Structure-based focusing using pharmacophores derived from the active site of 17beta-hydroxysteroid dehydrogenase.

Structure-based focusing constitutes a powerful approach to design libraries of compounds with a given biological profile. Computers with special software can be used to analyse the large amount of data usually available for the compounds. Pharmacophores can be used to identify new compounds that present a specific arrangement of features responsible for a certain type of activity. When available, information about the 3D structure of a biological target can also be included in the building of pharmacophore models. These pharmacophores can then be used as queries to search and/or focus large compound libraries. Multiple pharmacophores were generated from the 3D structure 17beta-hydroxystreoid dehydrogenase type1 complexed with different inhibitors. The validity of these pharmacophores was assessed against a test database containing known active and inactive 17beta-hydroxystreoid dehydrogenase type1 inhibitors. The most selective models were then used to search commercial databases for new structural lead molecules. This approach has allowed us to identify a few new compounds possessing structural features common to flavonoids, a structural class of compounds known to contain good inhibitors of 17beta-hydroxystreoid dehydrogenase type1 enzyme. A structure-based focusing approach is demonstrated to be a meaningful and powerful technique for identifying new lead candidates, which can be taken into the lead optimization process.

Molecular mechanism for agonist-promoted alpha(2A)-adrenoceptor activation by norepinephrine and epinephrine.

We present a mechanism for agonist-promoted alpha(2A)-adrenergic receptor (alpha(2A)-AR) activation based on structural, pharmacological, and theoretical evidence of the interactions between phenethylamine ligands and alpha(2A)-AR. In this study, we have: 1) isolated enantiomerically pure phenethylamines that differ both in their chirality about the beta-carbon, and in the presence/absence of one or more hydroxyl groups: the beta-OH and the catecholic meta- and para-OH groups; 2) used [(3)H]UK-14,304 [5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine; agonist] and [(3)H]RX821002 [2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline; antagonist] competition binding assays to determine binding affinities of these ligands to the high- and low-affinity forms of alpha(2A)-AR; 3) tested the ability of the ligands to promote receptor activation by measuring agonist-induced stimulation of [(35)S]GTPgammaS binding in isolated cell membranes; and 4) used automated docking methods and our alpha(2A)-AR model to predict the binding modes of the ligands inside the alpha(2A)-AR binding site. The ligand molecules are sequentially missing different functional groups, and we have correlated the structural features of the ligands and ligand-receptor interactions with experimental ligand binding and receptor activation data. Based on the analysis, we show that structural rearrangements in transmembrane helix (TM) 5 could take place upon binding and subsequent activation of alpha(2A)-AR by phenethylamine agonists. We suggest that the following residues are important in phenethylamine interactions with alpha(2A)-AR: Asp113 (D(3.32)), Val114 (V(3.33)), and Thr118 (T(3.37)) in TM3; Ser200 (S(5.42)), Cys201 (C(5.43)), and Ser204 (S(5.46)) in TM5; Phe391 (F(6.52)) and Tyr394 (Y(6.55)) in TM6; and Phe411 (F(7.38)) and Phe412 (F(7.39)) in TM7.

"RKKH" peptides from the snake venom metalloproteinase of Bothrops jararaca bind near the metal ion-dependent adhesion site of the human integrin alpha(2) I-domain.

Integrin alpha(1)beta(1) and alpha(2)beta(1) are the major cellular receptors for collagen, and collagens bind to these integrins at the inserted I-domain in their alpha subunit. We have previously shown that a cyclic peptide derived from the metalloproteinase domain of the snake venom protein jararhagin blocks the collagen-binding function of the alpha(2) I-domain. Here, we have optimized the structure of the peptide and identified the site where the peptide binds to the alpha(2) I-domain. The peptide sequence Arg-Lys-Lys-His is critical for recognition by the I-domain, and five negatively charged residues surrounding the "metal ion-dependent adhesion site" (MIDAS) of the I-domain, when mutated, show significantly impaired binding of the peptide. Removal of helix alphaC, located along one side of the MIDAS and suggested to be involved in collagen-binding in these I-domains, does not affect peptide binding. This study supports the notion that the metalloproteinase initially binds to the alpha(2) I-domain at a location distant from the active site of the protease, thus blocking collagen binding to the adhesion molecule in the vicinity of the MIDAS, while at the same time leaving the active site free to degrade nearby proteins, the closest being the beta(1) subunit of the alpha(2)beta(1) cell-surface integrin itself.

