Predicting pharmacokinetics of drugs using physiologically based modeling--application to food effects.

Our knowledge of the major mechanisms underlying the effect of food on drug absorption allows reliable qualitative prediction based on biopharmaceutical properties, which can be assessed during the pre-clinical phase of drug discovery. Furthermore, several recent examples have shown that physiologically based absorption models incorporating biorelevant drug solubility measurements can provide quite accurate quantitative prediction of food effect. However, many molecules currently in development have distinctly sub-optimal biopharmaceutical properties, making the quantitative prediction of food effect for different formulations from in vitro data very challenging. If such drugs reach clinical development and show undesirable variability when dosed with food, improved formulation can help to reduce the food effect and carefully designed in vivo studies in dogs can be a useful guide to clinical formulation development. Even so, such in vivo studies provide limited throughput for screening, and food effects seen in dog cannot always be directly translated to human. This paper describes how physiologically based absorption modeling can play a role in the prediction of food effect by integrating the data generated during pre-clinical and clinical research and development. Such data include physicochemical and in vitro drug properties, biorelevant solubility and dissolution, and in vivo pre-clinical and clinical pharmacokinetic data. Some background to current physiological absorption models of human and dog is given, and refinements to models of in vivo drug solubility and dissolution are described. These are illustrated with examples using GastroPlus to simulate the food effect in dog and human for different formulations of two marketed drugs.

Three-dimensional structure of fibrolase, the fibrinolytic enzyme from southern copperhead venom, modeled from the X-ray structure of adamalysin II and atrolysin C.

The fibrinolytic enzyme from southern copperhead snake venom, fibrolase, contains 1 mole of zinc per mole of protein, belongs to the major family of metalloproteinases known as the metzincins, and has been shown to degrade fibrin clots in vitro and in vivo. The purpose of this study was to develop a 3-dimensional model of fibrolase to investigate the geometry of conserved and variable sequences between members of the snake venom metalloproteinases. When compared to atrolysin C (form D) or adamalysin II (metzincins with completely different substrate specificity), fibrolase has approximately 60% overall sequence identity and nearly 100% sequence similarity in the active site. We used the crystal structure of adamalysin II to build a 3-dimensional homology model of fibrolase. Three disulfide bonds were constructed (the highly conserved disulfide bond [118-198] was maintained from the adamalysin II structure and 2 new disulfide bonds were introduced between residues 158-182 and 160-165). We used Sculpt 2.5 and HyperChem 5.0 to "dock" a substrate fragment octapeptide (HTEKLVTS), and a water molecule into the active site cleft. We calculated the differential average homology profile for fibrolase compared to 8 hemorrhagic and 5 nonhemorrhagic metzincins. We then determined the sequence regions that might be responsible for their substrate specificity. Our 3-dimensional homology model shows that the variable sequences lie on the periphery of the identified active site region containing the His triangle; this indicates that substrate specificity may depend on surface residues that are not directly associated with the active site.

Pressure-sensitive and -insensitive coupling in gamma-aminobutyric acid(A) receptors.

RATIONALE: Previous behavioral and biochemical studies suggest that allosteric coupling processes initiated by benzodiazepines, barbiturates and neuroactive steroids can be sub-categorized on the basis of their sensitivities to antagonism by increased atmospheric pressure. However, biochemical evidence supporting this hypothesis was limited to single concentration studies in long sleep (LS) mice. OBJECTIVE: The present paper addresses these issues by extending biochemical investigation of pressure effects on allosteric modulators across a range of concentrations that allosterically enhance gamma-aminobutyric acid (GABA)A receptor function and alter behavior using two mouse genotypes. In addition, the effects of pressure on ligand binding were explored to further investigate the mechanism of pressure antagonism of allosteric modulation. METHODS: The effects of 12 times normal atmospheric pressure (ATA) of helium-oxygen gas (heliox) on allosteric modulation of GABA(A) receptor function and [3H]flunitrazepam binding was tested in LS and C57BL mouse brain membranes (microsacs) using chloride flux and high-affinity binding assays. RESULTS: In both genotypes, exposure to 12 ATA heliox antagonized the allosteric enhancement of GABA(A) receptor function by flunitrazepam (0.1-10 microM) and pentobarbital (0.1-50 microM) but did not affect allosteric modulation by 3alpha-hydroxy-5beta-pregnan-20-one (0.1-1 microM). Pressure did not affect benzodiazepine receptor affinity (Kd) or the number of benzodiazepine receptors (Bmax). THE RESULTS: (1) confirm that there are differences in sensitivity to pressure antagonism of allosteric coupling among GABA(A) allosteric modulators; (2) demonstrate that these differences are not concentration or genotype dependent; (3) add evidence that pressure antagonizes allosteric modulation by uncoupling the receptor and (4) support the hypothesis that allosteric modulation of receptor function can be sub-categorized on the basis of sensitivity to pressure antagonism.

