Identification of CYP3A4 and CYP2C8 as the major cytochrome P450 s responsible for morphine N-demethylation in human liver microsomes.

1. The aim was to identify the cytochrome P450 (CYP) enzymes responsible for the N-demethylation of morphine in vitro. 2. In human liver microsomes, normorphine formation followed Michaelis-Menten kinetics with mean Km and Vmax of 12.4 +/- 2.2 mM and 1546 +/- 121 pmol min(-1) mg(-1), respectively. In microsomes from a panel of 14 human livers phenotyped for 10 CYP enzymes, morphine N-demethylation correlated with testosterone 6beta-hydroxylation (r=0.91, p<0.001) and paclitaxel 6-alpha hydroxylation (r=0.72, p<0.001), two specific markers of CYP3A4 and CYP2C8, respectively. Normorphine formation decreased when incubated in the presence of troleandomycin or quercetin (by 46 and 33-36%, respectively), which further corroborates the contribution of CYP3A4 and CYP2C8. 3. Among eight recombinant human CYP enzymes tested, CYP3A4 and CYP2C8 exhibited the highest intrinsic clearance. More than 90% of morphine N-demethylation could be accounted for via the action of both CYP3A4 and CYP2C8. 4. The in vitro findings suggest that hepatic CYP3A4, and to a lesser extent CYP2C8, play an important role in the metabolism of morphine into normorphine.

New diarylmethylpiperazines as potent and selective nonpeptidic delta opioid receptor agonists with increased In vitro metabolic stability.

Nonpeptide delta opioid agonists are analgesics with a potentially improved side-effect and abuse liability profile, compared to classical opioids. Andrews analysis of the NIH nonpeptide lead SNC-80 suggested the removal of substituents not predicted to contribute to binding. This approach led to a simplified lead, N, N-diethyl-4-[phenyl(1-piperazinyl)methyl]benzamide (1), which retained potent binding affinity and selectivity to the human delta receptor (IC(50) = 11 nM, mu/delta = 740, kappa/delta > 900) and potency as a full agonist (EC(50) = 36 nM) but had a markedly reduced molecular weight, only one chiral center, and increased in vitro metabolic stability. From this lead, the key pharmacophore groups for delta receptor affinity and activation were more clearly defined by SAR and mutagenesis studies. Further structural modifications on the basis of 1 confirmed the importance of the N, N-diethylbenzamide group and the piperazine lower basic nitrogen for delta binding, in agreement with mutagenesis data. A number of piperazine N-alkyl substituents were tolerated. In contrast, modifications of the phenyl group led to the discovery of a series of diarylmethylpiperazines exemplified by N, N-diethyl-4-[1-piperazinyl(8-quinolinyl)methyl]benzamide (56) which had an improved in vitro binding profile (IC(50) = 0.5 nM, mu/delta = 1239, EC(50) = 3.6 nM) and increased in vitro metabolic stability compared to SNC-80.

N,N-Diethyl-4-(phenylpiperidin-4-ylidenemethyl)benzamide: a novel, exceptionally selective, potent delta opioid receptor agonist with oral bioavailability and its analogues.

The design, synthesis, and pharmacological evaluation of a novel class of delta opioid receptor agonists, N, N-diethyl-4-(phenylpiperidin-4-ylidenemethyl)benzamide (6a) and its analogues, are described. These compounds, formally derived from SNC-80 (2) by replacing the piperazine ring with a piperidine ring containing an exocyclic carbon carbon double bond, were found to bind with high affinity and exhibit excellent selectivity for the delta opioid receptor as full agonists. 6a, the simplest structure in the class, exhibited an IC(50) = 0.87 nM for the delta opioid receptors and extremely high selectivity over the mu receptors (mu/delta = 4370) and the kappa receptors (kappa/delta = 8590). Rat liver microsome studies on a selected number of compounds show these olefinic piperidine compounds (6) to be considerably more stable than SNC-80. This novel series of compounds appear to interact with delta opioid receptors in a similar way to SNC-80 since they demonstrate similar SAR. Two general approaches have been established for the synthesis of these compounds, based on dehydration of benzhydryl alcohols (7) and Suzuki coupling reactions of vinyl bromide (8), and are herewith reported.