Three-dimensional models of alpha(2A)-adrenergic receptor complexes provide a structural explanation for ligand binding.

We have compared bacteriorhodopsin-based (alpha(2A)-AR(BR)) and rhodopsin-based (alpha(2A)-AR(R)) models of the human alpha(2A)-adrenengic receptor (alpha(2A)-AR) using both docking simulations and experimental receptor alkylation studies with chloroethylclonidine and 2-aminoethyl methanethiosulfonate hydrobromide. The results indicate that the alpha(2A)-AR(R) model provides a better explanation for ligand binding than does our alpha(2A)-AR(BR) model. Thus, we have made an extensive analysis of ligand binding to alpha(2A)-AR(R) and engineered mutant receptors using clonidine, para-aminoclonidine, oxymetazoline, 5-bromo-N-(4, 5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK14,304), and norepinephrine as ligands. The representative docked ligand conformation was chosen using extensive docking simulations coupled with the identification of favorable interaction sites for chemical groups in the receptor. These ligand-protein complex studies provide a rational explanation at the atomic level for the experimentally observed binding affinities of each of these ligands to the alpha(2A)-adrenergic receptor.

Chloroethylclonidine and 2-aminoethyl methanethiosulfonate recognize two different conformations of the human alpha(2A)-adrenergic receptor.

The substituted cysteine-accessibility method and two sulfhydryl-specific reagents, the methane-thiosulfonate derivative 2-aminoethyl methanethiosulfonate (MTSEA) and the alpha(2)-adrenergic receptor (alpha(2)-AR) agonist chloroethylclonidine (CEC), were used to determine the relative accessibility of engineered cysteines in the fifth transmembrane domain of the human alpha(2A)-AR (Halpha2A). The second-order rate constants for the reaction of the receptor with MTSEA and CEC were determined with the wild type Halpha2A (cysteine at position 201) and receptor mutants containing accessible cysteines at other positions within the binding-site crevice (positions 197, 200, and 204). The rate of reaction of CEC was similar to that of MTSEA at residues Cys-197, Cys-201, and Cys-204. The rate of reaction of CEC with Cys-200, however, was more than 5 times that of MTSEA, suggesting that these compounds may interact with two different receptor conformations. MTSEA, having no recognition specificity for the receptor, likely reacts with the predominant inactive receptor conformation (R), whereas the agonist CEC may stabilize and react preferentially with the active receptor conformation (R*). This hypothesis was consistent with three-dimensional receptor-ligand models, which further suggest that alpha(2A)-AR activation may involve the clockwise rotation of transmembrane domain 5.

A peptide inhibiting the collagen binding function of integrin alpha2I domain.

Integrin alpha2 subunit forms in the complex with the beta1 subunit a cell surface receptor binding extracellular matrix molecules, such as collagens and laminin-1. It is a receptor for echovirus-1, as well. Ligands are recognized by the special "inserted" domain (I domain) in the integrin alpha2 subunit. Venom from a pit viper, Bothrops jararaca, has been shown to inhibit the interaction of platelet alpha2beta1 integrin with collagen because of the action of a disintegrin/metalloproteinase named jararhagin. The finding that crude B. jararaca venom could prevent the binding of human recombinant ralpha2I domain to type I collagen led us to study jararhagin further. Synthetic peptides representing hydrophilic and charged sequences of jararhagin, including the RSECD sequence replacing the well known RGD motif in the disintegrin-like domain, were synthesized. Although the disintegrin-like domain derived peptides failed to inhibit ralpha2I domain binding to collagen, a basic peptide from the metalloproteinase domain proved to be functional. In an in vitro assay, the cyclic peptide, CTRKKHDNAQC, was shown to bind strongly to human recombinant alpha2I domain and to prevent its binding to type I and IV collagens and to laminin-1. Mutational analysis indicated that a sequence of three amino acids, arginine-lysine-lysine (RKK), is essential for ralpha2I domain binding, whereas the mutation of the other amino acids in the peptide had little if any effect on its binding function. Importantly, the peptide was functional only in the cyclic conformation and its affinity was strictly dependent on the size of the cysteine-constrained loop. Furthermore, the peptide could not bind to alpha2I domain in the absence of Mg2+, suggesting that the conformation of the I domain was critical, as well. Cells could attach to the peptide only if they expressed alpha2beta1 integrin, and the attachment was inhibited by anti-integrin antibodies.