Predicting the impact of physiological and biochemical processes on oral drug bioavailability.

Recent advances in computational methods applied to the fields of drug delivery and biopharmaceutics will be reviewed with a focus on prediction of the impact of physiological and biochemical factors on simulation of gastrointestinal absorption and bioavailability. Our application of a gastrointestinal simulation for the prediction of oral drug absorption and bioavailability will be described. First, we collected literature data or we estimated biopharmaceutical properties by application of statistical methods to a set of 2D and 3D molecular descriptors. Second, we integrated the differential equations for an advanced compartmental absorption and transit (ACAT) model in order to determine the rate, extent, and approximate gastrointestinal location of drug liberation (for controlled release), dissolution, passive and carrier-mediated absorption, and saturable metabolism and efflux. We predict fraction absorbed, bioavailability, and C(p) vs. time profiles for common drugs and compare those estimates to literature data. We illustrate the simulated impact of physiological and biochemical processes on oral drug bioavailability.

Biopharmaceutics of transmucosal peptide and protein drug administration: role of transport mechanisms with a focus on the involvement of PepT1.

Non-invasive delivery of peptide and protein drugs will soon become a reality. This is due partly to a better understanding of the endogenous transport mechanisms, including paracellular transport, endocytosis, and carrier-mediated transport of mucosal routes of peptide and protein drug administration. This paper focuses on work related to the elucidation of structure-function, intracellular trafficking, and regulation of the intestinal dipeptide transporter, PepT1.

In vivo and in vitro hyperbaric studies in mice suggest novel sites of action for ethanol.

The present study uses increased atmospheric pressure as an ethanol antagonist to test the hypothesis that allosteric coupling pathways in the GABA(A) receptor complex represent initial sites of action for ethanol. This was accomplished using behavioral and in vitro measures to determine the effects of pressure on ethanol and other GABAergic drugs in C57BL/6 and LS mice. Behaviorally, exposure to 12 times normal atmospheric pressure (ATA) of a helium-oxygen gas mixture (heliox) antagonized loss of righting reflex (LORR) induced by the allosteric modulators ethanol and pentobarbital, but did not antagonize LORR induced by the direct GABA agonist 4,5,6,7-tetrahydroisoxazolo-pyridin-3-ol (THIP). Similarly, exposure to 12 ATA heliox antagonized the anticonvulsant effects verses isoniazid of ethanol, diazepam and pentobarbital. Biochemically, exposure to 12 ATA heliox antagonized potentiation of GABA-activated 36Cl-uptake by ethanol, flunitrazepam and pentobarbital in LS mouse brain preparations, but did not alter GABA-activated 36Cl- uptake per se. In contrast to its antagonist effect versus other allosteric modulators, pressure did not antagonize these behavioral or in vitro effects induced by the neuroactive steroid, 3alpha-hydroxy-5beta-pregnan-20-one (3alpha,5beta-P). These findings add to evidence that pressure directly and selectively antagonizes drug effects mediated through allosteric coupling pathways. The results fit predictions, and thus support the hypothesis that allosteric coupling pathways in GABA(A) receptors represent initial sites of action for ethanol. Collectively, the results suggest that there may be common physicochemical and underlying structural characteristics that define ethanol sensitive regions of receptor proteins and/or their associated membranes that can be identified by pressure within (e.g., GABA(A)) and possibly across (e.g., GABA(A), NMDA, 5HT3) receptors.