Oxidative bioactivation of the lactol prodrug of a lactone cyclooxygenase-2 inhibitor.

The lactol derivative of a lactone cyclooxygenase-2 inhibitor (DFU) was evaluated in vivo and in vitro for its potential suitability as a prodrug. DFU-lactol was found to be 10 to 20 times more soluble than DFU in a variety of aqueous vehicles. After administration of DFU-lactol at 20 mg kg-1 p.o. in rats, a Cmax of 7.5 microM DFU was reached in the plasma. After oral administration, the ED50s of DFU-lactol in the carrageenan-induced paw edema and lipopolysaccharide-induced pyresis assays in rats are comparable with the ED50s observed when dosing with DFU. Incubations of DFU-lactol with rat and human hepatocytes demonstrated that the oxidation of DFU-lactol can be mediated by liver enzymes and that a competing pathway is direct glucuronidation of the DFU-lactol hydroxyl group. Assays with subcellular fractions from rat liver indicated that most of the oxidation of DFU-lactol occurs in the cytosolic fraction and requires NAD(P)+. Human liver cytosol can also support the oxidation of DFU-lactol to DFU when NAD(P)+ is added to the incubations. Fractionation of human liver cytosolic proteins showed that at least three enzymes are capable of efficiently effecting the oxidation of DFU-lactol to DFU. Incubations with commercially available dehydrogenases suggest that alcohol and hydroxysteroid dehydrogenases are involved in this oxidative process. These data together suggest that lactols may represent useful prodrugs for lactone-containing drugs.

Yeast transcript elongation factor (TFIIS), structure and function. I: NMR structural analysis of the minimal transcriptionally active region.

TFIIS is a general transcription elongation factor that helps arrested RNA polymerase II elongation complexes resume transcription. We have previously shown that yeast TFIIS (yTFIIS) comprises three structural domains (I-III). The three-dimensional structures of domain II and part of domain III have been previously reported, but neither domain can autonomously stimulate transcription elongation. Here we report the NMR structural analysis of residues 131-309 of yTFIIS which retains full activity and contains all of domains II and III. We confirm that the structure of domain II in the context of fully active yTFIIS is the same as that determined previously for a shorter construct. We have determined the structure of the C-terminal zinc ribbon domain of active yTFIIS and shown that it is similar to that reported for a shorter construct of human TFIIS. The region linking domain II with the zinc ribbon of domain III appears to be conformationally flexible and does not adopt a single defined tertiary structure. NMR analysis of inactive mutants of yTFIIS support a role for the linker region in interactions with the transcription elongation complex.

Refinement of an in vitro cell model for cytochrome P450 induction.

Induction of cytochromes P450 (P450s) by drugs can lead to drug-drug interactions. Primary hepatocytes have been reported to retain inducible P450s. To optimize the use of primary hepatocytes for predicting induction of P450 (CYP 3A and 2B) expression in vivo, both culture conditions and expression of induction potentials were investigated. In rat hepatocytes, basal CYP 3A1/2 expression was better maintained in cells cultured on Matrigel compared with collagen when low concentrations of dexamethasone were used. However, CYP 3A1/2 induction was not affected by either matrix. In contrast, induction of CYP 2B1/2 by phenobarbital was markedly stronger in hepatocytes cultured on Matrigel. To further validate the in vitro model, Sprague-Dawley rats and isolated hepatocytes cultured on Matrigel were exposed to a series of compounds. In an attempt to minimize large variability between experiments, a novel approach for calculating induction potential was applied. In vitro results for CYP 3A1/2 and 2B1/2 induction correlated well with those observed in vivo. In contrast with rat hepatocytes, basal CYP 3A4 expression in human hepatocytes decreased rapidly in cells cultured on either Matrigel or collagen. However, CYP 3A4 inducibility was retained in cells cultured on either matrix. Interestingly, induction of CYP 3A4 in human hepatocytes by several model compounds did not correlate with the induction of CYP 3A1/2 in rat hepatocytes. This in vitro assay should facilitate the demand for a fast and reproducible method for addressing P450 induction by numerous compounds at the drug discovery stage.

