Computer modeling of 3D structures of cytochrome P450s.

The understanding of structure-function relationship of enzymes requires detailed information of their three-dimensional structure. Protein structure determination by X-ray and NMR methods, the two most frequently used experimental procedures, are often difficult and time-consuming. Thus computer modeling of protein structures has become an increasingly active and attractive option for obtaining predictive models of three-dimensional protein structures. Specifically, for the ubiquitous metabolizing heme proteins, the cytochrome P450s, the X-ray structures of four isozymes of bacterial origin, P450cam, P450terp, P450BM-3 and P450eryF have now been determined. However, attempts to obtain the structure of mammalian forms by experimental means have thus far not been successful. Thus, there have been numerous attempts to construct models of mammalian P450s using homology modeling methods in which the known structures have been used to various extents and in various strategies to build models of P450 isozymes. In this paper, we review these efforts and then describe a strategy for structure building and assessment of 3D models of P450s recently developed in our laboratory that corrects many of the weaknesses in the previous procedures. The results are 3D models that for the first time are stable to unconstrained molecular dynamics simulations. The use of this method is demonstrated by the construction and validation of a 3D model for rabbit liver microsomal P450 isozyme 2B4, responsible for the oxidative metabolism of diverse xenobiotics including widely used inhalation anesthetics. Using this 2B4 model, the substrate access channel, substrate binding site and plausible surface regions for binding with P450 redox partners were identified.

Intramolecular hydrogen bonding and conformational studies of bridged thebaine and oripavine opiate narcotic agonists and antagonists.

A conformational study of a series of oripavine derivatives is reported using the PCILO semiempirical quantum mechanical method. Low-energy conformers of carbinol substituents on C7-C19-R1R2OH are found with and without intramolecular hydrogen bonding to the C6-OCH3 group. The relative energies of these conformers depend on the R1 and R2 groups and the diastereoisomerism of the alcohol. The results are consistent with available NMR and IR studies of intramolecular hydrogen bonding and with crystallographic data. The importance of interaction between specific conformations of C19 carbinols and a lipophilic receptor site is suggested. A hypothesis is formulated to explain observed differences in pharmacological activity between diastereoisomers at C19 in the oripavine series and also to explain how these diastereoisomers alter the established pattern of N-substituent effects on relative agonist/antagonist potency found in other rigid opiates. By contrast, conformational studies of the C19 optical isomers of the C7-C8 etheno form of buprenorphine lead to the prediction of greatly reduced intrinsic potency differences between C19 diastereoisomers for this compound and for buprenorphine itself.

Quantum chemical studies of morphine-like opiate narcotics. Effect of N-substituent variations.

Quantum chemical calculations including extensive conformational variations are performed on three morphine-like analgesics with varying N-substituents using the PCILO and INDO methods. The three compounds, morphine, nalorphine, and N-phenethylmorphine, have been shown experimentally to exemplify opiate narcotic agonism, antagonism, and increased agonism, respectively. In this study, these properties are correlated with the electronic and conformational results. The electronic properties of the fused ring skeleton including specifically the cationic region around the nitrogen are relatively unaffected by varying N-substituents. The properties studied include net charges, bond polarities, and the nature and energy of the highest filled and lowest empty molecular orbitals. The conformational behavior appears to be the main cause of differing receptor binding and interaction with the active site and is discussed in these terms.

Quantum chemical studies of meperidine and prodine.

Extensive quantum chemical calculations have been made of the electronic distribution and conformational behavior of meperidine and desmethyl-, (+)-alpha, and (+)-beta-prodine using PCILO, a semiempirical molecular orbital method. For this series of opiates, a phenyl equatorial conformation was preferred over a phenyl axial one, with the equatorial conformer most favored in the most potent compounds. Using the low-energy equatorial conformer obtained for each compound, together with calculated net atomic charges, their observed potency variation could successfully be explained. From the results, these compounds appear to act at the morphine receptor with an identical piperidine rather than phenyl ring site.

Conformational studies of a family of related antimalarial phenanthrene amino alcohols.

A conformational study utilizing quantum chemical methods was performed on a family of antimalarial alpha-(piperidyl)-3,6-bis(trifluoromethyl)-9-phenanthrenemethanols whose structures differ by the placement of the substituent on either the 2,3, or 4 position of the piperidyl ring. The antimalarial activity of these 3-substituted compounds has been shown experimentally to be highly stereospecific while the 2-substituted compounds are not and the 4-substituted compounds are inactive. In this study, such observed differences in behavior are correlated with conformational results and a pharmacophore is postulated. The identity of the active racemate of the 3-piperidyl compound is predicted.

