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Voltage-gated sodium channels are targets for many toxins and medically important drugs. Despite decades of intensive studies in industry and academia, atomic mechanisms of action are still not completely understood. The major cause is a lack of high-resolution structures of eukaryotic channels and their complexes with ligands. In these circumstances a useful approach is homology modeling that employs as templates X-ray structures of potassium channels and prokaryotic sodium channels. On one hand, due to inherent limitations of this approach, results should be treated with caution. In particular, models should be tested against relevant experimental data. On the other hand, docking of drugs and toxins in homology models provides a unique possibility to integrate diverse experimental data provided by mutational analysis, electrophysiology, and studies of structure-activity relations. Here we describe how homology modeling advanced our understanding of mechanisms of several classes of ligands. These include tetrodotoxins and mu-conotoxins that block the outer pore, local anesthetics that block of the inner pore, batrachotoxin that binds in the inner pore but, paradoxically, activates the channel, pyrethroid insecticides that activate the channel by binding at lipid-exposed repeat interfaces, and scorpion alpha and beta-toxins, which bind between the pore and voltage-sensing domains and modify the channel gating. We emphasize importance of experimental data for elaborating the models.

An integral part of modern evolutionary biology is comparative analysis of structure and function of macromolecules such as proteins. The first and critical step to understand evolution of homologous proteins is their amino acid sequence alignment. However, standard algorithms fop not provide unambiguous sequence alignments for proteins of poor homology. More reliable results can be obtained by comparing experimental 3D structures obtained at atomic resolution, for instance, with the aid of X-ray structural analysis. If such structures are lacking, homology modeling is used, which may take into account indirect experimental data on functional roles of individual amino-acid residues. An important problem is that the sequence alignment, which reflects genetic modifications, does not necessarily correspond to the functional homology. The latter depends on three-dimensional structures which are critical for natural selection. Since alignment techniques relying only on the analysis of primary structures carry no information on the functional properties of proteins, including 3D structures into consideration is very important. Here we consider several examples involving ion channels and demonstrate that alignment of their three-dimensional structures can significantly improve sequence alignments obtained by traditional methods.

Study of spatial structure of biologically active guanidine derivatives by the method of molecular mechanics has shown that in an anticoccidial drug, 1,3-bis ( p-chlorobenzylidenamino)guanidine (Cl-BAG) the most preferable are convolute conformations, in which the chlorine atoms that are distant in the valent chain are approached to each other at a distance of 3.7 A. This indicates predisposition of the optimal conformations to form chelate complexes with ions of metals, which is confirmed by comparative spectrophotometric studies of the second derivative of differential UV-spectra of Cl-BAG in the presence and absence of calcium ions. Its derivative without chlorine (BAG) is unable to bind Ca2+ and has been shown to have no anticoccidial action, which associates the biological potency with the presence of calcium-binding ability of the compounds. The capability of Cl-BAG for chelation depends essentially on nature of the chelated metal ion. The antienzyme testing of inhibiting action of the guanidine derivatives toward cholinesterases of human erythrocytes, horse blood serum, mink brain and serum, optic ganglia of the Pacific squid Todarodes pacificus has revealed difference between the enzymes due to possibility of redistribution of the positive charge between the guanidinium fragment and amino groups and a change of the degree of charge delocalization.

Ion channels in cell membranes are targets for a multitude of ligands including naturally occurring toxins, illicit drugs, and medications used to manage pain and treat cardiovascular, neurological, autoimmune, and other health disorders. In the past decade, the x-ray crystallography revealed 3D structures of several ion channels in their open, closed, and inactivated states, shedding light on mechanisms of channel gating, ion permeation and selectivity. However, atomistic mechanisms of the channel modulation by ligands are poorly understood. Increasing evidence suggest that cationophilic groups in ion channels and in some ligands may simultaneously coordinate permeant cations, which form indispensible (but underappreciated) components of respective receptors. This review describes ternary ligand-metal-channel complexes predicted by means of computer-based molecular modeling. The models rationalize a large body of experimental data including paradoxes in structure-activity relationships, effects of mutations on the ligand action, sensitivity of the ligand action to the nature of current-carrying cations, and action of ligands that bind in the ion-permeation pathway but increase rather than decrease the current. Recent mutational and ligand-binding experiments designed to test the models have confirmed the ternary-complex concept providing new knowledge on physiological roles of metal ions and atomistic mechanisms of action of ion channel ligands.

