Bioinformatics and receptor mechanisms of psychotropic drugs.

One important aspect in biotechnology is gene discovery and target validation for drug discovery. Information from the human genome (HUGO) project may be used to deduce the amino acid sequence of all proteins produced in the human body. However, knowing the amino acid sequence of a protein is not the same as knowing its function. Identification of novel molecular targets for discovery of new, safer and more efficient therapeutic drugs from the human genome sequences requires multidisciplinary research efforts, including proteomics, structural biology and bioinformatics. In addition to possible effects on gene expression, most of the currently used therapeutic drugs either have enzymes or membrane proteins as their molecular targets of action. These membrane proteins include transporters of small molecules across cell membranes, ion channels, or receptors that convey signals from one side of a membrane to the other. Our research group as well as others have used computational techniques, along with biotechnology, molecular biology and other experimental techniques, to construct detailed 3-dimensional models of transporter proteins and G-protein coupled receptors (GPCRs), which are the molecular targets of action of psychotropic drugs. The models have been used to simulate the molecular dynamics and study the ligand binding and signal transduction mechanisms of these receptors. The use of bioinformatics, as exemplified in our modelling of GPCRs, is only one of the key factors for success in post-genomic research for new targets for therapeutic drugs.

Molecular dynamics of buspirone analogues interacting with the 5-HT1A and 5-HT2A serotonin receptors.

Three-dimensional (3-D) models of the human serotonin 5-HT1A and 5-HT2A receptors were constructed, energy refined, and used to study the interactions with a series of buspirone analogues. For both receptors, the calculations showed that the main interactions of the ligand imide moieties were with amino acids in transmembrane helix (TMH) 2 and 7, while the main interactions of the ligand aromatic moieties were with amino acids in TMH5, 6 and 7. Differences in binding site architecture in the region of highly conserved serine and tyrosine residues in TMH7 gave slightly different binding modes of the buspirone analogues at the 5-HT1A and 5-HT2A receptors. Molecular dynamics simulations of receptor-ligand interactions indicated that the buspirone analogues did not alter the interhelical hydrogen bonding patterns upon binding to the 5-HT2A receptor, while interhelical hydrogen bonds were broken and others were formed upon ligand binding to the 5-HT1A receptor. The ligand-induced changes in interhelical hydrogen bonding patterns of the 5-HT1A receptor were followed by rigid body movements of TMH2, 4 and 6 relative to each other and to the other TMHs, which may reflect the structural conversion into an active receptor structure.

A putative three-dimensional arrangement of the human serotonin transporter transmembrane helices: a tool to aid experimental studies.

The human serotonin transporter is the molecular target for selective serotonin reuptake inhibitor drugs which are being used for treatment of depression. A three-dimensional model of the membrane spanning parts of the transporter was constructed. The transporter was assumed to consist of 12 transmembrane alpha-helices. The model was based on published experimental data of cocaine binding to mutant transporters, amino acid sequence analysis, and interactive molecular graphics. The model suggests that a high affinity cocaine binding site is situated in a region of the model where Asp98 acts like an anchor, while a putative low affinity site is situated in another region with Glu508 as the anchoring amino acid. A series of docking experiments with various reuptake inhibitors were conducted, using interactive molecular graphics techniques combined with energy calculations and analysis of the transporter-ligand complexes. Experiments involving molecular mapping of ligand binding areas may benefit from using the current model in experimental design. From the current model, several amino acids were proposed as prime candidates for mutagenesis and subsequent ligand binding studies. Also for evaluation of results from site directed mutagenesis experiments with SERT and similar transporters we assume the model will be helpful.

Molecular dynamics of 5-HT1A and 5-HT2A serotonin receptors with methylated buspirone analogues.

In the present study experimentally determined ligand selectivity of three methylated buspirone analogues (denoted as MM2, MM5 and P55) towards 5-HT1A and 5-HT2A serotonin receptors was theoretically investigated on a molecular level. The relationships between the ligand structure and 5-HT1A and 5-HT2A receptor affinities were studied and the results were found to be in agreement with the available site-directed mutagenesis and binding affinity data. Molecular dynamics (MD) simulations of ligand-receptor complexes were performed for each investigated analogue, docked twice into the central cavity of 5-HT1A/5-HT2A, each time in a different orientation. Present results were compared with our previous theoretical results, obtained for buspirone and its non-methylated analogues. It was found that due to the presence of the methyl group in the piperazine ring the ligand position alters and the structure of the ligand-receptor complex is modified. Further, the positions of derivatives with pyrimidinyl aromatic moiety and quinolinyl moiety are significantly different at the 5-HT2A receptor. Thus, methylation of such derivatives alters the 3D structures of ligand-receptor complexes in different ways. The ligand-induced changes of the receptor structures were also analysed. The obtained results suggest, that helical domains of both receptors have different dynamical behaviour. Moreover, both location and topography of putative binding sites for buspirone analogues are different at 5-HT1A and 5-HT2A receptors.