Structure, function, and molecular modeling approaches to the study of the intestinal dipeptide transporter PepT1.

The proton-coupled intestinal dipeptide transporter, PepT1, has 707 amino acids, 12 putative transmembrane domains (TMD), and is of importance in the transport of nutritional di- and tripeptides and structurally related drugs, such as penicillins and cephalosporins. By using a combination of molecular modeling and site-directed mutagenesis, we have identified several key amino acid residues that effect catalytic transport properties of PepT1. Our molecular model of the transporter was examined by dividing it into four sections, parallel to the membrane, starting from the extracellular side. The molecular model revealed a putative transport channel and the approximate locations of several aromatic and charged amino acid residues that were selected as targets for mutagenesis. Wild type or mutagenized human PepT1 cDNA was transfected into human embryonic kidney (HEK293) cells, and the uptake of tritiated glycylsarcosine [3H]-(Gly-Sar) was measured. Michaelis-Menton analysis of the wild-type and mutated transporters revealed the following results for site-directed mutagenesis. Mutation of Tyr-12 or Arg-282 into alanine has only a very modest effect on Gly-Sar uptake. By contrast, mutation of Trp-294 or Glu-595 into alanine reduced Gly-Sar uptake by 80 and 95%, respectively, and mutation of Tyr-167 reduced Gly-Sar uptake to the level of mock-transfected cells. In addition, preliminary data from fluorescence microscopy following the expression of N-terminal-GFP-labeled PepT1Y167A in HEK cells indicates that the Y167A mutation was properly inserted into the plasma membrane but has a greatly reduced Vmax.

Proton-driven dipeptide uptake in primary cultured rabbit conjunctival epithelial cells.

PURPOSE: To characterize proton-driven carrier-mediated dipeptide uptake in primary cultured conjunctival epithelial cells of the pigmented rabbit using beta-alanyl-L-histidine (L-carnosine) as a model dipeptide substrate. METHODS: Uptake of tritiated L-carnosine was monitored using conjunctival epithelial cells on days 6 through 8 in culture on a filter support. The structural features of dileucine stereoisomers and cephalexin contributing to interaction with the dipeptide transporter were evaluated by computer modeling and inhibition of tritiated L-carnosine uptake. RESULTS: Uptake of L-carnosine by primary cultured conjunctival epithelial cells in the presence of an inwardly directed proton gradient showed directional asymmetry (favoring apical uptake by a factor of five), temperature dependence, and saturability correlated with substrate concentration, with a Michaelis-Menten constant (Km) of 0.3 +/- 0.03 mM and a maximum uptake rate (Vmax) of 22.0 +/- 1.0 picomoles per milligram protein per minute. L-Carnosine uptake was optimal at pH 6.0 and was reduced by 60% and 35%, respectively, by 50 microM p-trifluoromethoxyphenylhydrazone (a proton ionophore) and by acid preloading with 50 mM NH4Cl. The constituent amino acids did not inhibit L-carnosine uptake. L-Carnosine uptake was inhibited, however, from 50% to 80% by other dipeptides and structurally similar drugs such as bestatin, beta-lactam antibiotics, and angiotensin-converting enzyme inhibitors. The LL, LD, or DL forms of the dipeptide Leu-Leu inhibited tritiated L-carnosine uptake by approximately 60%, 40%, and 70%, respectively. By contrast, the DD form did not inhibit uptake. Results from computer modeling suggest that an appropriate dipeptide N-terminal to C-terminal distance and a favorable orientation of the side chains may be important for substrate interaction with the conjunctival dipeptide transporter. CONCLUSIONS: Uptake of the dipeptide L-carnosine in primary cultured pigmented rabbit conjunctival epithelial cells is probably mediated by a proton-driven dipeptide transporter. This transporter may be used for optimizing the uptake of structurally similar peptidomimetic drugs.