Quantum chemical studies of N-substituent variation in the oxymorphone series of opiate narcotics.

Quantum chemical calculations were performed on six N-derivatives of oxymorphone including N-methyl- (oxymorphone), N-allyl- (naloxone), N-dimethylallyl- (nalmexone), N-methylcyclopropyl- (naltrexone), N-methylcyclobutyl-(nalbuphone), and N-phenethylnoroxymorphone using the PCILO method. The object of the study was to identify conformational features of the N-substituents which might be responsible for the intrinsic observed pharmacological properties of opiate agonism and antagonism. Both axial and equatorial N-substituent conformers were considered, as well as possible interactions of the C14-OH group with such substituents. Variations of agonist/antagonist potency ratios within this series could not be explained by differing relative energies of equatorial and axial conformations or by varying rates of interconversion between the two. Direct effects of the C14-OH group on conformations of N-substituents also could not account for their relative agonist/antagonist potencies. Consistent with a previous hypothesis, the observed potencies and binding data could be explained most consistently by the availability of several low-energy equatorial conformations of N-substituents and their interactions with the C14-OH group through a common anionic receptor site.

Quantum chemical studies of methadone.

Quantum chemical calculations were performed on the flexible methadone molecule to test the hypothesis that it structurally mimics the fused ring structure of morphine. In these calculations using the semiempirical, PCILO method, protonated and nonprotonated conformations were considered representative of different types of intramolecular interaction at the morphine receptor. Calculated energies for these conformations were compared to those calculated for protonated and nonprotonated extended chain and crystal structure conformers. Lowest energy conformations showed intramolecular bonding but the resultant molecular geometries were not very morphine-like. A comparison of the structure of methadone to that of meperidine seemed equally as valid.

Effects of addition of a 2-methyl group to ethyl nipecotates (beta-meperidines) on receptor affinities and opiate agonist/antagonist activities.

A series of 2-methyl-3-carbethoxy-3-(m-hydroxyphenyl)piperidine opiates (13a-d) with N-substituent variations have been synthesized, and their receptor affinities and in vivo agonist and antagonist activities and energy-conformational profiles have been determined. These are racemates of the alpha-epimer at the C-2 position, with a methyl group cis to the 3-phenyl group. One of the main goals of this study was to compare the conformational and pharmacological behavior of these 2-methyl "beta-meperidine" analogues to their 2-desmethyl racemic counterparts (14a-c) previously reported in the literature. The 2-desmethyl and 2-methyl analogues were found to have very similar phenyl equatorial conformers as their lowest energy forms with the addition of a 2-methyl group diminishing conformational flexibility. The presence of the 2-methyl group appears to diminish affinity at the mu-receptor and also to somewhat diminish already weak antinociceptic agonist activity. Given the similarity in lowest energy conformation, this reduction is most likely caused by the unfavorable interaction of the methyl group itself with a local mu-receptor binding site. Superposition of the phenol OH and protonated amine nitrogen NH of either 2-methyl enantiomer of 13a in its lowest energy conformer with the same OH and NH groups of metazocine, used as a high affinity rigid analogue, leads to reasonable overlap. However, the N-substituents and the piperidine and phenyl rings do not overlap in this proposed pharmacophore, perhaps accounting for the rather poor affinities found for these 3-phenylpiperidines and the lack of N-substituent modulation of affinity and efficacy as in fused ring opioids.

Analgesics. 1. Synthesis and analgesic properties of N-sec-alkyl- and N-tert-alkylnormorphines.

A series of N-sec- and N-tert-alkylnormorphines was synthesized and evaluated for analgesic potency, antagonist activity, and opiate receptor binding. Computer-assisted conformational analysis profiles were utilized to assist in the selection of compounds for synthesis and correlation of receptor events with in vivo observations. N-tert-Alkylnormorphines 5a-c were devoid of agonist activity; however, some sec-alkyl analogues showed interesting mixed agonist-antagnoist actions. N-sec-Butyl- and N-(alpha-methylally)normorphine were separated into R and S isomers, which exhibited quantitative pharmacological differences. The N-sec-butyl S isomer 10a showed analgesia approximating morphine with nalorphine-like antagonist activity. Preliminary testing indicates only slight evidence for physical dependence with this compound.

Rational design, discovery, and synthesis of a novel series of potent growth hormone secretagogues.