Theoretical models of interactions between buspirone analogues and 5-HT1A and 5-HT2A serotonin receptor subtypes.

In present study the structure-selectivity relationship of buspirone and six of its analogues towards 5-HT1A and 5-HT2A serotonin receptors was investigated on molecular level. Molecular mechanics energy minimisation and advanced molecular dynamics (MD) simulations allowed us to perform a dynamic structural analysis of transmembrane helical domains of the human 5-HT1A and 5-HT2A receptors and investigate the ligand-induced changes of the entire structure of the ligand-receptor complex. The obtained results suggest, that helical and extracellular domains of both receptors have different topography of the putative binding sites and also different dynamical behaviour. The results of this study are consistent with experimental site-directed mutagenesis data and binding affinities of examined ligands towards both serotonin receptors.

A World Wide Web-service to aid the development of AMBER parameters using analogy to standard parameters.

A World-Wide Web service has been constructed to assist the development of force field parameters extending the AMBER force fields. This service extracts parameters from the standard AMBER force field parameter databases. From a Web-based interface the user can choose between bond, angle and torsional parameters with certain constraints on element and/or atom hybridization. The software constructed for the purpose of finding appropriate parameters will locate standard AMBER force field parameters matching the user specification. For bond and angle parameters a scatter plot of the reference values against force constants is provided. This service has been produced to assist in extraction and evaluation of parameters that may be useful for molecules other than proteins and nucleic acids.

Molecular modeling of the endogenous peptide Leu-Ser-Ala-Leu, pindolol, 5-hydroxytryptamine and their interactions with the human 5-hydroxytryptamine1B (5-HT1B) receptor.

Molecular modeling techniques were used to build a three-dimensional model of the human 5-HT1B receptor. The receptor model was used to examine receptor interactions of 5-hydroxytryptamine (serotonin), (S)pindolol and of the tetrapeptide Leu-Ser-Ala-Leu (LSAL), which recently has been shown to interact specifically with the 5-HT1B receptor. We have assumed that the NH3(+)-LSAL-COO- form of the tetrapeptide is the biologically active, and propose that a negatively charged residue conserved among various species homologues of the 5-HT1B receptor may act as a counter-ion for the positively charged N-terminus of the tetrapeptide. The strongest LSAL-receptor interactions were obtained after molecular dynamics simulations that were started with the N-terminus of LSAL positioned close to Asp352 in transmembrane helix 7. The model suggests that Asp352 in transmembrane helix 7 may act as a counter-ion for the positively charged N-terminus, and that the side chains of Tyr109 (transmembrane helix 2) and Trp125 (transmembrane helix 3) may form hydrogen bonds with the negatively charged C-terminus of LSAL.

[Virtual reality in medical education]. / Simulert virkelighet innen utdanning av medisinere.

Virtual reality technology has found new applications in industry over the last few years. Medical literature has for several years predicted a break-through in this technology for medical education. Although there is a great potential for this technology in medical education, there seems to be a wide gap between expectations and actual possibilities at present. State of the technology was explored by participation at the conference "Medicine meets virtual reality V" (San Diego Jan. 22-25 1997) and a visit to one of the leading laboratories on virtual reality in medical education. In this paper we introduce some of the basic terminology and technology, review some of the topics covered by the conference, and describe projects running in one of the leading laboratories on virtual reality technology for medical education. With this information in mind, we discuss potential applications of the current technology in medical education. Current virtual reality systems are judged to be too costly and their usefulness in education too limited for routine use in medical education.

GPCRDB: an information system for G protein-coupled receptors.

The GPCRDB is a G protein-coupled receptor (GPCR) database system aimed at the collection and dissemination of GPCR related data. It holds sequences, mutant data and ligand binding constants as primary (experimental) data. Computationally derived data such as multiple sequence alignments, three dimensional models, phylogenetic trees and two dimensional visualization tools are added to enhance the database's usefulness. The GPCRDB is an EU sponsored project aimed at building a generic molecular class specific database capable of dealing with highly heterogeneous data. GPCRs were chosen as test molecules because of their enormous importance for medical sciences and due to the availability of so much highly heterogeneous data. The GPCRDB is available via the WWW at http://www.gpcr.org/7tm

The ligand-binding site of buspirone analogues at the 5-HT1A receptor.