Molecular identification of a role for tyrosine 167 in the function of the human intestinal proton- coupled dipeptide transporter (hPepT1).

hPepT1 is a proton-coupled peptide transporter that mediates the absorption of di- and tripeptides. Here we show that tyrosine 167 (Y167) in transmembrane domain 5 (TMD5) of this 12-transmembrane spanning protein contributes to its transport function. We identified this particular amino acid by a computer model of the arrangement of the TMDs of hPepT1 and investigated its role by site-directed mutagenesis and dipeptide uptake studies. [3H]Gly-sar uptake in cells transiently transfected with Y167A-hPepT1 was abolished completely, even though the level of Y167A-hPepT1 expression by Western blot analysis and cell surface expression by immunofluorescence microscopy was similar to those of the wild type. Therefore, mutation affected transport function, but apparently not the steady-state protein level or trafficking of the transporter to the plasma membrane. Moreover, mutation of Y167 into phenylalanine, serine, or histidine all abolished gly-sar uptake in transfected HEK 293 cells. Taken together, these findings suggest that Y167 plays an essential role in hPepT1 function, perhaps due to the unique chemistry of its phenolic side chain.

Characterization of the anticonvulsant properties of ganaxolone (CCD 1042; 3alpha-hydroxy-3beta-methyl-5alpha-pregnan-20-one), a selective, high-affinity, steroid modulator of the gamma-aminobutyric acid(A) receptor.

Ganaxolone (CCD 1042) is a 3beta-methyl-substituted analog of the endogenous neuroactive steroid 3alpha-hydroxy-5alpha-pregnan-20-one. Ganaxolone inhibited binding of the gamma-aminobutyric acid (GABA)A receptor-chloride channel ligand t-[35S]butylbicyclophosphorothionate (IC50 of 80 nM) and enhanced binding of the benzodiazepine site ligand [3H]flunitrazepam (EC50 of 125 nM) and the GABA site ligand [3H]muscimol (EC50 of 86 nM), consistent with activity as a positive allosteric modulator of the GABA(A) receptor. Electrophysiological recordings showed that, whereas nanomolar concentrations of ganaxolone potentiated GABA-evoked chloride currents in Xenopus oocytes expressing the human GABA(A) receptor subunits alpha1beta1gamma2L, alpha2beta1gamma2L or alpha3beta1gamma2L, direct activation of chloride flux occurred to a limited extent only at micromolar concentrations. Ganaxolone was effective in nontoxic doses against clonic convulsions induced by s.c. pentylenetetrazol administration in mice and rats (ED50 values of 4.3 and 7.8 mg/kg i.p., respectively). Ganaxolone also exhibited potent anticonvulsant activity against seizures induced by s.c. bicuculline (ED50 of 4.6 mg/kg i.p.), i.p. TBPS (ED50 of 11.7 mg/kg i.p.) and i.p. aminophylline (ED50 of 11.5 mg/kg i.p.) in mice. Although ganaxolone effectively blocked tonic seizures induced by maximal electroshock in mice (ED50 of 29.7 mg/kg i.p.), it did so only at doses that produced ataxia on the Rotorod (TD50 of 33.4 mg/kg i.p.). Conversely, ganaxolone was a potent anticonvulsant against fully kindled stage 5 seizures induced by corneal kindling in rats (ED50 of 4.5 mg/kg i.p.), producing these effects at doses well below those that resulted in ataxia (TD50 of 14.2 mg/kg i.p.). The seizure threshold, as determined by an increase in the dose of i.v. infused pentylenetetrazol required to induce clonus, was also significantly elevated by nontoxic doses of ganaxolone in mice. In summary, these data indicate that ganaxolone is a high-affinity, stereoselective, positive allosteric modulator of the GABA(A) receptor complex that exhibits potent anticonvulsant activity across a range of animal procedures. The profile of anticonvulsant activity obtained for ganaxolone supports clinical evaluation of this drug as an antiepileptic therapy with potential utility in the treatment of generalized absence seizures as well as simple and complex partial seizures.

Synthesis and in vitro activity of 3 beta-substituted-3 alpha-hydroxypregnan-20-ones: allosteric modulators of the GABAA receptor.

