Ab initio modelling of the N-terminal domain of the secretin receptors

G protein coupled receptors of the secretin family are activated by peptide hormones of about 30 residues in length. There is considerable sequence homology within both the hormone and receptor families. The receptors possess in addition to the integral membrane domain a characteristic extracellular domain of about 120 residues in length, having conserved cysteine residues, which are involved in disulphide bridge formation, and tryptophanes, which have been shown to be critical for hormone binding. This extracellular domain does not have detectable homology to any known protein fold. In order to be able to propose a structure for this domain we have used ab initio prediction methods combined with constraints based on experimental results for the disulphide connectivity. The results of computational tools for predicting secondary structure and accessibility, together with ligand binding and mutational data and other structural considerations were used in the ab initio protein folding programs DRAGON and GADGET and also the simpler program RAMBLE, which was able to explore different permutations of disulphide bond connectivity, tryptophan side chain orientation and chain topology. The methods generated a limited number of plausible models but no single unique solution was found under the constraints. One of these was refined into a full atomic model that contained a possible peptide binding site comprising the most conserved residues.     

Use of capillary electrophoresis in drug quality assessment of synthetic porcine secretin

The purity profile for porcine secretin attributable to contamination by equilibrium products such as aspartoyl(3) secretin has been shown to be dependent on the pH of the analytical system. Capillary zone electrophoresis (CZE) methods have been developed for the efficient separation of synthetic porcine secretin, its equilibrium products and other impurities in aqueous solutions at both acidic and alkaline pH. These conditions are more representative of those used for the reconstitution and administration of porcine secretin, and good results cannot be achieved using HPLC due to poor peak shape above pH 5.8. The influence of various CZE operational parameters was systematically examined. The methods were validated for accuracy, precision, linearity, LOD and LOQ. A comparative evaluation of the stability of test solutions was determined using CZE and HPLC over a range of pH values. HPLC and CZE methods produced similar results at low pH.     

Effect of dose, pH, and osmolarity on nasal absorption of secretin in rats. III. In vitro membrane permeation test and determination of apparent partition coefficient of secretion

Nasal absorption of secretin in rats was enhanced in an acid solution and the maximum absorption was observed at a sodium chloride solution molarity of 0.462. In order to predict how changes in the secretin molecule would affect its absorption through the nasal mucosa independently of structural changes in the epithelial membrane, an artificial membrane permeation test was conducted, and the apparent partition coefficient between octanol and a test solution was determined. The concentration of secretin was measured using high performance liquid chromatography. The amount of secretin that permeated through an artificial membrane was hardly affected by changes in pH, which suggest that the size of the secretin molecule was not changed. The apparent partition coefficient, however, increased as the pH of the test solution rose from 3.81 to 7.0, which suggested that the hydrophobicity of secretin was enhanced. In relation to the osmolarity of the test solution, the amount of permeation was hardly affected by the concentration of sodium chloride, but the partition coefficient increased with the concentration of the sodium chloride solution. It was supposed that the size of the secretin molecule was not changed in spite of the increasing hydrophobicity, and the nasal absorption of secretin at a sodium chloride molarity of 0.462 was dependent on a change in the epithelial cells. When sorbitol was used as an osmoregulatory agent, the apparent partition coefficient hardly varied as the osmolarity of the solution was increased, whereas the amount of permeation decreased, and these findings reflected the nasal absorption in rats.     

Predicting the effects of amino acid replacements in peptide hormones on their binding affinities for class B GPCRs and application to the design of secretin receptor antagonists

Computational prediction of the effects of residue changes on peptide-protein binding affinities, followed by experimental testing of the top predicted binders, is an efficient strategy for the rational structure-based design of peptide inhibitors. In this study we apply this approach to the discovery of competitive antagonists for the secretin receptor, the prototypical member of class B G protein-coupled receptors (GPCRs). Proteins in this family are involved in peptide hormone-stimulated signaling and are implicated in several human diseases, making them potential therapeutic targets. We first validated our computational method by predicting changes in the binding affinities of several peptides to their cognate class B GPCRs due to alanine replacement and compared the results with previously published experimental values. Overall, the results showed a significant correlation between the predicted and experimental ΔΔG values. Next, we identified candidate inhibitors by applying this method to a homology model of the secretin receptor bound to an N-terminal truncated secretin peptide. Predictions were made for single residue replacements to each of the other nineteen naturally occurring amino acids at peptide residues within the segment binding the receptor N-terminal domain. Amino acid replacements predicted to most enhance receptor binding were then experimentally tested by competition-binding assays. We found two residue changes that improved binding affinities by almost one log unit. Furthermore, a peptide combining both of these favorable modifications resulted in an almost two log unit improvement in binding affinity, demonstrating the approximately additive effect of these changes on binding. In order to further investigate possible physical effects of these residue changes on receptor binding affinity, molecular dynamics simulations were performed on representatives of the successful peptide analogues (namely A17I, G25R, and A17I/G25R) in bound and unbound forms. These simulations suggested that a combination of the α-helical propensity of the unbound peptide and specific interactions between the peptide and the receptor extracellular domain contribute to their higher binding affinities.     

List of publications of Prof. Dr. H. J. Seifert

List of publications of Prof. Dr. H. J. Seifert     

S.C.F.M.O. study of Hafner's hydrocarbons

SCF MO calculations have been made for Hafner's hydrocarbons using neglect of differential overlap after the manner ofPople andPariser andParr. ^* transitions have been calculated by a limited configuration interaction method. The results show that agreement between predicted spectra and observed spectra is quite good for the heptalene derivative and reasonably good for the pentalene derivative. It is suggested that both molecules are aromatic in contradiction to predictions by Craig's rule.

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Zusammenfassung SCF MO-Rechnungen mit zero differential overlap nach der Methode vonPople, Pariser undParr wurden fürHafner's Kohlenwasserstoff angestellt. Mittels begrenzter Konfigurationswechselwirkung (wurden)die ^*-Übergänge berechnet, wobei die Ergebnisse im Fall des Heptalen-Derivates verhältnismäßig gut, in dem des Pentalen-Derivates jedoch nur ungefähr mit dem Experiment übereinstimmen. Darüber hinaus sprechen die Resultate im Gegensatz zur Craig'schen Regel dafür, daß beide Moleküle aromatischen Charakter haben.

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Résumé Nous avons effectué des calculs SCF MO pour les hydrocarbures deHafner, en négligeant le recouvrement différentiel d'aprèsPople etPariser etParr. Quelques transitions ^* ont été calculées par interaction de configurations limitée. L'accord avec l'expérience est assez bon pour le dérivé de l'heptalène, et raisonable pour le dérivé du pentalène. Les deux molécules semblent être aromatiques, contrairement à la règle de Craig.

     

60th Birthday of Prof. Dr. Dr. h.c. Heino Finkelmann

This section contains news of the macromolecular community all over the world. Articles about, for example, people, projects, and market trends are welcome. Suggestions should be sent to the editorial office of the Macromolecular journals, preferably by E‐mail to macromol@wiley‐vch.de. The editorial office decides which articles will be published.     

Aromatic annelation. I. Synthesis of pyridines.

The preparation of substituted pyridines from cycloalkanones by elaboration of an aromatic ring is described.