In the joint experimental and computational efforts reported here to obtain novel chemical entities as growth hormone secretagogues (GHSs), a small database of peptides and non-peptides known to have GHS activity was used to generate and assess a 3D pharmacophore for this activity. This pharmacophore was obtained using a systematic and efficient procedure, "DistComp", developed in our laboratory. The 3D pharmacophore identified was then used to search 3D databases to explore chemical structures that could be novel GHSs. A number of these were chosen for synthesis and assessment of their ability to release growth hormone (GH) from rat pituitary cells. Among the compounds tested, those with a benzothiazepin scaffold were discovered with micromolar activity. To facilitate lead optimization, a second program, a site-dependent fragment QSAR procedure was developed. This program calculates a library of chemical and physical properties of "fragments" or chemical components in a known pharmacophore and determines which, if any, of these properties are important for the observed activity. The combined use of the 3D pharmacophore and the results of the site-dependent fragment QSAR analysis led to the discovery and synthesis of a novel series of potent GHSs, a number of which had nanomolar in vitro activity.

Effect of structural modifications in the C7-C11 region of the retinoid skeleton on biological activity in a series of aromatic retinoids.

A series of conformationally restricted analogues of (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)propenyl ] benzoic acid--(E)-4-[1-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-2 - propenyl]benzoic acid, (E)-4-[3-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-2-bu ten- 2-yl]benzoic acid, trans-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) cyclopropyl]benzoic acid, 4-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-anthracenyl)benzoic acid, 6-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-2- naphthalenecarboxylic acid, 6-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthalenyl)-2- naphthalenecarboxylic acid and 6-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-5-methyl-2- naphthalenecarboxylic acid--were synthesized and screened for retinoid biological activity. Comparison of the conformers of these analogues generated by molecular mechanics calculations with the biological activity profiles of these compounds indicates that geometric constraints required for high biological activity are imposed on the bridge joining the two aromatic ring systems by the retinoid receptor.

Computer-assisted mechanistic structure-activity studies: application to diverse classes of chemical carcinogens.

In the first part of this paper we have indicated how the techniques and capabilities of theoretical chemistry, together with experimental results, can be used in a mechanistic approach to structure-activity studies of toxicity. In the second part, we have illustrated how this computer-assisted approach has been used to identify and calculate causally related molecular indicators of relative carcinogenic activity in five classes of chemical carcinogens: polycyclic aromatic hydrocarbons and their methyl derivatives, aromatic amines, chloroethanes, chloroalkenes and dialkyl nitrosamines. In each class of chemicals studied, candidate molecular indicators have been identified that could be useful in predictive screening of unknown compounds. In addition, further insights into some mechanistic aspects of chemical carcinogenesis have been obtained. Finally, experiments have been suggested to both verify the usefulness of the indicators and test their mechanistic implications.

Quantum chemical calculations on the two-step mechanism of proflavin binding to DNA.

Quantum chemical calculations on the binding of proflavin to DNA lead to a model in which the outside binding to a phosphate group leads to an induced fit in the intercalation receptor site. The calculations suggest hydrogen bonding of the amine groups of the outside bound proflavin to the anionic oxygen of the backbone phosphate. The resulting partial neutralization facilitates the conformational transitions required for intercalation. The results are consistent with the observed preference of proflavin for dCpdG over dGpdC sequences and with the observed kinetics of the binding reaction.

Thermodynamics of ligand binding to the cloned delta-opioid receptor.

The goal of this study was to determine the relative contribution of entropy and enthalpy to the free energies of binding to recombinant mouse delta-opioid receptors for the peptide agonist, DPDPE ([D-Pen2,D-Pen5]enkephalin), the peptide antagonist, TIPP(psi) (Tyr-Tic(psi)[CH2NH]Phe-Phe-OH), the nonpeptide agonist, SNC80 ((+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl )-3-methoxybenzyl]-N,N-diethylbenzamide), and the nonpeptide antagonist, naltrindole. Competitive binding studies were carried out using [3H]naltrindole at 0 degrees C, 12 degrees C, 25 degrees C and 37 degrees C, the affinities calculated and van't Hoff plots constructed for each ligand. The temperature dependence of binding and van't Hoff plots indicated that the entropy contribution is the major component of the free energy, for all four ligands, independent of its activity or chemical nature.

A molecular model for an anionic opiate mu-receptor: affinity and activation of morphine conformers.