The biologically relevant conformation of substance P is likely to be dictated by the lipid milieu wherein the hormone would interact with its receptor. Assuming that specific constraints to the hormone structure may be imparted by its interaction with Ca2+ ions in the low dielectric lipid medium, the interaction of substance P and its inactive analog, Ala7-substance P, has been characterized in a lipid-mimetic solvent. Circular dichroism (CD) and NMR spectral methods were employed to study the conformation of the free and Ca2+-bound forms of the peptides and the conformational changes that occur on Ca2+ binding. The results show that both peptides assume a helical structure in the non-polar solvent used, a mixture of acetonitrile and trifluoroethanol. The N-terminal region is, however, less ordered in the analog peptide compared with the native hormone. Ca2+ addition causes significant conformational changes in both the peptides. However, while substance P binds two Ca2+ ions in a cooperative manner, Ala7-substance P binds only one Ca2+ ion with a relatively weaker affinity. Computations of the minimum-energy conformations of the free and Ca2+-bound peptides were performed using interproton distances derived from nuclear Overhauser enhancement spectra of the two peptides, as well as the information provided by changes in proton chemical shifts caused by Ca2+ addition. Taken together, the results of this study suggest that differences in the interaction of substance P and Ala7-substance P with Ca2+ in the non-polar milieu, which in turn leads to differences in their Ca2+-bound conformations, may be the basis for the differences in their biological potencies.

17 beta-Estradiol (E2) is a potent stimulator of certain forms of breast cancer. The final step of E2 biosynthesis is catalyzed by the estrogenic 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD1), which is an important target for anti-cancer drugs. X-ray crystallography indicated that the binding site for the steroids has a tunnel-like shape. We have used a Monte Carlo-Minimization (MCM) protocol to explore possibilities of interactions of E2 with the binding site tunnel of 17 beta-HSD1. The enzyme was represented by flexible residues having at least one atom within 6 A from either E2 or NADP (as seen in a crystal ternary complex) and by rigid residues having at least one atom within 10 A from E2 or NADP. Special constraints were used to pull the substrate 10 A along the tunnel with 1 A step; the complex was MCM-optimized at each position of the steroid. The optimal binding mode of E2 in 17 beta-HSD agrees with the crystallographic data; however, wide and flat minima of the MCM profile suggest alternative modes of the steroid binding. The advance of the steroid along the tunnel is accompanied by essential conformational rearrangements of the enzyme side chains, noticeable rotation of the substrate along its longitudinal axis, and certain conformational deformations of the substrate. The contributions of the enzyme residues and of the steroid atoms to the intermolecular energy were estimated.

A conformational variability of the collagen triple helix was studied with the methods of molecular mechanics. The Rich-Crick model with one hydrogen bond per tripeptide fragment or the model with two hydrogen bonds per tripeptide fragment were used for tripeptides forming the primary structure of the protein. Imino acid and amino acid residues were located in the second position of the tripeptide fragments in the first and second cases, respectively. Conformations on domain boundaries, which had alternating structures with one and two hydrogen bonds per tripeptide, were particularly studied. Essentially all types of collagen backbone composed of amino acid residues most frequently occurring in this protein were considered. A new model was suggested that combined elements of the Rich-Crick model and our new approach. This was shown to be stereochemically valid, energetically advantageous, and consistent with the experimental data. It was conclusively demonstrated that the primary structure of collagen determines its tertiary structure.

