Biomolecular self-assembly can be used as a powerful tool for nanoscale engineering. In this paper, we describe the development of building blocks for nanobiotechnology, which are based on the fusion of streptavidin to a crystalline bacterial cell surface layer (S-layer) protein with the inherent ability to self-assemble into a monomolecular protein lattice. The fusion proteins and streptavidin were produced independently in Escherichia coli, isolated, and mixed to refold and purify heterotetramers of 1:3 stoichiometry. Self-assembled chimeric S-layers could be formed in suspension, on liposomes, on silicon wafers, and on accessory cell wall polymer containing cell wall fragments. The two-dimensional protein crystals displayed streptavidin in defined repetitive spacing, and they were capable of binding D-biotin and biotinylated proteins. Therefore, the chimeric S-layer can be used as a self-assembling nanopatterned molecular affinity matrix to arrange biotinylated compounds on a surface. In addition, it has application potential as a functional coat of liposomes.

The human histamine H 3 receptor (hH 3 R) is a G-protein coupled receptor (GPCR), which modulates the release of various neurotransmitters in the central and peripheral nervous system and therefore is a potential target in the therapy of numerous diseases. Although ligands addressing this receptor are already known, the discovery of alternative lead structures represents an important goal in drug design. The goal of this work was to study the hH 3 R and its antagonists by means of molecular modelling tools. For this purpose, a strategy was pursued in which a homology model of the hH 3 R based on the crystal structure of bovine rhodopsin was generated and refined by molecular dynamics simulations in a dipalmitoylphosphatidylcholine (DPPC)/water membrane mimic before the resulting binding pocket was used for high-throughput docking using the program GOLD. Alternatively, a pharmacophore-based procedure was carried out where the alleged bioactive conformations of three different potent hH 3 R antagonists were used as templates for the generation of pharmacophore models. A pharmacophore-based screening was then carried out using the program Catalyst. Based upon a database of 418 validated hH 3 R antagonists both strategies could be validated in respect of their performance. Seven hits obtained during this screening procedure were commercially purchased, and experimentally tested in a [ 3 H]N a -methylhistamine binding assay. The compounds tested showed affinities at hH 3 R with K i values ranging from 0.079 to 6.3 lM.

The first antagonists known for the histamine H3 receptor were mono-substituted imidazole-containing compounds like thioperamide. Meanwhile numerous novel leads have been developed possessing improved affinities, selectivities, specificities, and pharmacokinetic properties. Scope and limitations of this promising class are discussed concerning their structure-activity relationships as well as pharmacological and potential therapeutic aspects.

G-protein coupled receptors (GPCRs) are a protein family of outstanding pharmaceutical interest. GPCR homology models, based on the crystal structure of bovine rhodopsin, have been shown to be valuable tools in the drug-design process. The initial model is often refined by molecular dynamics (MD) simulations, a procedure that has been recently discussed controversially. We therefore analyzed MD simulations of bovine rhodopsin in order to identify contacts that could serve as constraints in the simulation of homology models. Additionally, the effect of an N-terminal truncation, the nature of the membrane mimic, the influence of varying protonation states of buried residues and the importance of internal water molecules was analyzed. All simulations were carried out using the program-package GROMACS. While N-terminal truncation negatively influenced the overall protein stability, a stable simulation was possible in both solvent environments. As regards the protonation state of titratable sites, the experimental data could be reproduced by the program UHBD (University of Houston Brownian Dynamics), suggesting its application for studying homology models of GPCRs. A high flexibility was observed for internal water molecules at some sites. Finally, interhelical hydrogen-bonding interactions could be derived, which can now serve as constraints in the simulations of GPCR homology models.

IntroductionHuman histamine H 3 receptors (hH 3 R) are predominantly expressed in the CNS where they are believed to modulate the release of histamine and other neurotransmitters, prompting interest in this target for several important therapeutic applications. Intense research is currently ongoing in order to identify and optimise suitable drugs [1]. Computational chemistry tools, such as virtual library design or virtual high throughput screening can efficiently support experimental studies, potentially resulting in an acceleration of the drug design process. In order to apply these techniques, information on the binding site is required, which can be provided either via a crystal structure or a homology model when a crystal structure of the target protein is not available. Our goal was to generate a model of the hH 3 R binding site, test its specificity for known hH 3 R antagonists and apply this model for deriving new lead structures for targeting the hH 3 R. Materials and methods Generation of a model of the hH 3 R binding siteA model of the hH 3 R was generated based on the X-ray structure of bovine rhodopsin [2] lacking only the 3rd intracellular loop region. In order to find optimal parameters for the simulation of hH 3 R/ligand complexes, extensive calculations with the reference structure of bovine rhodopsin, testing the influence of various conditions (e.g. protonation states, membrane models) were carried out [unpublished results]. Based on these results and on available mutational data of family A GPCRs, a homology model of the hH 3 R was generated and simulated by molecular dynamics in a phospholipid/H 2 O environment. Docking studiesA total of 313 validated H 3 receptor ligands of both the imidazole and non-imidazole type and 469 drug-like compounds randomly picked from the Maybridge Database were docked using the program GOLD Inflamm. res. 54, Supplement 1 (2005) S50-S51[3] with standard default settings for a '3-times speed up' genetic algorithm, and the corresponding GoldScores were evaluated. All compounds were treated as being in the protonation state under physiological condition. All imidazole groups were considered in their protonated form as there is evidence that imidazole-containing H 3 receptor antagonists interact with a conserved aspartic acid D114 in helix 3 [4]. In proximity to this residue the pK a of the imidazole group can considerably shift, so that the protonated form is favored. In order to reduce the bias of docking programs towards higher molecular weight compounds we multiplied the resulting GoldScores by the correction term 1/√N, where N is the number of non-hydrogen atoms [5]. Figure 1 shows the docking-geometry for ligand FUB759 [6] in the hH 3 R binding pocket. In this predicted binding situation a salt bridge is formed between the piperidine nitrogen and aspartic acid D114 in helix 3 that is highly conserved within the family of rhodopsin-like GPCRs. The quinoline moiety of FUB759 and likewise other hydrophobic tails can be accommodated in a cleft between helix 5 and...