In this study, the minimal neglect of differential overlap, hydrogen bonding corrected (MNDO/H) method was used to construct an explicit three component mu-opioid receptor binding site composed of formate (Glu-, Asp-), H2O (Ser, Thr) and NH4+ (Lys+, Arg+) which has optimum interactions with the protonated amine and polar oxygen regions of morphine, respectively. These moieties are common to most classes of opioids and are thought to be involved in key interactions of the protonated form with the receptor leading to recognition and activation. Two predominant conformers of morphineH+, N-Me (equatorial) and N-Me (axial) were used as templates for construction of the binding site. In these studies, a plausible recognition mechanism involving electrostatic interaction between the protonated amine and an anionic receptor site, together with a proton-donating phenolic group and proton-accepting polar oxygens was characterized. The role of hydration of both the receptor site and morphine in determining affinity was also explicitly considered. The results strongly indicate that the high-affinity binding of morphineH+ to an 'anionic' receptor site is due mainly to a large entropy term resulting from expulsion of H2O from the receptor site upon introduction of morphine. A possible mechanism of receptor activation was also explored involving proton transfer from morphine to the receptor. Two results obtained support the plausibility of this mechanism: the barrier to proton transfer is reduced by receptor interaction with the polar oxygens and a conformational change occurs in the model receptor during this process.

Binding of 1,4-benzodiazepines to a novel [3H]Ro15-4513 binding site in the rat spinal cord.

An alpidem-insensitive benzodiazepine binding site in the rat spinal cord has recently been identified in our laboratory. We report here the binding of 23 1,4-benzodiazepines to this site using [3H]Ro15-4513 (ethyl-8-azido-6-dihydro-5-methyl-4H-imidazo[1,2- a][1,4]benzodiazepine-3-carboxylate) in the presence of 65 microM alpidem (6-chloro-2-(4-chlorophenyl)-N,N- dipropylimidazo[1,2-a]pyridine-3-acetamide). This binding site displays a wide affinity for 1,4-benzodiazepines, most of which show much higher affinity for benzodiazepine receptors in various brain regions and transfected cell systems. The highest affinity ligands are: brotizolam (1-bromo-4-(2-chlorophenyl)-9-methyl-6H-thieno[3,2- f][1,2,4]triazolo[4,3-a][1,4]diazepine) (4.3 nM), Ro15-4513 (5.0 nM), Ro42-8773 (7-chloro-3-[3-(cyclopropylmethoxy)-1-propynyl]-4,5-dihydro- 5-methyl-6H-imidazo[1,5-a][1,4]benzodiazepine-6-one) (5.7 nM), Ro16-6028 (t-butyl (s)-8-bromo-11,12,13,13a-tetrahydro-9-oxo-9H- imidazo[1,5-a][1,4]benzodiazepine-1-carboxylate) (5.9 nM) and triazolam (8-chloro-6-(2-chlorophenyl)-1-methyl-4H- [1,2,4]triazolo[4,3-a][1,4]benzodiazepine) (7.9 nM). The structural feature common to these compounds is an imidazo- or triazolo-ring on the 1- and 2-position of the benzodiazepine. However, the presence of this feature does not guarantee high affinity binding as Ro15-1788 (8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5- a][1,4]benzodiazepine-3-carboxylic acid ethyl ester) (100 nM) and Ro23-0364 (6-[2-chlorophenyl]-4H- imidazo[1,5-a][1,4]benzodiazepine-3-carboxamide) (360 nM) display much lower affinity for this site. Studies are currently underway to investigate the functional significance of this unusual benzodiazepine binding site.

Evidence for mu1-opioid receptor involvement in fentanyl-mediated respiratory depression.

Several fentanyl analogs (Bagley et al., 1989, J. Med. Chem. 32, 663) were compared to fentanyl and morphine for their effects on respiratory depression as determined by arterial blood gas (pH, pCO2 and pO2) measurements. Fentanyl (0.1 mg/kg), morphine (10 mg/kg), #16 (1-phenethyl-4-[N-(pyridin-2-yl)-N-(methoxymethylcarbonyl)amino] piperidine, 1 mg/kg), #17 (1-phenethyl-4-[N-(pyridin-2-yl) -N-(2-furoyl)amino]piperidine, 0.5 mg/kg) and #29 (1-phenethyl-4-[N- (pyrimidin-2-yl)-N-(methoxy-methylcarbonyl) amino]piperidine, 10 mg/kg) produced significant respiratory depression in rats. Pretreatment with the mu1-opioid receptor selective antagonist, naloxonazine (10 mg/kg), blocked the respiratory effect of fentanyl and its analogs, but not that of morphine. The results suggest that the mu1-opioid receptor plays an important role in the respiratory effects of fentanyl and its analogs. Hence, the mechanism of fentanyl-induced respiratory depression appears to be distinct from that produced by morphine. The most likely explanation for this difference is the possible contribution of muscle rigidity and catalepsy to the observed changes in blood gas parameters caused by the fentanyl analogs, while the respiratory depression of morphine, measured by these same parameters, appears to be independent of its effect on muscle rigidity.

