beta 2 gamma 2 subunits was about half that of hippocampal neurons or HEK-293 cells transfected with alpha 1 beta 2 gamma 2. The lower sensitivity of alpha 6 beta 2 gamma 2 receptors for (+)-DHP and (+)-BI-1 is accounted for by their greater apparent affinity for GABA. When the GABA concentration was decreased to 0.5 microM to take into account the four-fold higher apparent affinity of alpha 6 beta 2 gamma 2 receptors, these receptors exhibited enhancement similar to alpha 1 beta 2 gamma 2 receptors. These results indicate that both native and recombinant GABAA receptors have enantioselective sites at which neurosteroids and benz[e]indenes modulate GABA responses, and that differences in agonist affinity contribute to apparent differences in steroid sensitivity among GABAA receptors.

Animals↗

PubMed1996

Enantioselectivity of steroid-induced gamma-aminobutyric acidA receptor modulation and anesthesia.

Neuroactive steroids have been postulated to cause anesthesia by binding to unique steroid recognition sites on gamma-aminobutyric acid (GABA) receptors and modulating GABA receptor function. Steroids interact with these sites diastereoselectively, but it is unknown whether steroid sites show enantioselectivity. To address this issue, we synthesized enantiomers to (+)-3alpha-hydroxy-5alpha-androstane-17beta-carbonitrile and (+)-3alpha-hydroxy-5alpha-pregnan-20-one. In this study, we show that potentiation of GABA-mediated currents and gating of the GABA(A) channel by steroids, as well as steroid-induced anesthesia in tadpoles and mice, is enantioselective, with the (+)-enantiomers exhibiting significantly greater potency in all assays. The correlation between the effects of steroid enantiomers on channel behavior and their effects as anesthetics provides strong evidence that GABA(A) receptors play a predominant role in steroid-induced anesthesia. The enantiomers also provide a tool to probe the relative contributions of direct chloride channel activation versus potentiation of GABA-elicited currents to the induction of anesthesia. Studies examining the effects of combinations of (+)- and (-)-3alpha-hydroxy-5alpha-androstane-17beta-carbonitrile were consistent with the hypothesis that potentiation of GABA-activated currents contributes to steroid-induced anesthesia but indicated that direct steroid activation of GABA(A) receptors is not mechanistically important in producing anesthesia.

Androstanols↗

PubMed1995

Neurosteroid analogues. 3. The synthesis and electrophysiological evaluation of benz[e]indene congeners of neuroactive steroids having the 5 beta-configuration.

A series of 7-(2-hydroxyethyl)benz[e]indene analogues of 3 alpha-hydroxy-5 beta-pregnan-20-one (7), a neuroactive steroid known to be a positive allosteric modulator of GABAA receptor function, was prepared. Electrophysiological measurements carried out on cultured rat hippocampal neurons were used to evaluate the modulatory effects of the analogues on GABAA receptor function. Analogues were tested for their ability to potentiate 1 microM GABA-mediated chloride currents and for their ability to directly gate chloride currents at this ligand-gated ion channel. Active analogues typically enhanced GABA-mediated currents at concentrations below those required to directly gate chloride currents. The dose-response relationships for potentiation of 1 microM GABA-mediated chloride currents were studied for [3S-(3 alpha, 3a alpha, 5a beta, 7 beta, 9a alpha, 9b beta)]-1- [dodecahydro-7-(2-hydroxyethyl)-3a-methyl-1H-benz[e]inden-3- yl]ethanone (3), steroid 7, 3 alpha-hydroxy-5 alpha-pregnan-20-one (5), and the analogous 7 alpha-(2-hydroxyethyl)benz[e]indene analogue of steroid 5 (compound 1). Compound 3 was the most active potentiator (EC50 = 0.017 microM) of GABA-mediated current. The direct gating actions of compound 3 were not observed at a concentration of 1 microM, but were observed at a concentration of 10 microM.

Animals↗

PubMed1995

Localization of tolbutamide binding sites on human serum albumin using titration calorimetry and heteronuclear 2-D NMR.

The sulfonylureas are a class of oral hypoglycemic agents used to treat type II diabetes mellitus, and tolbutamide is a "first generation" member of this family. It is a nonpolar, weakly acidic drug that binds to serum albumin in the circulation. In the present study, we have examined the interactions of tolbutamide with human serum albumin by isothermal titration calorimetry and heteronuclear multiple-quantum coherence NMR spectroscopy. Calorimetric titrations revealed that tolbutamide binds to albumin at three independent sites with the same or comparable affinity. This result was independently confirmed by NMR experiments which resolved three resonances at 1H chemical shifts of 2.07, 2.11 and 2.14 ppm, corresponding to [methyl-13C]tolbutamide bound to three discrete binding sites. The binding affinity quantitated by calorimetry (Kd = 21 +/- 9 microM at pH 7.4, 37 degrees C) was approximately 5 times lower than the most frequently reported value. Tolbutamide titrations of albumin complexed with three other drugs whose binding sites have been localized by X-ray crystallography (salicylate, clofibric acid, and triiodobenzoic acid) demonstrated direct competition for common binding sites. NMR experiments with samples containing [methyl-13C]tolbutamide and these competing drugs permitted assignment of the resonances at 2.07 and 2.14 ppm to tolbutamide bound to the aspirin sites in albumin subdomains IIIA and IIA, respectively. These findings permit the first assignment of tolbutamide binding sites to specific locations on the albumin molecule within the context of the recently published crystal structure of human serum albumin. In addition, this information provides a molecular basis for predicting unfavorable drug interactions involving tolbutamide in patients with type II diabetes.

Binding Sites↗

PubMed1995

Synthesis of (17R)- and (17S)-17-hydroxy-14, 15-secoandrost-4-en-15-yn-3-one and the X-ray crystal structure of the (17S)-diastereomer.

