A molecular mechanics study of the effect of substitution in position 1 on the conformational space of the oxytocin/vasopressin ring

Summary The effect of the substitution in position 1 on the low-energy conformations of the oxytocin/vasopressin 20-membered ring was investigated by means of molecular mechanics. Three representative substitutions were considered: -mercapto-,-dimethyl)propionic acid (Dmp), (-mercapto-,-cyclopentamethylene)propionic acid (Cpp), both forming strong antagonists, and (,-dimethyl--mercapto)propionic acid (-Dmp), forming analogs of strongly reduced biological activity, with the -mercaptopropionic (Mpa) residue taken as reference. Both ECEPP/2 (rigid valence geometry) and AMBER (flexible valence geometry) force fields were employed in the calculations. Three basic types of backbone conformations were taken into account which are distinguished by the type of -turn at residues 3 and 4: 1/III, II, and I/III, all types containing one or two intra-annular hydrogen bonds. The allowed (ring-closed) disulfide-bridge conformations were searched by an algorithm formulated in terms of scanning the disulfide-bridge torsional angle C-S-S-C. The ECEPP/2 and AMBER energies of the obtained conformations were found to be in reasonable agreement. Two of the low-energy conformers of the [Mpa¹]-compound agreed very well with the cyclic part of the two conformers found in the crystal structure of [Mpa¹]-oxytocin. An analysis of the effect of -substitution on relative energies showed that the conformations with the N-C-CH₂-CH₂ (₁) and C-CH₂-CH₂-S (₁) angles of the first residue around (–100°, 60°) and (100°, –60°) are not affected; this in most cases implies a left-handed disulfide bridge. In the case of -substitution the allowed values of ₁ are close to ± 60°. This requirement, being in contradiction to the one concerning -substitution, could explain the very low biological activity of the -substituted analogs. The conformational preferences of substituted compounds can largely be explained by the analysis of local interactions within the first residue. Based on the selection of the conformations which are low in energy for both the reference and -substituted compounds, two distinct types of possible binding conformations were proposed, the first one being similar to the crystal conformer with a left-handed disulfide bridge, the second one having a right-handed bridge, but a geometry different from that of the crystal conformer with the right-handed bridge. The first type of disulfide-bridge arrangement is equally favorable for both I/III and II types of backbone structure, while the second one is allowed only for the II type of backbone. No conformation of the I/III type has a low enough energy to be considered as a possible binding conformation for all of the active compounds studied in this work.     

Novel and Sensitive Noncompetitive Enzyme Immunoassay (Hetero-Two-Site Enzyme Immunoassay) for Arginine Vasopressin

Abstract

A novel and sensitive noncompetitive enzyme immunoassay (hetero-two-site enzyme immunoassay) for arginine vasopressin in plasma is described. Plasma (0.3 ml) was diluted 1.3-fold with an appropriate buffer and filtered by centrifugation in a micro-concentrator with polysaccharide membrane to eliminate plasma proteins. Arginine vasopressin in plasma filtrates was biotinylated and trapped onto anti-arginine vasopressin IgG-coated polystyrene balls. After washing the polystyrene balls to eliminate other biotinylated substances, the biotinylated arginine vasopressin was eluted from the polystyrene balls with HCl and was reacted with anti-arginine vasopressin Fab′-peroxidase conjugate. The complex formed was trapped onto streptavidin-coated polystyrene balls. Peroxidase activity bound to the polystyrene balls was assayed by fluorometry. The detection limit of arginine vasopressin was 11 fg (10 amol)/tube. This was 45-fold lower than that by competitive enzyme immunoassay using the same antiserum as used in this study and 9 to 400-fold lower than those previously reported by competitive radioimmunoassays. The assay range of arginine vasopressin in plasma was 0.14–140 ng /l using 100 μl of plasma filtrates corresponding to 75 u1 o f plasma. Plasma levels of arginine vasopressin i n 8 healthy subjects aged 25–41 yr with, ad libitum water in take and normal activity approximately 4 h after breakfast were 0.72 ± 0.22 (SD) ng /l (range, 0.42–1.04 ng /l).     

Review: Aptamers in microfluidic chips

This review, covering reports published from 2002 to August 2010, shows how aptamers have made significant contributions in the improvements of microfluidic chips for affinity extraction, separations and detections. Furthermore, microfluidic chip methods for studying aptamer-target interactions and performing aptamer selections have also been summarized. Accordingly, research vacancies and future development trends in these areas are discussed.     

Synthesis and biological evaluation of novel phenothiazine derivatives as non-peptide arginine vasopressin V2 receptor antagonists

A series of novel phenothiazine derivatives was synthesized and tested for arginine vasopressin receptor antagonist activity. They were synthesized as novel arginine vasopressin receptor antagonists from phenothiazine as a scaffold via successive acylation, reduction and acylation reactions. Their structures were characterized by 1HNMR, 13CNMRandHRMS, and biological activitywas evaluated by in vitro and in vivo studies. The in vitro binding assay indicated that several compounds are potent selective V2 receptor antagonists. Compounds with promising binding affinity to V2 receptors were selected to conduct the in vivo diuretic studies on Sprague-Dawley rats. Among them, 1n, 1r, 1t and 1v exhibited excellent diuretic activity, especially 1r and 1v. Therefore, 1r and 1v are potent novelAVP V2receptor antagonist candidates.     