Two naturally occurring metabolites of progesterone, 3 alpha-hydroxy-5 alpha- and 5 beta-pregnan-20-one (1 and 2), are potent allosteric modulators of the GABAA receptor. Their therapeutic potential as anxiolytics, anticonvulsants, and sedative/hypnotics is limited by rapid metabolism. To avoid these shortcomings, a series of 3 beta-substituted derivatives of 1 and 2 was prepared. Small lipophilic groups generally maintain potency in both the 5 alpha- and 5 beta-series as determined by inhibition of [35S]TBPS binding. In the 5 alpha-series, 3 beta-ethyl, -propyl, -trifluoromethyl and -(benzyloxy)methyl, as well as substituents of the form 3 beta-XCH2, where X is Cl, Br, or I or contains unsaturation, show limited efficacy in inhibiting [35S]TBPS binding. In the 5 beta-series, the unsubstituted parent 2 is a two-component inhibitor, whereas all of the 3 beta-substituted derivatives of 2 inhibit TBPS via a single class of binding sites. In addition, all of the 3-substituted 5 beta-sterols tested are full inhibitors of [35S]TBPS binding. Electrophysiological measurements using alpha 1 beta 2 gamma 2L receptors expressed in oocytes show that 3 beta-methyl- and 3 beta-(azidomethyl)-3 alpha-hydroxy-5 alpha-pregnan-20-one (6 and 22, respectively) are potent full efficacy modulators and that 3 alpha-hydroxy-3 beta-(trifluoromethyl)-5 alpha-pregnan -20-one (24) is a low-efficacy modulator, confirming the results obtained from [35S]TBPS binding. These results indicate that modification of the 3 beta-position in 1 and 2 maintains activity at the neuroactive steroid site on the GABAA receptor. In animal studies, compound 6 (CCD 1042) is an orally active anticonvulsant, while the naturally occurring progesterone metabolites 1 and 2 are inactive when administered orally, suggesting that 3 beta-substitution slows metabolism of the 3-hydroxyl, resulting in orally bioavailable steroid modulators of the GABAA receptor.

In vitro and in vivo activity of 16,17-dehydro-epipregnanolones: 17,20-bond torsional energy analysis and D-ring conformation.

PURPOSE: Certain neuroactive pregnane steroids (also known as "epalons") are allosteric modulators of the GABA, receptor and have been shown to be potent anticonvulsants, anxiolytics, sedative/hypnotics, and anesthetic agents. The purpose of this study was to calculate the structural consequences of introduction of a double bond in the 16,17-position and to determine if this modification would selectively reduce sedative activity, but maintain the potent anticonvulsant activity of neuroactive steroids. METHODS: We have studied the biochemical and behavioral effects of introducing a 16,17 double bond into the naturally occurring neuroactive steroids, 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha,5 alpha-P) and 3 alpha-hydroxy-5 beta-pregnan-20-one (3 alpha,5 beta-P) and three synthetic neuroactive steroid derivatives, 3 alpha-hydroxy-3 beta-methyl-5 alpha-pregnan-20-one (3 alpha,3 beta Me,5 alpha-P), 3 alpha-hydroxy-5 alpha-androstane (3 alpha, 5 alpha-A), and alphaxalone (3 alpha,5 alpha-11-one-P). RESULTS: The 16-ene analogs of most of these neuroactive steroids were found to be 7- and 16-fold less potent in inhibiting [35S]TBPS binding to GABAA receptors and in a similar fashion, had reduced anticonvulsant and sedative potency in proportional amounts. The exception was the androstane (3 alpha,5 alpha-A) without a 17-acetyl group, that had virtually identical IC50 and ED50 values for the saturated and unsaturated derivatives. Calculation of the torsional energy profile for each of the 17-acetyl side chain conformations showed that the conformational energy minima found in the alpha,beta-unsaturated keto systems, produce an orientation of the 20-keto group that is rotated by 165 degrees when compared to the non-conjugated acetyl group (determined by X-ray crystallography and its minimum energy conformation). CONCLUSIONS: The modified orientation of the 20-keto group of neuroactive steroids containing a 16-ene, provides an explanation for their decreased biological activity overall, but did not lead to an enhanced protective index.

Low level hyperbaric antagonism of diazepam's locomotor depressant and anticonvulsant properties in mice.