Specificity improvement of a recombinant anti-testosterone Fab fragment by CDRIII mutagenesis and phage display selection.

The monoclonal antibodies so far developed by hybridoma technology have not had high enough specificity or affinity to distinguish the closely related steroid hormones in routine clinical assays. We have employed random mutagenesis and phage display approaches to improve the specificity of one anti-testosterone monoclonal antibody (3-C4F5). The affinity of the antibody is 0.3 x 10(9) M(-1) and the cross-reactivities with most of the related steroids are low. However, the antibody cross-reacts about 1% with dehydroepiandrosterone sulfate (DHEAS) and owing to the high DHEAS serum concentration this is about 1000-fold too high for clinical immunoassays. The complementarity-determining regions (CDRs) of the heavy and light chains, which were predicted by molecular modelling to be in close contact with the testosterone (TES) ligand, were randomized and mutant Fab libraries were cloned into a phagemid vector. Binders were selected by a competitive panning procedure. By combining the identified light and heavy chain CDRIII mutations the TES affinity was preserved at the wild-type level but DHEAS cross-reactivity was decreased to 0.03%. An important finding was that by the competitive panning procedure the overall binding specificity of the 3-C4F5 antibody was refined, since the cross-reactivities to related steroids were also significantly decreased in the combined mutant.

Structure of the Ca(2+)-free Gla domain sheds light on membrane binding of blood coagulation proteins.

Reversible membrane binding of gamma-carboxyglutamic acid (Gla)-containing coagulation factors requires Ca(2+)-binding to 10-12 Gla residues. Here we describe the solution structure of the Ca(2+)-free Gla-EGF domain pair of factor x which reveals a striking difference between the Ca(2+)-free and Ca(2+)-loaded forms. In the Ca(2+)-free form Gla residues are exposed to solvent and Phe 4, Leu 5 and Val 8 form a hydrophobic cluster in the interior of the domain. In the Ca(2+)-loaded form Gla residues ligate Ca2+ in the core of the domain pushing the side-chains of the three hydrophobic residues into the solvent. We propose that the Ca(2+)-induced exposure of hydrophobic side chains is crucial for membrane binding of Gla-containing coagulation proteins.

Molecular dynamics simulation of fungal cellulose-binding domains: differences in molecular rigidity but a preserved cellulose binding surface.

A total of 23 fungal cellulose-binding domain (CBD) sequences were aligned. Structural models of the cellulose-binding domain of an exoglucanase (CBHII) and of three endoglucanases (EGI, EGII and EGV) from Trichoderma reesei cellulases were homology modelled based on the NMR structure of the fungal cellobiohydrolase CBHI, from the same organism. The completed models and the known structure of the CBHI cellulose-binding domain were refined by molecular dynamics simulations in water. All four models were found to be very similar to the structure of the CBHI cellulose-binding domain and sequence comparison indicated that in general the three-dimensional structures of fungal cellulose-binding domains are very similar. In all the CBDs studied, two disulphide bridges apparently stabilize the polypeptide fold. From the models, and additional disulphide bridge was predicted in EGI and CBHII, and in eight further CBDs from other organisms. Three highly conserved aromatic residues on the hydrophilic side of the wedge make this surface flat. This surface is expected to make contact with the substrate. Three invariant amino acids, Gln7, Asn29 and Gln34, on this flat face are in suitable positions for hydrogen bonding with the cellulose surface. Analysis of the differences in the protein surface properties indicated that the endoglucanases tend to be more hydrophilic than the exoglucanases. The largest structural variation was found around positions 12-16. The fungal CBD sequences are discussed in relation to variations in function and pH dependence. Comparison of the modelled structures with experimental binding data for the CBHI and EGI allowed the formulation of a qualitative relationship to cellulose affinity.(ABSTRACT TRUNCATED AT 250 WORDS)

Introduction of lysine residues on the light chain constant domain improves the labelling properties of a recombinant Fab fragment.