Elongation factor TFIIS contains three structural domains: solution structure of domain II.

Transcription elongation by RNA polymerase II is regulated by the general elongation factor TFIIS. This factor stimulates RNA polymerase II to transcribe through regions of DNA that promote the formation of stalled ternary complexes. Limited proteolytic digestion showed that yeast TFIIS is composed of three structural domains, termed I, II, and III. The two C-terminal domains (II and III) are required for transcription activity. The structure of domain III has been solved previously by using NMR spectroscopy. Here, we report the NMR-derived structure of domain II: a three-helix bundle built around a hydrophobic core composed largely of three tyrosines protruding from one face of the C-terminal helix. The arrangement of known inactivating mutations of TFIIS suggests that two surfaces of domain II are critical for transcription activity.

Mass spectrometric characterization of sequence-specific complexes of DNA and transcription factor PU.1 DNA binding domain.

Electrospray ionization mass spectrometry (ESI-MS) has been used to study the noncovalent interaction of the 13.5-kDa DNA binding domain of PU.1 (PU.1-DBD) with specific double-stranded DNA (dsDNA) target molecules. Mixtures of PU.1-DBD protein and wild-type target DNA sequence yielded ESI-MS spectra showing only protein-dsDNA complex ions of 1:1 stoichiometry and free dsDNA. When PU.1-DBD protein, wild type target DNA, and a mutant target DNA lacking the consensus sequence were mixed, only the 1:1 complex with the wild-type DNA was observed, consistent with gel electrophoresis mobility shift assay results, demonstrating the observation of sequence-specific protein-dsDNA complexes using ESI-MS.

Refolding of bovine pancreatic trypsin inhibitor via non-native disulphide intermediates.

The disulphide folding pathway of bovine pancreatic trypsin inhibitor (BPTI), especially at the two-disulphide stage, has been dissected by replacing one or two particular cysteine residues by serine. This restricts which disulphide species are possible, and the observed kinetics of disulphide-coupled folding reveal the roles of the remaining species. The results obtained confirm the kinetic roles in the original BPTI pathway of the two specific two-disulphide intermediates with non-native second disulphide bonds, (30-51, 5-14) and (30-51, 5-38). Moreover, the rates of folding through each of these intermediates are shown to account quantitatively for the rate of folding of the normal protein; therefore, essentially all the molecules refold through these two particular intermediates. They are amongst the most productive on the folding pathway, and their roles are readily explicable on the basis of their conformations.

Thermodynamic analysis of the structural stability of the tetrameric oligomerization domain of p53 tumor suppressor.

The structural stability of an amino acid fragment containing the oligomerization domain (residues 303-366) of the tumor suppressor p53 has been studied using high-precision differential scanning calorimetry (DSC) and circular dichroism spectroscopy (CD). Previous NMR solution structural determinations have revealed that the fragment forms a symmetric 29.8 kDa tetramer composed of a dimer of dimers (p53tet) [Lee, W., Harvey, T. S., Yin, Y., Yau, P., Litchfield, D., & Arrowsmith, C. H. (1994) Nature Struct. Biol. 1, 877-890]. Thermal unfolding of the tetramer is reversible and can be described as a two-state transition in which the folded tetramer is converted directly to unfolded monomers (N4<==>4U). According to the DSC and CD data, the population of intermediate species consisting of folded monomers or dimers is insignificant, indicating that isolated dimeric or monomeric structures have a much lower stability than the dimer and do not become populated during thermal denaturation under the conditions studied. The transition temperature of unfolding is found to be highly dependent on protein concentration and to follow the expected behavior for a tetramer that dissociates upon unfolding. Experiments conducted at pH 4.0 in 25 mM sodium acetate at a tetramer concentration of 145.8 microM have a transition temperature (Tm) of 75.3 degrees C while at 0.5 microM the value drops to 39.2 degrees C. The enthalpy change of unfolding at 60 degrees C is 26 kcal (mol of monomer)-1 with a heat capacity change of 387 cal (K.mol of monomer)-1. The stability of p53tet is dependent on pH and salt concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct calorimetric analysis of the enzymatic activity of yeast cytochrome c oxidase.