Exposure to 12 atmospheres absolute (12 ATA) helium oxygen gas (heliox) (low level hyperbaric exposure) antagonizes the behavioral effects of ethanol and n-propanol, but not morphine. These and other results indicate that the mechanism of the antagonism is direct (pharmacodynamic) and selective. Our study further investigates the selectivity of low level hyperbaric antagonism by testing its effectiveness against diazepam, a high affinity binding drug that acts via allosteric modulation of GABAA receptors. C57BL/6J mice received injections i.p. of vehicle or diazepam, and were then exposed to 1 ATA air, 1 ATA heliox or 12 ATA heliox. Exposure to 12 ATA heliox antagonized the locomotor depressant effect of 4 and 6 mg/kg, but not 8 mg/kg diazepam. Hyperbaric exposure also antagonized the anticonvulsant effect of 8 and 24 mg/kg, but not 4 mg/kg, diazepam vs. 300 mg/kg isoniazid. Exposure to 12 ATA heliox did not significantly affect blood concentrations of diazepam or its metabolite n-desmethyl diazepam. The pharmacological characteristics of the antagonism (direct, surmountable, rightward shift in diazepam's dose-response curve) closely matched those seen in previous studies for hyperbaric antagonism of ethanol. The results add to the evidence that low level hyperbaric exposure is a direct, mechanistic antagonist that selectively antagonizes drugs that act via perturbation or allosteric modulation of receptor function. Moreover, the results suggest that allosteric coupling pathways, which transduce binding events on ligand-gated ion channels, may represent initial sites of action for ethanol.

The neuroactive steroid 3 alpha-hydroxy-5 beta-pregnan-20-one is a two-component modulator of ligand binding to the GABAA receptor.

Neuroactive steroids allosterically inhibit [35S]t-butylbicyclophosphorothionate ([35S]TBPS) and enhance [3H]flunitrazepam binding to the GABAA receptor complex. In the presence of 5 microM GABA, 3 alpha-hydroxy-5 beta-pregnan-20-one (3 alpha, 5 beta-P) inhibits [35S]TBPS binding with high- (IC50 21-32 nM) and low- (IC50 24-63 microM) affinity components in bovine cortical, cerebellar, and hippocampal membranes. The percentage of high-affinity sites ranges from 53% in cortex to 65% in cerebellum and hippocampus. However, 3 alpha, 5 beta-P is a single-site inhibitor in thalamus (IC50 43 nM). In the absence of GABA, similar affinities for the high- and low-affinity components were detected, although the percentages of high-affinity sites were reduced. Similarly, 3 alpha, 5 beta-P enhances [3H]flunitrazepam binding with high- (EC50 44-58 nM) and low- (EC50 2-13 microM) affinity components which account for 71-77% and 23-29% of the sites, respectively, in cortex, cerebellum and hippocampus. 3 alpha, 5 beta-P is a single-site enhancer in thalamus (EC50 80 nM). In contrast to 3 alpha,5 beta-P, 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha,5 alpha-P) is a single site modulator of [35S]TBPS and [3H]flunitrazepam binding in all regions examined. These data provide pharmacological evidence consistent with receptor heterogeneity for neuroactive steroids.

Calculations of antibody-antigen interactions: microscopic and semi-microscopic evaluation of the free energies of binding of phosphorylcholine analogs to McPC603.

The study of antibody-antigen interactions should greatly benefit from the development of quantitative models for the evaluation of binding free energies in proteins. The present work addresses this challenge by considering the test case of the binding free energies of phosphorylcholine analogs to the murine myeloma protein McPC603. This includes the evaluation of the differential binding energy as well as the absolute binding energies and their corresponding electrostatic contributions. Four different approaches are examined: the Protein Dipoles Langevin Dipoles (PDLD) method, the semi-microscopic PDLD (PDLD/S) method, a free energy perturbation (FEP) method based on an adiabatic charging procedure and a linear response approximation that accelerates the FEP calculation. The PDLD electrostatic calculations are augmented by estimates of the relevant hydrophobic and steric contributions. The determination of the hydrophobic energy involves an approach which considers the modification of the effective surface area of the solute by local field effects. The steric contributions are analyzed in terms of the corresponding reorganization energies. This treatment, which considers the protein as a harmonic system, views the steric forces as the restoring forces for the electrostatic interactions. The FEP method is found to give unreliable results with regular cut-off radii and starts to give quantitative results only in very expensive treatment with very large cut-off radii. The PDLD and PDLD/S methods are much faster than the FEP approach and give reasonable results for both the relative and absolute binding energies. The speed and simplicity of the PDLD/S method make it an effective strategy for interactive docking studies and indeed such an option is incorporated in the program MOLARIS. A component analysis of the different energy contributions of the FEP treatment and a similar PDLD analysis indicate that electrostatic effects provide the largest contribution to the differential binding energy, while the hydrophobic and steric contributions are much smaller. This finding lends further support to the idea that electrostatic interactions play a major role in determining the antigen specificity of McPC603.