A three-dimensional model of the 5-HT1A receptor in man was constructed by molecular-modelling techniques and used to study the molecular interactions of a series of buspirone analogues with the 5-HT1A receptor by molecular-mechanical-energy minimization and molecular-dynamics simulations. The receptor has seven trans-membrane alpha helices (TMHs) organized according to the electron-density-projection map of visual rhodopsin, and includes all loops between TMHs and the N- and C-terminal parts. The best fit between the buspirone analogues and the receptor model was obtained with the quinolinyl part of the ligand molecules interacting with amino acids in TMH6, the imide group interacting with amino acids in TMH2, TMH3 and TMH7, and the carbonyl groups hydrogen-bonded with Ser86 and Ser393. The ligand-binding rank order deduced from the experimentally determined inhibition constant was reproduced by calculation of receptor-binding energies of the buspirone analogues. The models suggest that steric hindrance and repulsive forces between the receptor and the imide group of the buspirone analogues are the most important determinants of ligand-binding affinity for discriminating between these ligands.

A database of mutants and effects of site-directed mutagenesis experiments on G protein-coupled receptors.

A database system and computer programs for storage and retrieval of information about guanine nucleotide-binding protein (G protein) -coupled receptor mutants and associated biological effects have been developed. Mutation data on the receptors were collected from the literature and a database of mutants and effects of mutations was developed. The G protein-coupled receptor, family A, point mutation database (GRAP) provides detailed information on ligand-binding and signal transduction properties of more than 2130 receptor mutants. The amino acid sequences of receptors for which mutation experiments have been reported were aligned, and from this alignment mutation data may be retrieved. Alternatively, a search form allowing detailed specification of which mutants to retrieve may be used, for example, to search for specific amino acid substitutions, substitutions in specific protein domains or reported biological effects. Furthermore, ligand and bibliographic oriented queries may be performed. GRAP is available on the Internet (URL: http://www-grap.fagmed.uit.no/GRAP/+ +homepage.html) using the World-Wide Web system.

Molecular modelling of UH-301 and 5-HT(1a) receptor interactions.

A three-dimensional model of the human 5-HT(1a) receptor was constructed by molecular modelling, and the molecular and electronic structures of (R)- and (S)-5-fluoro-8-hydroxy-2-(dipropylamino)tetralin (UH-301) and of (R)- and (S)-8-hydroxy-2-(dipropylamino)tetralin (8-OH-DPAT) were examined by molecular mechanics and quantum mechanics calculations and molecular dynamics simulations. The receptor model has seven transmembrane alpha-helices (TMHs), organized according to a projection map of visual rhodopsin, and includes all loops between helices and the N- and C-terminal parts. Interactions of UH-301 and 8-OH-DPAT with the 5-HT(1a) receptor were examined by molecular dynamics simulations and energy minimization of receptor-ligand complexes. 8-OH-DPAT had lower electrostatic potentials around the hydroxyl group and stronger hydrogen bonding to the receptor model than had UH-301. The simulations indicated that the 5-HT(1a) receptor agonists, (R)- and (S)-8-OH-DPAT and (R)-UH-301, interacted with the receptor at a site closer to Asp82 in TMH2 than did (S)-UH-301, which is a 5-HT1a receptor antagonist. Simulations of receptor-ligand complexes indicated that Asp82, Asp116, Serl99, Thr2OO and Ile385 are essential for binding of both agonist and antagonist to the receptor.

A putative model of the dopamine transporter.

A three-dimensional model of the human dopamine transporter was constructed by molecular modeling techniques from its amino acid sequence, based on sequence analysis of this and 9 other transporter proteins. The model has 12 membrane spanning alpha-helices arranged in two 7-helical bundles, loops between helices and amino- and carboxy termini. The molecular electrostatic potentials were mainly negative at the synaptic side and positive in the cytoplasmic domains of the transporter model, strongly positive in some of the transmembrane domains, and strongly negative in other transmembrane domains. The model suggests specific binding sites for dopamine and cocaine, a functional role for chloride ions, and accounts for known structure-activity relationships of cocaine analogs at the dopamine transporter.

Molecular dynamics of the 5-HT1a receptor and ligands.

A 3-D model of the human 5-HT1a receptor was constructed from its amino acid sequence by computer graphics techniques, molecular mechanics calculations and molecular dynamics simulations. The model has seven alpha-helical membrane spanning segments, which form a central core containing a putative ligand binding site. Electrostatic potentials 1.4 A outside the water accessible surface were mainly negative on the synaptic side of the receptor model and at the postulated ligand binding site, and positive in the cytoplasmic domains. The negative electrostatic potentials around the synaptic domains indicate that positively charged ligands are attracted to the receptor by electrostatic forces. Molecular dynamics simulations of the receptor model with serotonin, ipsapirone, R(-)-methiothepin or S(+)-methiothepin in the central core suggested that up to 22 different amino acid residues may form a ligand binding pocket, and contribute to the specificity of ligand recognition and binding.