(17R,S)-17-Hydroxy-14,15-secoandrost-4-en-15-yn-3-one has been shown previously to be a mechanism-based inactivator of rat liver 3 alpha-hydroxysteroid dehydrogenase. This manuscript describes the synthesis of this diastereomeric 14,15-secosteroid from [2S-2 alpha,4a alpha,4b beta,10a beta)]- 1,2,3,4a,4b,7,9,10,10a-decahydro-2,4b-dimethyl-7-oxo-2-phenanth renecarboxylic acid methyl ester. The separation of this diastereomeric 14,15-secosteroid into (17R)- and (17S)-17-hydroxy-14,15-secoandrost-4-en-15-yn-3-one was accomplished by HPLC separation of the (S)-1-[(4-methylphenyl)sulphonyl]-2-pyrrolidinecarboxylate derivatives on a silica column. The crystal structure of (17S)-17-hydroxy-14,15-secoandrost-4-en-15-yn-3-one was then solved by X-ray diffraction analysis to establish unambiguously the absolute configuration of the diastereomeric 14,15-secosteroid.

3-Hydroxysteroid Dehydrogenases↗

PubMed1995

Synthesis of (17R)- and (17S)-17-hydroxy-14, 15-secoandrost-4-en-15-yn-3-one and the X-ray crystal structure of the (17S)-diastereomer.

(17R,S)-17-Hydroxy-14,15-secoandrost-4-en-15-yn-3-one has been shown previously to be a mechanism-based inactivator of rat liver 3 alpha-hydroxysteroid dehydrogenase. This manuscript describes the synthesis of this diastereomeric 14,15-secosteroid from [2S-(2 alpha,4a alpha,4b beta, 10a beta)]- 1,2,3,4a,4b,7,9,10,10a-decahydro-2,4b-dimethyl-7-oxo-2-phenanth renecarboxylic acid methyl ester. The separation of this diastereomeric 14,15-secosteroid into (17R)- and (17S)-17-hydroxy-14,15-secoandrost-4-en-15-yn-3-one was accomplished by HPLC separation of the (S)-1-[(4-methylphenyl)sulphonyl]-2-pyrrolidinecarboxylate derivatives on a silica column. The crystal structure of (17S)-17-hydroxy-14,15-secoandrost-4-en-15-yn-3-one was then solved by X-ray diffraction analysis to establish unambiguously the absolute configuration of the diastereomeric 14,15-secosteroid.

3-Hydroxysteroid Dehydrogenases↗

PubMed1995

Interaction of picrotoxin with GABAA receptor channel-lining residues probed in cysteine mutants.

We used the substituted-cysteine-accessibility method to identify the channel-lining residues in a region (257-261) near the putative cytoplasmic end of the M2 membrane-spanning segment of the rat gamma-aminobutyric acid type A (GABAA) receptor alpha 1 subunit. The residues alpha 1Val257 and alpha 1Thr261 were accessible to charged, sulfhydryl-specific reagents applied extracellularly in both the open and closed states. The accessibility of alpha 1V257C and alpha 1T261C in the closed state implies that the gate must be at least as close to the cytoplasmic end of the channel as alpha 1Val257. Also, the positively charged reagent methanethiosulfonate ethylammonium penetrated from the extracellular end of the channel to alpha 1T261C, with which it reacted, indicating that the anion-selectivity filter is closer to the cytoplasmic end of the channel than this residue is. Co-application of picrotoxin prevented the sulfhydryl reagents from reacting with alpha 1V257C but did not prevent reaction with the more extracellular residue alpha 1T261C. Picrotoxin protection of alpha 1V257C may be due to steric block by picrotoxin bound in the channel at the level of alpha 1Val257; however, if this protection is allosteric, it is not due to the induction of the resting closed state in which alpha 1V257C was accessible to sulfhydryl reagent.

Amino Acid Sequence↗

PubMed1995

Dual modulation of the gamma-aminobutyric acid type A receptor/ionophore by alkyl-substituted gamma-butyrolactones.

Alkyl-substituted gamma-butyrolactones (GBLs) and gamma-thiobutyrolactones exhibit convulsant or anticonvulsant activity, depending on the alkyl substituents. alpha-Substituted lactones with small alkyl substituents are anticonvulsant and potentiate gamma-aminobutyric acid (GABA)-mediated chloride currents, whereas beta-substituted compounds are usually convulsant and block GABAA currents. We have now found that this distinction is not so clear-cut, in that some compounds can both block and augment GABAA currents, but with different time courses. For example, alpha,alpha-diisopropyl-GBL (alpha-DIGBL) potentiates exogenous GABA currents in cultured rat hippocampal neurons but diminishes GABA-mediated inhibitory postsynaptic currents. A more detailed analysis demonstrates a triphasic effect of alpha-DIGBL on GABA currents, with a rapid inhibitory phase, a slower potentiating phase, and then an "off response" when the GABA/alpha-DIGBL perfusion is stopped. Thus, alpha-DIGBL can inhibit and potentiate GABA currents with kinetically different time courses. Inhibition is more rapid, but at steady state potentiation dominates. Using a simplified model of the GABAA receptor/ionophore, we have simulated our experimental observations with alpha-DIGBL. Another lactone, beta-ethyl-beta-methyl-gamma-thiobutyrolactone, also has dual actions, with inhibition predominating at low concentrations and potentiation predominating at high concentrations. We propose two distinct GBL modulatory sites on the GABAA receptor, i.e., an inhibitory "picrotoxin" site and an enhancing "lactone site." New information on the structure of the GABAA receptor/ionophore may allow the molecular dissection of these two sites.

4-Butyrolactone↗