Differential responses of expressed recombinant human gamma-aminobutyric acidA receptors to neurosteroids.

Neuroactive steroids, in particular 3 alpha-hydroxypregnanes, are allosteric modulators of the gamma-aminobutyric acidA (GABAA) receptor. Regionally selective expression of receptor subunit subtypes may account for differential responsiveness of tissues to GABAergic inhibition and neurosteroid modulatory effects. The effect of 5 alpha-pregnan-3 alpha-ol-20-one (epiallopregnanolone) on heterotropic cooperativity on the GABAA receptor complex has been studied in three subtypes of expressed recombinant human receptors and in rat brain and spinal cord. Steroid potentiation of [3H]flunitrazepam binding was greatest for the alpha 3 beta 1 gamma 2 receptor complex, whereas alpha 1 beta 1 gamma 2 and alpha 2 beta 1 gamma 2 complexes showed less than 100% enhancement in binding. Previous studies suggest that the spinal cord is devoid of alpha 1, whereas cerebellum is rich in alpha 1 subunits. Correspondingly, a differential enhancement of [3H]flunitrazepam binding in spinal cord (51%) versus cerebellum (28%) was also observed. The structure of neuroactive steroids is important in determinikng the extent of neuromodulatory activity. The 5 beta-pregnanes,5 beta-pregnan-3 alpha-ol-20-one (epipregnanolone) and 5 beta-pregnan-3 alpha,21-diol-20-one (5 beta-tetrahydrodeoxycorticosterone), were both less potent than their corresponding 5 alpha derivatives. A 3 alpha hydroxyl group is essential for neuromodulatory activity in the expressed receptors, as demonstrated by the observation that 5 alpha-pregnan-3 beta-ol-20-one (allopregnanolone) and 4-pregnen-3, 20-dione (progesterone) were both inactive.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of yeast transcription factor IIIC with dimeric Schizosaccharomyces pombe tRNA(Ser)-tRNA(Met) genes.

A unique tRNA(Ser)-tRNA(Met) tandem gene arrangement was characterized previously from Schizosaccharomyces pombe. Three alleles exist in which a tRNA(Ser) gene is separated by 7 base pairs from an initiator tRNA(Met) gene. Promotion of transcription occurs only within the tRNA(Ser) gene, yielding a dimeric precursor transcript. Using nuclease protection and gel retardation assays, we have analyzed how the Saccharomyces cerevisiae RNA polymerase III transcription factor C (TFIIIC) interacts with this dimeric gene template. The primary interaction site of TFIIIC with the tRNA(Ser) gene is at the 3'-internal control region (ICR), which can be distinguished kinetically from its weaker interaction with the 5'-ICR of the gene. We examined a variety of point mutations and double mutations within the tRNA(Ser) gene which reduce transcription. We found that changes in highly conserved nucleotides within the ICRs reduce TFIIIC binding up to 7-fold compared with the parent suppressor gene. The interaction of TFIIIC with the tRNA(Ser) gene does not sterically prevent stable binding of TFIIIC to the 3'-ICR of the tRNA(Met) gene. However, the affinity of binding of TFIIIC to the dimeric template is 7-fold higher than to the tRNA(Met) gene, alone, demonstrating that the tRNA(Met) gene contains intrinsically weak promoter elements. This may contribute to the inability of the tRNA(Met) gene to independently direct transcription from its ICR elements.