Unraveling the identity of benzodiazepine binding sites in rat hipppocampus and olfactory bulb.

The goals of the work reported here were (i) to identify distinct GABA(A)/benzodiazepine receptors in the rat hippocampus and olfactory bulb using receptor binding assays, and (ii) to determine the affinities and selectivities of benzodiazepine receptor ligands from structurally diverse chemical families at each site identified. These studies were aided by the use of software AFFINITY ANALYSIS SYSTEM, developed in our laboratory for analysis of receptor binding data that allows the determination of receptor heterogeneity using non-selective radioligands. Saturation binding assays using [3H]RO15-4513 (ethyl 8-azido-6-dihydro-5-methyl-6-oxo-4H-imidazo[1, 5-a]-[1,4]benzodiazepine-3-carboxylate) revealed two binding sites in each of these two tissues. The higher affinity site corresponds to alpha(5) subunit-containing GABA(A) receptor and the lower affinity site to a combination of alpha(1), alpha(2), and alpha(3) subunit-containing receptors. These results should be useful in the challenging task of identifying the various functional GABA(A) receptors in the central nervous system, and in providing a link between receptor affinities and in vivo activities of the GABA(A)/benzodiazepine receptor ligands studied.

Determinants of recognition of ligands binding to benzodiazepine receptor/GABA(A) receptors initiating sedation.

Complementary behavioral and computational studies of 21 structurally diverse, gamma-amino butyric acid (GABA)(A) benzodiazepine receptor ligands that influence spontaneous locomotor activity have been performed in this work. This behavioral endpoint is a well-accepted indicator of sedation particularly for GABA(A)/benzodiazepine receptor ligands. The goal of the work presented here is the identification and assessment of the minimum requirements for ligand recognition of GABA(A)/benzodiazepine receptors leading to activity at the sedation endpoint embedded in a common 3D pharmacophore for recognition. Using the experimental results, together with a systematic computational procedure developed in our laboratory, a five-component 3D pharmacophore for recognition of the GABA(A) receptor subtypes associated with the sedative behavioral response has been developed consisting of: two proton-accepting moieties, a hydrophobic region, a ring with polar moieties and an aromatic ring in a common geometric arrangement in all ligands having an effect at the sedation endpoint. To provide further evidence that the 3D pharmacophore developed embodied common requirements for receptor recognition, a pharmacophore analysis was performed for agonists, inverse agonists and antagonists separately. Each of the resulting pharmacophores contained the same five moieties at comparable distances to those found for the pharmacophore generated using all of them together. This result confirms that this pharmacophore constitutes a recognition pharmacophore representing required features in the overlapping portion of their binding sites. The reliability of this 3D pharmacophore was then assessed in several ways. First, it was determined that ligands that had no effect at the sedation endpoint did not comply with the pharmacophore requirements. Second, four benzodiazepine receptor ligands known to have an effect at the sedation endpoint, but not used in the pharmacophore development were found to satisfy the requirements of this pharmacophore. Third, the geometric and chemical requirements embedded in this pharmacophore were used to search 3D databases resulting in the identification of benzodiazepine receptor ligands known to affect sedation, but not included in the pharmacophore development. Finally, a 3D-quantitative structure analysis procedure (QSAR) model was developed based upon the ligands in the training set superimposed at their sedation pharmacophore points. The 3D-QSAR model shows good predictivity for binding of these ligands to receptor subtypes containing alpha1 but not alpha5 subunits. The pharmacophore developed for the sedation endpoint thus provides a predictive binding model for diverse ligand binding to alpha1 containing receptor subtypes.

Evidence for more than two central benzodiazepine receptors in rat spinal cord.

Competitive binding assays were performed in rat spinal cord membranes at 0 degrees C against [3H]Ro15-1788. The displacement curves of Ro15-1788 and alpidem produced pseudo-Hill slopes of 1.0 and 0.5, respectively. In addition, 5-10% of the specifically bound [3H]Ro15-1788 could not be displaced by greater than 100 microM of alpidem. These two pieces of evidence strongly suggest the presence of at least three central benzodiazepine binding sites in the spinal cord.