Drastic changes in the CD and fluorescence spectra of oxytocin [cyclo(Cys1-Tyr2-Ile3-Gln4-Asn5-Cys6)-Pro7-Leu8-Gly 9-NH2] occur on binding Ca2+ in trifluoroethanol (Ananthanarayanan and Brimble, preceding paper). To further characterize the conformation of the Ca(2+)-bound hormone, we carried out 1H-nmr measurements in deuterated trifluorethanol of oxytocin and its 1:1 Ca2+ complex. The one-dimensional nmr data identified residues involved in Ca2+ binding and the extent of their perturbation on Ca2+ addition. The 3JNH-CH coupling constants and two-dimensional nuclear Overhauser effect (NOE) spectral cross peaks confirmed the helical nature of the Ca2+ complex deduced from CD data. Interproton distances in the free hormone and its Ca2+ complex were estimated from the respective NOE data. Apparent global minimum-energy conformations of free and Ca2+ bound oxytocin were computed using the Monte Carlo with energy minimization protocol, with and without incorporating the NOE-derived distance constraints. Taken together, our results show Ca2+ binding to oxytocin to be a two-step process. The binding of the first Ca2+ brings the otherwise extended tail segment of oxytocin closer to the ring moiety so that it wraps around the cation. This causes the maximal extent of change in all the spectral parameters. The subsequent formation of the 2:1 Ca-oxytocin complex results in the tail detaching itself away from the ring so as to bind the second Ca2+ ion. This leads to further spectral changes in the hormone molecule. The tail segment plays a major role in both steps. These observations may be useful in understanding the structural basis of oxytocin action.

All equilibrium conformations of 12 anti-acetylcholinesterase organophosphorus inhibitors were calculated by the molecular mechanics method. The accessibility of the phosphorus atom of the inhibitors for interactions with the nucleophilic group at the enzyme active centre was estimated. The conformers with the phosphorus atom sterically accessible from the side opposite to the breaking ester bond were classified as productive. A correlation was revealed between the activity of the inhibitors and the population of their productive conformation.

All equilibrium conformations of a series of N-alkyl-α-alkyl derivatives of noradrenaline and their synthetic precursors—phenylaminoketones—have been calculated. It has been shown that spasmodic changes in bronchodilating activity of the catecholamines, observed with the increase in α-alkyl substituent size, may arise from the difference in the ratio of diastereoisomers produced by reduction of aminoketones.

Theoretical conformational analysis of a series of alpha 1-adrenoceptor activators and inhibitors including 6-fluoronoradrenaline, methoxamine, phenoxydenzamine, piperoxane, and WB-4101 has been carried out by classical semi-empirical method. The conformational energy minimization was performed in the space of the majority of torsional and bond angles. The selection of productive conformations was made according to the following criteria: 1) low conformational energy; 2) similarity of the nitrogen atoms and phenyl rings spatial disposition in all ligands; 3) accessibility for intermolecular interactions of the functional moieties in all ligands. The productive conformation of alpha 1-adrenoceptor endogenous activator, noradrenaline, has the Ph-C-C-N fragment in the perpendicular (-)-gaushe conformation, and ethanolamine side chain beta-hydroxyl in trans arrangement relative to the meta hydroxyl of the catechol ring. A topographic model for the receptor was suggested, its components being the arylophilic, nucleophilic, hydrophobic, and proton acceptor groups, along with the binding region for catechol hydroxyls, situated against the most accessible sides of the functional moieties of the ligands. Structure-activity relationships for a series of alpha 1-adrenoceptor ligands are discussed in the light of the proposed model. Both common and characteristic features of the alpha 1-adrenoceptor model are considered in comparison with the earlier suggested beta 2-adrenoceptor model.

The spatial structure of methylamide N-acetyl-L-argine was studied taking into account the non-valent and electrostati interactions, the torsion energy, and the distorsion of valency angles. Calculation of the favourable conformations of the molecule was carried out with the use of all the combinations of angles phi, psi, chi1 divided by chi4 as an intital approximation. These correspond to the low energy forms of the main chain and to the minima of the torsion potentials of the side chain. Conformational possibilities of arginine and lysine were compared. The calculated stable conformation of N-acetyl-L-arginine-methylamide are compared with the geometry of arginine residues in the proteins with known structure.

By using classical potential functions, nonbound interaction energy calculations are carried out on the deoxydinucleoside phosphate complexes dUpdA:dUpdG and dUpdU:dGpdA. All dihedral and bond angles, except those of the nitrogen bases, are varied. On the basis of the calculations possible conformations of nucleic acid fragments containing G:U base pair are proposed. Dihedral angles of the conformations are close to those of regular Watson-Crick helices while interaction energies are 3-4 kcal/mole higher than that of A:U containing fragment conformations.

By means of molecular mechanics, theoretical conformational analysis has been made of 19 substrates of butyrylcholinesterase - acetylcholine derivatives with different structure of the ammonium group. It was concluded that the anionic point is located in the cavity of the enzymic molecule. Dimensions and shape of this cavity were established which provide satisfactory correlation between its filling by substrate conformers and the rate of their enzymic hydrolysis. Some suggestions were made with respect to the mechanism of the effect of non-productive sorbtion of the substrates on the rate of their enzymic hydrolysis.

The paper reviews some problems of theoretical investigation of the spatial of adrenergic drugs and their receptors, citing results recently obtained by the authors. It includes the chapters as follows: Introduction; Conformation - activity relationships of ligands; Theoretical conformational analysis; Noradrenaline; Classification and some models of adrenergic receptors; Problems of ligands productive conformation selection and the description of receptor topography; Principles of choice of ligands; Criteria for productive conformation selection; Productive conformations of beta 2-adrenergic agonists and beta 2-receptor topography; Productive conformations of alpha 1-adrenergic receptor ligands and alpha 1-receptor topography; Structure-activity relationships of some ligands in the light of adrenergic beta 2- and alpha 1-receptors topography; Possible role of calcium ion in the interaction of adrenergic agonists with receptors.

The spatial structure of the methylamide of N-acetyl-L-lysine has been analysed taking into account non-bonded and electrostatic interactions, torsional energy, bond angles distortion and hydrogen bonding. Conformational capacities of the backbone and mutual dependence of spatial structures of the backbone and the side chain was described by conformational maps obtained by energy minimisation, the dihedral angles and the bond angles of the side chain being varied for every phi, psi point. Every possible combination for phi, psi, x1-x5-angles was used corresponding to the stable form of the backbone and to torsion potential minima of the initial approximations in the calculation of preferred conformations of the molecule. Comparisons are made between stable forms of the methylamide of N-acetyl-L-lysine and Lys residues in proteins with known structure.

Conformational possibilities of pirrolidine analogues of acetylcholine beta-(N-methyl pirrolidinium)-ethyl ester of acetic acid and beta-(N-ethyl pirrolidinium)-ethyl ester of acetic acid and beta-(N-ethyl pirrolidinium)-ethyl ester of acetic acid were investigated by the method of atomic potentials. The conformational energy was considered as a sum of non-bonded and electrostatical interactions, torsional energy and distortions of bond angles. It has been shown that the replacement of the nitrogen methyl group to ethyl group results in decrease of the average barrier height between two gauche conformations of the O--C--C--N fragment. Comparison of conformational properties of some cholinesterase substrates permit to draw a suggestion that the barrier height influences the rate of the enzymatic hydrolysis.

All equilibrium conformations of twenty-three acetylcholinesterase effectors were calculated by the molecular mechanics method, nonbonded interactions, torsion energy and energy of bond angles deformation being taken into account. In a series of conformationally flexible derivatives of acetylcholine the correlation was found between hydrolysis rate and population of the completely extended tt-conformation. In a series of cyclic analogues of acetylcholine the high hydrolysis rate occurs only for substrates sterically corresponding to tt-conformation of acetylcholine with regard to disposition of ammonium group, carbonyl oxygen and carbonyl carbon. The hydrolysis rate of acetylcholine derivatives with elongated chain between acetyl and cationic groups is directly proportional to the population of the conformations similar to tt-conformation of acetylcholine. It is concluded that tt-conformation of acetylcholine is productive for acetylcholinesterase hydrolysis.

All equilibrium conformations for nine ligands, essentially different in structure, of the noradrenaline carrier through the synaptosomal membrane of rat hypothalamus were calculated by semi-empirical method. Among these compounds were amphetamine, methylphenidate, tricyclic antidepressants. The conformational energy minimization was performed in the space of torsional and bond angles. Geometrical characteristics of the conformers were determined in the cartesian coordinate system fixed relative to the benzene ring and nitrogen atom of the ligand. The selection of biologically active (productive) conformations was made according to the following criteria: 1) low conformational energy; 2) similarity of the nitrogen atoms and phenyl rings spatial disposition in all ligands; 3) accessibility for intermolecular interactions of the same sides of functional groupings in all ligands. The above criteria enabled the productive conformations for all ligands to be chosen unambiguously. The productive conformation of noradrenaline was found to have the Ph-C-C-N fragment in perpendicular trans-conformation. A topographic model for the carrier active site was suggested, its components being the nucleophilic and two arylophilic groups situated against the most accessible sides of the functional moieties of the productively bound ligands.