The local minima method (LMM) of pharmacophore determination: a protocol for predicting the bioactive conformation of small, conformationally flexible molecules.

Software has been developed for potential energy surface analysis and the local minima method of pharmacophore determination. LMM is rigorous and systematic and employs multiple conformations which are the local minima from the potential energy surface of each compound in the data set. It produces a series of possible pharmacophores from a postulated set of pharmacophore elements. The best pharmacophore is then determined by performing a comparative molecular field analysis (CoMFA) on each one. The pharmacophore which produces the most self-consistent model is deemed the best. Local minima on the gas-phase potential energy surface are shown to be a reasonably close approximation to protein bound conformations, and these conformations can be found through systematic conformational searches followed by minimization of the local minima. LMM was used to develop a 3D-QSAR model for dopamine beta-hydroxylase (DBH) inhibitors which was highly predictive (predictive R2 = 0.71 and standard error of predictions = 0.41). The model predicted that the phenyl and thienyl series of inhibitors were acting as bioisosteres. Examination of compounds overlayed in the model indicated a possible hydrogen bond acceptor in the DBH active site. Three tyrosine residues previously labeled by mechanism based inhibitors may be acting as the acceptor and therefore represent excellent candidates for site-directed mutagenesis studies.

Modeling of competitive phosphono amino acid NMDA receptor antagonists.

A pharmacophore for the phosphono amino acid antagonists of the NMDA receptor has been developed using computer-based molecular modeling techniques. An important feature of this model is that a single binding site is proposed for the phosphonic acid moiety. All competitive antagonists we have examined incorporating amino acid and phosphonate groups in their structure fit the pharmacophore in energetically accessible conformations.

Molecular design and modeling of protein-heparin interactions.

The methods and approaches taken to investigate heparin-apoE peptide interactions have involved a series of steps, including (1) identification of the heparin-binding domains of apoE, (2) determination of the minimal amino acid sequence regions involved in heparin binding, heparin-induced conformational changes, and stability of apoE peptide structures in solution, (3) modeling of these peptide and oligosaccharide structures, and (4) examination of their behavior during molecular dynamics calculations to determine if the modeled complexes simulate the results of the solution study. The heparin-binding regions of apoE were determined by fragmentation of the protein and identification of the heparin-binding fragments by ligand-blotting procedures using 125I-labeled heparin. Studies with synthetic peptide fragments of various lengths and dot-blot procedures with 125I-labeled heparin identified the minimal residues critical for heparin-binding and CD studies established the prominent secondary structures of these domains. These studies also showed that heparin binds to the apoE(211-243) and apoE(129-169) regions to induce and stabilize beta-strand and alpha-helical peptide conformations. Secondary structure algorithms were used to identify the specific residues with the highest probabilities of forming alpha-helix and beta-strand structures. Based on the predictive algorithms, the apoE(211-234) and apoE(129-159) structures were built using the Insight program and their molecular interactions with various heparin oligosaccharide models were investigated by molecular dynamics. In agreement with the solution studies in the presence of salt, the molecular dynamics studies showed that the oligosaccharides stabilized the beta-strand and alpha-helical peptide configurations against simulated thermal denaturations. Further modeling studies are in progress to examine the mechanism of the heparin-induced increase in ordered structure of these peptides.

1-(Thienylalkyl)imidazole-2(3H)-thiones as potent competitive inhibitors of dopamine beta-hydroxylase.

1-(2-Thienylalkyl)imidazole-2(3H)-thiones (5a-k) are competitive inhibitors of dopamine beta-hydroxylase (DBH) and demonstrate the utility of thiophene in the design of potent competitive inhibitors of this enzyme. The structure-activity relationships for these compounds are discussed and compared with those of 1-phenylalkyl-imidazole-2(3H)-thiones (1). With the aid of molecular modeling, an idealized active-site conformer is proposed and an explanation for the difference in activity between the phenyl (1) and thienyl (5) DBH inhibitors is presented. The difference in activity is consistent with our proposal that thiophene may not always be a bioisostere for phenyl. The inhibitor of most interest, 1-[2-(2-thienyl)ethyl]imidazole-2(3H)-thione (5g), was selected for study in the spontaneously hypertensive rat. The changes in dopamine and norepinephrine levels that resulted from oral administration of 5g correlated with the reduction of blood pressure.

Molecular modeling of protein-glycosaminoglycan interactions.

Forty-nine regions in 21 proteins were identified as potential heparin-binding sites based on the sequence organizations of their basic and nonbasic residues. Twelve known heparin-binding sequences in vitronectin, apolipoproteins E and B-100, and platelet factor 4 were used to formulate two search strings for identifying potential heparin-binding regions in other proteins. Consensus sequences for glycosaminoglycan recognition were determined as [-X-B-B-X-B-X-] and [-X-B-B-B-X-X-B-X-] where B is the probability of a basic residue and X is a hydropathic residue. Predictions were then made as to the heparin-binding domains in endothelial cell growth factor, purpurin, and antithrombin-III. Many of the natural sequences conforming to these consensus motifs show prominent amphipathic periodicities having both alpha-helical and beta-strand conformations as determined by predictive algorithms and circular dichroism studies. The heparin-binding domain of vitronectin was modeled and formed a hydrophilic pocket that wrapped around and folded over a heparin octasaccharide, yielding a complementary structure. We suggest that these consensus sequence elements form potential nucleation sites for the recognition of polyanions in proteins and may provide a useful guide in identifying heparin-binding regions in other proteins. The possible relevance of protein-glycosaminoglycans interactions in atherosclerosis is discussed.

Conformational energy differences between side chain alkylated analogues of the hallucinogen 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane.

Theoretical conformational energy calculations were carried out for the (+) and (-) isomers of the hallucinogen 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM, STP). Energies were also calculated for two analogues of DOM, 1-amino-1-(2,5-dimethoxy-4-methylbenzyl)cyclopropane and 1-(2,5-dimethoxy-4-methylphenyl)-2-methyl-2-aminopropane. This method utilized classical, empirical potential-energy functions. A previously proposed active conformational region was studied. Compounds could be ranked in order of potency based on relative conformational energies in this region. Measurement of 13C spin--lattice relaxation times (T1) for the two alpha, alpha-disubstituted DOM analogues confirmed theoretical predictions of very restricted conformational freedom for the dimethyl compound but more flexibility for the cyclopropane analogue.

Conformational analysis of pyridoxal Schiff's bases. Nuclear magnetic resonance studies of the conformations about the C4-C4', Calpha-Cbeta, and N-Calpha bonds of the pyridoxal Schiff's bases of amino acids.

The solution conformations of a series of pyridoxal-amino acid Schiff's bases were analyzed using 13C and 1H nuclear magnetic resonance techniques. The 13C--1H coupling constants were assigned based on model compounds and isotopic labeling. The predominant conformation of the the C4--C4' bond was found to be "cis" based on nuclear Overhauser effect (NOE) measurements and the "simultaneous" upfield shift of both H4' and H5' in the Schiff's bases of aromatic amino acids. Going from the monoanion (pD 8.2) to the dianion (pD 12.3), changes in these two effects suggested an increasing contribution of the "trans" conformer. The conformation of the N--Calpha bond was found to be approximately the same for all the Schiff's bases studied based on the long-range coupling constants 3J(C4'--Halpha) of these compounds, and the NOE studies indicate that there is a close spatial relationship between H4' and Halpha. The conformations of the Calpha--Cbeta bond of the Schiff's bases of aromatic amino acids were determined by stereospecific deuterium labeling at the beta position. A pi--pi interaction between the aromatic ring and the pi system of pyridoxal was observed which disappeared upon saturation of the aromatic ring.

Conformation-reactivity relationship for pyridoxal Schiff's bases. Rates of racemization and alpha-hydrogen exchange of the pyridoxal Schiff's bases of amino acids.

The role of stereoelectronic effects in controlling the reaction specificity of biological reactions involving pyridoxal phosphate-amino acid Schiff's bases was tested with nonenzymatic models. The rates of racemization and Halpha exchange of a series of pyridoxal-amino acid Schiff's bases were determined. The order of these rates does not parallel the predictions based solely on electronic or steric effect, but parallels the proportions of the reactive conformers (e.g., conformers with the Calpha--Halpha bond orthogonal to the pi system) estimated by CPK models. The special reactivity of the phenylalanine Schiff's base was consistent with a special conformation in which some type of pi--pi interaction increases the proportion of exchangeable conformers, thus further substantiating the role of conformation in governing the reactivity of the Calpha--Halpha bond. Furthermore, semiempirical calculations of the conformation about the Calpha00N bond were performed using the CAMSEA conformational analysis program. The results of conformational calculations are consistent with the results of conformational analysis by nuclear magnetic resonance. The order of reactivity of the the Calpha--Halpha bond of the SB dianion, pH 12.0, predicted by calculation based on stereoelectronic effects, though not quantatively parallel to the observed rate constants, is qualitatively in agreement with the experimental results.