The kinetic and thermodynamic parameters associated with the enzymatic reaction of yeast cytochrome c oxidase with its biological substrate, ferrocytochrome c, have been measured by using a titration microcalorimeter to monitor directly the rate of heat production or absorption as a function of time. This technique has allowed determination of both the energetics and the kinetics of the reaction under a variety of conditions within a single experiment. Experiments performed in buffer systems of varying ionization enthalpies allow determination of the net number of protons absorbed or released during the course of the reaction. For cytochrome c oxidase the intrinsic enthalpy of reaction was determined to be -16.5 kcal/mol with one (0.96) proton consumed for each ferrocytochrome c molecule oxidized. Activity measurements at salt concentrations ranging from 0 to 200 mM KCl in the presence of 10 mM potassium phosphate, pH 7.40, and 0.5 mM EDTA display a biphasic dependence of the electron transferase activity upon ionic strength with a peak activity observed near 50 mM KCl. The ionic strength dependence was similar for both detergent-solubilized and membrane-reconstituted cytochrome c oxidase. Despite the large ionic strength dependence of the kinetic parameters, the enthalpy measured for the reaction was found to be independent of ionic strength. Additional experiments involving direct transfer of the enzyme from low to high salt conditions produced negligible enthalpy changes that remained constant within experimental error throughout the salt concentrations studied (0-200 mM KCl). These results indicate that the salt effect on the enzyme activity is of entropic origin and further suggest the absence of a major conformational change in the enzyme due to changes in ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)

Thermal stability of membrane-reconstituted yeast cytochrome c oxidase.

The thermal dependence of the structural stability of membrane-reconstituted yeast cytochrome c oxidase has been studied by using different techniques including high-sensitivity differential scanning calorimetry, differential detergent solubility thermal gel analysis, and enzyme activity measurements. For these studies, the enzyme has been reconstituted into dimyristoylphosphatidylcholine (DMPC) and dielaidoylphosphatidylcholine (DEPC) vesicles using detergent dialysis. The phospholipid moiety affects the stability of the enzyme as judged by the dependence of the denaturation temperature on the lipid composition of the bilayer. The enzyme is more stable when reconstituted with the 18-carbon, unsaturated phospholipid (DEPC) than with the 14-carbon saturated phospholipid (DMPC). In addition, the shapes of the calorimetric transition profiles are different in the two lipid systems, indicating that not all of the subunits are affected equally by the lipid moiety. The overall enthalpy change for the enzyme denaturation is essentially the same for the two lipid reconstitutions (405 kcal/mol of protein for the DMPC and 425 kcal/mol for the DEPC-reconstituted enzyme). In both systems, the van't Hoff to calorimetric enthalpy ratios are less than 0.2, indicating that the unfolding of the enzyme cannot be represented as a two-state process. Differential detergent solubility experiments have allowed us to determine individual subunit thermal denaturation profiles. These experiments indicate that the major contributors to the main transition peak observed calorimetrically are subunits I and II and that the transition temperature of subunit III is the most affected by the phospholipid moiety. Experiments performed at different scanning rates indicate that the thermal denaturation of the enzyme is a kinetically controlled process characterized by activation energies on the order of 40 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)