Molecular dynamics of serotonin and ritanserin interacting with the 5-HT2 receptor.

A three-dimensional model of the serotonin (5-hydroxytrytamine; 5-HT) 5-HT2 receptor was constructed from the amino acid sequence by molecular graphics techniques, molecular mechanics energy calculations and molecular dynamics simulations. The receptor model has 7 alpha helical segments which form a membrane-spanning duct with a putative ligand binding site. Most of the synaptic domains and the ligand binding site were surrounded by negative electrostatic potentials, suggesting that positively charged ligands are attracted to the receptor by electrostatic forces. The cytoplasmic domains, except the C-terminal tail, had mainly positive electrostatic potentials. The molecular dynamics of the receptor-ligand complex was examined in 100 ps simulations with 5-HT or ritanserin at a postulated binding site. During the simulations the helices moved from an initial circular arrangement into a more oval arrangement, and became slightly tilted relative to each other. The protonated ligands neutralized the negative electrostatic potentials around Asp 120 and Asp 155 in the central core of the receptor. 5-HT had only weak interactions with Asp 155 but strong interactions with Asp 120 during the simulations, with the amino group of 5-HT tightly bound to the carboxylic side chain of Asp 120. Ritanserin showed similarly strong interactions with Asp 120 and Asp 155 during the simulations.

Molecular structure and dynamics of the four 10-hydroxynortriptyline isomers.

The three-dimensional structures, molecular conformations, and electrostatic potentials of the R-E-, S-E-, R-Z-, and S-Z-isomers of 10-hydroxynortriptyline were examined by computer graphics, molecular mechanical energy calculations, and molecular dynamics simulations in vacuo and in aqueous solution. Molecular models of the isomers, based on the structure of nortriptyline, were refined by energy minimization and used as starting points in the simulations. R-E- and S-Z-10-hydroxynortriptyline formed intramolecular hydrogen bonds between the side-chain nitrogen atom and the hydroxyl group during the simulations in vacuo, and had the side chain folded over the ring system in the minimum energy conformations. Intramolecular hydrogen bonding was not observed for R-Z- and S-E-10-hydroxynortriptyline, which had extended side chains in the minimum energy conformations and stronger negative molecular electrostatic potentials around the hydroxyl group than the R-E- and S-Z-isomers.

Molecular modeling of antipsychotic drugs and G protein coupled receptors.

The three dimensional structure, electrostatic potentials and molecular dynamics of a series of tricyclic antipsychotic drugs and metabolites were examined by computer graphics and molecular modeling techniques. Three dimensional models of the 5-HT1A and 5-HT2 receptor and of the dopamine D2 receptor were constructed from the amino acid sequences. The receptor models have strongly negative electrostatic potentials around the synaptic domains and a postulated ligand binding site. This indicates that protonated ligands are attracted to these receptors by electrostatic forces. Pharmacologically inactive trans(E)-thioxanthenes and phenothiazine ring sulphoxides had strongly negative electrostatic potentials around a part of the ring system. This may weaken their electrostatic interactions with the D2 receptor, and be the reason for their lack of potency in D2 receptor binding and related pharmacological tests. Molecular dynamics simulations in aqueous solution demonstrated that both the side chains and the tricyclic ring systems of the drugs are highly flexible, and move between different conformations in picoseconds.

Molecular dynamics of dopamine at the D2 receptor.

A three-dimensional model of the dopamine D2 receptor, assumed to be a target of antipsychotic drug action, was constructed from its amino acid sequence. The model was based on structural similarities within the super-family of guanine nucleotide-binding regulatory (G) protein-coupled neuroreceptors and has seven alpha-helical transmembrane segments that form a central core with a putative ligand-binding site. The space between two residues postulated to be involved in agonist binding, Asp-80 and Asn-390, perfectly accommodated an anti-dopamine molecule. Molecular electrostatic potentials were mainly negative on the synaptic side of the receptor model and around aspartate residues lining the central core and positive in the cytoplasmic domains. The docking of dopamine into a postulated binding site was examined by molecular dynamics simulation. The protonated amino group became oriented toward negatively charged aspartate residues in helix 2 and helix 3, whereas the dopamine molecule fluctuated rapidly between different anti and gauche conformations during the simulation. The receptor model suggests that protonated ligands are attracted to the binding site by electrostatic forces and that protonated agonists may induce conformational changes in the receptor, leading to G-protein activation, by increasing the electrostatic potentials near Asp-80.