Structure and conformation of Arg8 vasopressin modified analogs

Fourier‐transform Raman and infrared spectra were acquired for four arginine vasopressin (AVP) analogs containing L ‐diphenylalanine (Dpa): [Dpa²]AVP, [Cpa¹,Dpa²]AVP, [Dpa³]AVP, and [Cpa¹,Dpa³]AVP (where Cpa denotes 1‐mercaptocyclohexaneacetic acid). We compared and analyzed these spectra. In addition, the Raman spectra were compared to the corresponding surface‐enhanced Raman scattering spectra recorded in an aqueous silver colloidal dispersion. Silver colloidal dispersions prepared by the simple borohydride reduction of silver nitrate were used as substrates. The geometry of these molecules etched on the silver surface was deduced from the observed changes in the intensity enhancement, breadth, and shift in wavenumber of the Raman bands in the spectra of the bound versus free species. Based on the obtained data, adsorption mechanisms were proposed for each case, and the suggested adsorbate structures were compared. All the molecules were thought to adsorb onto a silver surface via a phenyl ring, free electron pairs on the sulfur atom, and CO and  CONH‐bonds. However, the orientation of these fragments on the colloidal silver surface and the strength of the interactions with this surface are different. For [Dpa³]AVP and [Cpa¹,Dpa³]AVP, a strong interaction among the—CCN‐peptide fragment and the colloidal silver surface occurs. Copyright © 2011 John Wiley & Sons, Ltd.     

Synthesis and biological evaluation of novel derivatives of desloratadine

Series of novel derivatives of desloratadine designed as arginine vasopressin receptor antagonists were synthesized and structurally characterized by melting points,~1H NMR and HRMS.Their in vivo diuretic activities were evaluated on rats,and several target compounds showed promising diuretic results, especially compounds 8,18,27 and 31.Further in vitro bonding assay and cAMP assay showed that these compounds had a higher affinity to vasopressin V2 receptor than VI a receptor.Our studies indicated that desloratadine may be an active substructure for novel arginine vasopressin receptor antagonist development.     

Conformational differences of oxytocin and vasopressin as observed by fluorescence anisotropy decays and transient effects in collisional quenching of tyrosine fluorescence

We used gigahertz frequency-domain fluorometry to examine the tyrosyl fluorescence intensity and anisotropy decays of the single-tyrosine cyclic peptide hormones oxytocin and vasopressin. Acrylamide quenching and a distance-dependent quenching model for collisional quenching were used to evaluate the extent of tyrosyl exposure to the quencher and to provide increased resolution of the picosecond anisotropy decays. Analysis of the intensity decays using a lifetime distribution model shows different distributions for oxytocin and vasopressin. We found that the tyrosyl fluorescence of lysine-vasopressin, as revealed both by the lifetime Stern-Volmer plots and from the quenching analysis, is quenched more effectively than oxytocin. ForN-acetyltyrosinamide (NATyrA), oxytocin, and lysine-vasopressin, we recovered apparent diffusion coefficients for quenching of 4.7×10^–6, 0.44×10^–6, and 4.3×10^–6 cm²/s, respectively, the lower value for oxytocin suggesting a shielded environment for its tyrosyl residue. Tyrosyl anisotropy decays were recovered by global analysis of progressively quenched samples. Compared with oxytocin, vasopressin displayed a longer correlation time for overall rotational diffusion and a higher amplitude for picosecond segmented motions of its tyrosyl residue. All the data are consistent with a more extended and flexible solution structure for vasopressin than for oxytocin.Dedicated to Professor Alfons Kawski on the occasion of his 65th birthday.     

Molecular modeling of the human vasopressin V2 receptor/agonist complex

The V2 vasopressin renal receptor (V2R), which controls antidiuresis in mammals, is a member of the large family of heptahelical transmembrane (7TM) G protein-coupled receptors (GPCRs). Using the automated GPCR modeling facility available via Internet (http://expasy.hcuge.ch/swissmod/SWISS-MODEL.html) for construction of the 7TM domain in accord with the bovine rhodopsin (RD) footprint, and the SYBYL software for addition of the intra- and extracellular domains, the human V2R was modeled. The structure was further refined and its conformational variability tested by the use of a version of the Constrained Simulated Annealing (CSA) protocol developed in this laboratory. An inspection of the resulting structure reveals that the V2R (likewise any GPCR modeled this way) is much thicker and accordingly forms a more spacious TM cavity than most of the hitherto modeled GPCR constructs do, typically based on the structure of bacteriorhodopsin (BRD). Moreover, in this model the 7TM helices are arranged differently than they are in any BRD-based model. Thus, the topology and geometry of the TM cavity, potentially capable of receiving ligands, is in this model quite different than it is in the earlier models. In the subsequent step, two ligands, the native [arginine⁸]vasopressin (AVP) and the selective agonist [d-arginine⁸]vasopressin (DAVP) were inserted, each in two topologically non-equivalent ways, into the TM cavity and the resulting structures were equilibrated and their conformational variabilities tested using CSA as above. The best docking was selected and justified upon consideration of ligand-receptor interactions and structure-activity data. Finally, the amino acid residues were indicated, mainly in TM helices 3-7, as potentially important in both AVP and DAVP docking. Among those Cys¹¹², Val¹¹⁵-Lys¹¹⁶, Gln¹¹⁹, Met¹²³ in helix 3; Glu¹⁷⁴ in helix 4; Val²⁰⁶, Ala²¹⁰, Val²¹³-Phe²¹⁴ in helix 5; Trp²⁸⁴, