Superimposition-based protocol as a tool for determining bioactive conformations. I. Application to ligands of the glycinergic receptor (GlyR).

The natural templates (NT) approach, which is a superimposition-based protocol that has been successfully employed in several studies, is here applied to ligands of the glycine ligand-gated ion channel receptor. Bioactive conformations for glycine and its analogs were obtained using strychnine (a natural and specific competitive antagonist) as template. Experimental evidence was used to guide the superimposition protocol. Three essential regions have been defined in strychnine's structure that serve as a pharmacophore for agonist and antagonist activities. Reasonable alignments of known ligands were found in the majority of the cases. Molecular mechanics (i.e., conformational searches for the relatively flexible ligands) and molecular dynamics (for relatively rigid ligands such as strychnine and 5,6,7,8-tetrahydro-4H-isoxazolo[3,4-d]azepin-3-ol) were used to assess the energetic accessibility of the proposed bioactive conformations.

Superimposition-based protocol as a tool for determining bioactive conformations. II. Application to the GABA(A) receptor.

The natural templates (NT) superimposition method is used to determine the pharmacophoric requirements of the A subtype of the gamma-aminobutyric acid (GABA) receptor. Bioactive conformations for antagonists and agonists are found by superimposing them on a relatively rigid alkaloid bicuculline, which itself is a competitive antagonist at this ligand-gated ion channel receptor. As has been usual in the application of this modeling method, consideration of available experimental data is the cornerstone for obtaining realistic models. The identification of two substructural fragments of bicuculline permitted classification of the ligands. Analysis of the antagonists and agonists with respect to the two substructural fragments revealed two bioactive conformations of the highly flexible GABA molecule, one of which is extended with the nonhydrogenic atoms roughly coplanar torsional angles of -37 and -179 degrees at N-C-C-C and C-C-C-C (carboxyl), respectively. The second bioactive compound is clearly non planar (torsional angles of -81 and -109 degrees at N-C-C-C and C-C-C-C (carboxyl), respectively).

A computational model of the nicotinic acetylcholine binding site.

We have derived a model of the nicotinic acetylcholine binding site. This was accomplished by using three known agonists (acetylcholine, nicotine and epibatidine) as templates around which polypeptide side chains, found to be part of the receptor cavity from published molecular biology studies, are allowed to flow freely in molecular dynamics simulations and mold themselves around these templates. The resulting supramolecular complex should thus be a complement, both in terms of steric effects as well as electronic effects, to the agonists and it should be a good estimation of the true receptor cavity structure. The shapes of those minireceptor cavities equilibrated rapidly on the simulation time scale and their structural congruence is very high, implying that a satisfactory model of the nicotinic acetylcholine binding site has been achieved. The computational methodology was internally tested against two rigid and specific antagonists (dihydro-beta-erytroidine and erysoidine), that are expected to give rise to a somewhat differently shaped binding site compared to that derived from the agonists. Using these antagonists as templates there were structural reorganizations of the initial receptor cavities leading to distinctly different cavities compared to agonists. This indicates that adequate times and temperatures were used in our computational protocols to achieve equilibrium structures for the agonists. Overall, both minireceptor geometries for agonists and antagonists are similar with the exception of one amino acid (ARG209).

Arylpiperazines with serotonin-3 antagonist activity: a comparative molecular field analysis.

Comparative molecular field analysis (CoMFA) is applied to antagonists of the 5-HT3 receptor. Analysis is done separately on three published sets of arylpiperazines and on a combination of the three sets. d-Tubocurarine, a conformationally restricted 5-HT3 ligand, is used as a template to assist in selecting the conformation of the antagonists for CoMFA alignment. Two forms of the arylpiperazines (neutral and protonated) and three different kinds of calculated charges (Gasteiger-Hückel, AM1, and AM1 with solvation effect included) are compared. Protonated structures give better statistical results than the neutral species. The way in which charges are calculated does not greatly affect the results. In terms of molecular fields, the behavior in each separate set of compounds cannot be extrapolated to the combined set of 47 compounds. The average value of r2cv from PLS cross-validation on the combined set is 0.70 and varies between 0.56 and 0.80 depending on the orientation of the molecules in the coordinate system. The CoMFA model is tested on four compounds not in the training set: quipazine, N-methylquipazine, 4-phenyl-N-methylquipazine, and KB-6933. Mean agreement of experimental and predicted pKi values of the antagonists is 0.7 log unit. Novel structural modifications are interpreted by the CoMFA model.

The nicotinic cholinergic receptor: a theoretical model.

Based on published affinity-labeling and mutagenesis experiments describing the effect of changes in specific amino acids in molecular biological studies on the nicotinic acetylcholinergic receptor (nAChR), we have identified 12 amino acids which are important in functioning at the nicotinic cholinergic receptor. The work presented here provides an atomistic model of this important receptor based on our molecular modeling studies. We found five of these amino acids (TRP86, ASP89, TYR93, ASP138, and THR191) to be associated with the cationic end of acetylcholine (ACh), which is electron-deficient. Three other amino acids (ARG209, TYR190, and TYR198) are associated with the ester end, where an enhanced electron density is present. After hydrogen bonding between the two oxygen atoms at the ester end, and two of the guanidinium hydrogen atoms in ARG209. ASP200 hydrogen bonds to the other two hydrogen atoms of the guanidinium group, thus forming a pseudo-ring. Two aromatic amino acids (TRP149 and TYR151) then enhance the binding at the pseudo-ring through additional hydrogen bonding and charge-transfer complexation, with THR150 functioning to further stabilize this evolving charge-transfer complex. We postulate that this latter process allows the ion channel to twist, thus opening it. From the published amino acid sequence in the polypeptides at the 5HT-3, GABA, and glycine receptors (Maricq et al.: Science 254:432-437, 1991), we also speculate on which amino acids are involved in these three receptors.

Glycine and GABA receptors: molecular mechanisms controlling chloride ion flux.

We have been able to show that the three clearly identified atoms common to the inhibitory neurotransmitters glycine and GABA, that we previously hypothesized to serve as attachment points at the glycinergic and gabanergic receptor, can indeed interact through both electrostatic and hydrogen bonding to several amino acids, which have been identified in molecular biological investigations as both present and critical in the physiological functioning of key polypeptides common to these inhibitory receptors. In addition, amino acids also involved in stabilizing the interaction between the antagonists strychnine and R5135 at the glycinergic and gabanergic receptors, respectively, have been shown to fit our complex model. We identify in detail molecular mechanisms to explain how glycine and GABA initiate chloride ion movement from extraneuronal fluid in the synaptic cleft to intraneuronal volume. In addition, we also identify the molecular mechanisms involved in the blocking of chloride ion movement by strychnine at the glycinergic receptor and by R5135 at the gabanergic receptor. We also present two computer-generated color prints, one for the glycine receptor and one for the GABA receptor, which show the quantum mechanically geometry optimized complex formed between receptor side chains, i.e., the part of the amino acids in the polypeptide that interacts with the zwitterionic inhibitory neurotransmitters. These computer-generated color figures also show a) the important electrostatic and hydrogen bonding in these interactions, b) a van der Waals model of this complex to illustrate that no steric repulsions exist, and c) the molecular electrostatic potential energy map showing the electrostatic potentials of neurotransmitter bound to the receptor model. Finally, we show with computer calculations that the pseudo-rings, formed between the positive quanidinium group in arginine and one of the oxygen atoms in the carboxyl group in both glycine or GABA, result in a positive planar region which appears to be involved in a charge-transfer complex with aromatic benzene groups in amino acids such as phenylalanine and tryosine.

Comparison of binding mechanisms at cholinergic, serotonergic, glycinergic and GABAergic receptors.

Employing computational methods and published data from molecular biological studies involving amino acid sequences in the polypeptide receptors, the authors studied and compared how two excitatory neurotransmitters, ACh and 5-HT, and two inhibitory neurotransmitters, glycine and GABA, can bind to their respective recognition sites at CNS receptors. Models for each neurotransmitter interaction with specific amino acids are described and identified. Molecular mechanisms are identified that can explain how the binding process initiates ion flow through channels located within the postsynaptic membrane such that if the neurotransmitter is inhibitory, hyperpolarization occurs, and if excitatory, depolarization occurs. Although the theoretical work described indicates that there is a difference in molecular mechanisms operative at the anionic and cationic channels, and provides an explanation why the former is more specific, the molecular modeling data and the similarities of specific amino acids in the sequence in all four receptor polypeptides used to construct the four models support ACh, 5-HT, glycine and GABA as being members of the same ligand-gated ion channel superfamily.

Identifying agonistic and antagonistic mechanisms operative at the GABA receptor.

Based on our molecular modeling investigations of the glycinergic receptor, we expanded our studies to similarly investigate the GABAergic receptor. New data suggest there may exist a slightly different agonistic mechanism for the molecules described herein as compared to glycine. The origin of this is undoubtedly the fact that, while glycine has a positive and two negative binding sites, it is significantly shorter than GABA and the other GABA agonists. Clearly, discovery of more glycine agonists is needed to further clarify this point. Moreover, we find a remarkedly different antagonistic mechanism exists for this phylogenetically newer inhibitory system in the central nervous system (CNS) than recently reported for strychnine and eight weaker glycine antagonists. We used GABA and six agonists (muscimol, dihydromuscimol, THIP, isoguvacine, trans-3-aminocyclopentane-1-carboxylic acid, piperidine-4-sulfonic acid) and five antagonists (bicuculline-N15-methobromide, R5135, pitrazepin, iso-THAZ and securinine) to derive our conclusions. We found that each of the agonists have three clearly defined atoms that can serve as attachment points at the GABAA receptor site. One of the three attachment atoms includes a carbonyl or carboxylate oxygen. The role of the carbonyl or carboxylate atom is very important. First, we theorize that a rapid two-point attachment occurs (one from the positive end and one from one of the other two negative atoms on the ligand) at the recognition site in the receptor where GABA or a GABAergic agonist binds. The positive end of the agonist perhaps associates through hydrogen bonding to a beta-carboxyl group in one of the aspartate molecules in the polypeptide. The negative attachment points perhaps bind through hydrogen bonding to arginine molecules in this polypeptide. The second negative site in the agonist immediately triggers a conformational change by pulling together the aforementioned groups by electrostatic attraction, and hence opening the chloride channel. We propose the carbonyl oxygen is partly responsible for triggering the opening by formation of a double hydrogen bond to arginine. We postulate that this attraction is the first step inducing the conformational change. In the case of the GABA antagonists investigated, a fourth attachment site was not found. In fact only two sites have been identified similar to the group II glycine antagonists. Our data support a hypothesis for GABAergic antagonist activity which suggests that the antagonist simply binds to the recognition site and blocks the neurotransmitter, GABA, from entering this site thereby preventing the opening of the chloride channel; it just stays closed. This mechanism is different from the mechanism proposed for the large number of Group I glycine antagonists (Aprison et al.: J Neurosci Res 41: 259-269, 1995).

On identifying a second molecular antagonistic mechanism operative at the glycine receptor.

We used molecular modeling techniques to examine six reported antagonists of glycine with varying Ki values against strychnine. We found the data suggest two groups operating with different mechanisms. In group 1 (strychnine, brucine, Pitrazepin, and bicuculline methobromide) the antagonist contains two or three sites that can electrostatically bind to the three comparable groups of opposite charge in the recognition site where the natural neurotransmitter binds, thus opening the chloride channel. In addition, when in this position, the antagonist is able to also block the now opened chloride channel with a different portion of its structure. In many cases, this involves an interaction between a carbonyl group on the antagonist and the guanidinium group of arginine which is part of the polypeptide segment of the outer mouth of the chloride channel (Grenningloh et al., Nature 330:25-26, 1987). In group 2 (R5135 and 1,5-diphenyl-3,7-diazaadamantan-9-ol) the antagonist contains charged sites but when one of these molecules attaches to the recognition site, the chloride channel is not opened. In addition, R5135 contains a carbonyl group which attaches to arginine as pointed out in the text, whereas 1,5-diphenyl-3,7-diazaadamantan-9-ol contains a phenyl group that can block the channel.

On a molecular comparison of strong and weak antagonists at the glycinergic receptor.

Using molecular modeling techniques, we studied nine glycine antagonists in order to try to identify the molecular descriptors that characterize strychnine as a strong antagonist and N,N-dimethyl-muscimol, iso-THIA, THIA, N-methyl-THIP, iso-THAZ, THAZ, iso-THPO, and iso-THAO (see Experimental for chemical names) as weak glycine antagonists. We confirm that all nine compounds have the three-atom regions (two negative and one positive) that we have postulated are necessary to permit such compounds to attach to the recognition site in the glycinergic synapse. Furthermore, in the case of antagonists we have postulated the presence of a fourth atom that can attach to the top of the chloride ion channel. Each of the nine antagonists has such a fourth negative atom and the latter property gives each of these compounds their antagonistic characteristic. Further, only in the case of strychnine is there evidence that at its positively charged end does the positive charge extend to cover a region that could bind through electrostatic domains to a tertiary carboxyl group in an amino acid like aspartate. Published molecular biological data show that such an amino acid is present in the portion of the polypeptides identified in the glycine receptor. The bidentate binding is superior to the single site attachment that is present in the other eight weak glycine antagonists. In addition, the two negative atom sites in each antagonist are also in a position to participate in electrostatic binding through bidentate involvement with the positively charged guanidinium group of arginine. The latter amino acid also has been identified in the portion of the polypeptide chain at the glycine receptor. Finally, our molecular data predict that after strychnine, the eight weak glycine antagonists listed above are in order of decreasing potency, i.e., N,N-dimethyl-muscimol is the best of the weak antagonists and iso-THAO should be the weakest.

Identification of a second glycine-like fragment on the strychnine molecule.

Strychnine is a complex molecule that inhibits the physiological actions of glycine, an important inhibitory neurotransmitter in the spinal cord, brain stem, and other areas of many vertebrates. Since 1987, we have employed atomistic molecular modeling tools to find an explanation at the molecular level for how this antagonism works. We have located a second glycine-like fragment in the strychnine molecule that, when compared to glycine in a three pair atom analysis, provides an excellent topological and electronic charge congruence. The topological congruence in the second glycine-like fragment is much better than with the first fragment reported in 1987 when using a truncated strychnine molecule in the quantum mechanical analysis. A fourth negative atom, a characteristic of antagonists which we reported earlier (Aprison and Lipkowitz: J Neurosci Res 30:442-446, 1991; Aprison and Lipkowitz: J Neurosci Res 31:166-174, 1992) was found in strychnine. This result follows the pattern reported recently for the three weak glycine antagonists N,N-dimethylmuscimol, N-methyl-THIP, and iso-THAO, a bicyclic 5-isoxazolol zwitterion.

Quantum mechanical study and nuclear magnetic resonance measurements of some alpha-arylcarboxyalkyl acids as anti-inflammatory agents.

The CNDO/2 quantum mechanical conformation method of analysis, charge density and protonation energy calculations, as well as 13C and 1H NMR measurements were carried out for ibufenac, ibuprofen, methylibuprofen, and for a series of alpha-arylpropionic acids. It was found that the nature of the terminal lipophilic residue does not significantly influence the conformation of the alpha-arylcarboxyalkyl acid side chain. The preferred conformational angle, for the torsion of the phenyl-C alpha bond, was found to be 90, 120, and 180 degrees in ibufenac, ibuprofen, and methylibuprofen, respectively. This conformational angle is calculated to be the same in all the alpha-arylpropionic acids. The protonation energies of the alpha-arylpropionic acids are correlated with the anti-inflammatory activity. It was found that the smaller the protonation energy, the larger the anti-inflammatory activity.

Self-consistent field-molecular orbital (SCF-MO) calculations and nuclear magnetic resonance measurements for fosfomycin and related compounds.

In the present work, the mechanism of action of fosfomycin [(-)-(1R,2S)-(1,2-epoxypropyl)phosphonic acid] as an antibiotic agent is studied by "ab initio" quantum mechanical calculations and by 1H, 13C, and 31P NMR measurements. Attention is focused on the relative charge density and chemical shift of the C(2) atom of the epoxy ring, which seems to be closely related with the activity of this antibiotic. The theoretical results suggest that the sulfhydryl addition should be preceded by a necessary anchoring of the phosphonate moiety on a positive group of the receptor.

Conformational analysis of some alpha-phenylpropionic acids with anti-inflammatory activity.

CNDO/2 quantum mechanical conformational calculations, as well as 13C and 1H NMR measurements, have been carried out for the propionic acid residues of 2-(p-isobutylphenyl)propionic acid (ibuprofen) and 2-methyl-2-(p-isobutylphenyl)propionic acid. A relationship between the conformational angle of the propionic acid residue and the anti-inflammatory activity appears to exist. The more open the Ph--C alpha--COOH dihedral angle, the larger the anti-inflammatory activity.