Europium chelates provide a non-radioactive alternative for sensitive labelling of antibodies for diagnostic immunoassays. Lysine residues at antibody surfaces are ready targets for labelling by an isothiocyanate derivative of the europium chelate (Eu3+). Here the labelling efficiency of a recombinant anti-human alpha-fetoprotein (hAFP) Fab fragment has been improved by increasing its lysine content by protein engineering. Molecular modelling was used to identify three light chain constant domain surface arginine residues, R154, R187 and R210, which were mutated to lysine residues. The mutations did not influence the affinity of the lysine-enriched Fab fragment and its labelling efficiency was found to be approximately 40% higher than that of the wild-type Fab fragment. With low degree of labelling, the affinities of the two Fab fragments were identical and comparable with that of the original monoclonal anti-hAFP IgG. With a higher degree of labelling the affinities of both Fab fragments decreased more than that of the intact IgG since more lysine residues are available for labelling in the additional heavy chain constant domains of the larger molecule. Electrostatic adsorption and covalent immobilization of the Fab fragments were characterized by BIAcore and the lysine-enriched Fab fragment was found to be more efficiently immobilized to an activated carboxymethyl surface.

A novel, small endoglucanase gene, egl5, from Trichoderma reesei isolated by expression in yeast.

A method is presented for the isolation of genes encoding hydrolytic enzymes without any knowledge of the corresponding proteins. cDNA made from the organism of interest is cloned into a yeast vector to construct an expression library in the yeast Saccharomyces cerevisiae. Colonies producing hydrolytic enzymes are screened by activity plate assays. In this work, we constructed a yeast expression library from the filamentous fungus Trichoderma reesei and isolated a new beta-1,4-endoglucanase gene on plates containing beta-glucan. This gene, egl5, codes for a previously unknown small protein of 242 amino acids. Despite its small size, the protein contains two conservative domains found in Trichoderma cellulases, namely the cellulose-binding domain (CBD) and the linker region that connects the CBD to the catalytic core domain. Molecular modelling of the EGV CBD revealed some interesting structural differences compared to the CBD of the major cellulase CBHI from T. reesei. The catalytic core of EGV is unusually small for a cellulase and represents a new family of cellulases (Family K) and of glycosyl hydrolases (Family 45) together with the endoglucanase B of Pseudomonas fluorescens and the endoglucanase V of Humicola insolens on the basis of hydrophobic cluster analysis.

Modelling of the lignin peroxidase LIII of Phlebia radiata: use of a sequence template generated from a 3-D structure.

A model of the lignin peroxidase LIII of Phlebia radiata was constructed on the basis of the structure of cytochrome c peroxidase (CCP). Because of the low percentage of amino acid identity between the CCP and the lignin peroxidase LIII of Phlebia radiata, alignment of the sequences was based on the generation of a template from a knowledge of the 3-D structure of CCP and consensus sequences of lignin peroxidases. This approach gave an alignment in which all the insertions in the lignin peroxidase were placed at loop regions of CCP, with a 21.1% identity for these two proteins. The model was constructed using this alignment and the computer program COMPOSER, which assembles the model as a series of rigid fragments derived from CCP and other proteins. Manual intervention was required for some of the longer loop regions. The alpha-helices forming the structural framework, and especially the haem environment of CCP, are conserved in the LIII model and the core is close packed without holes. A possible site of the substrate oxidation at the haem edge of LIII is discussed.

Modelling of alpha 2-adrenergic ligands.

Six different ligands, which bind to alpha 2-adrenergic receptor were studied using molecular mechanics and quantum chemical methods. Superimposition was performed using low energy conformations resulting the common binding model. Both electrostatic and structural features of different compounds were compared. It is suggested, that the reason for agonism/antagonism is based on small structural differences in ligands. In agonists the distance between the cationic nitrogen and an aromatic ring plane was about 1 A shorter than in antagonists, whereas the distance between the same nitrogen and outmost part of ring atoms was about 1 A longer in agonists than in antagonists.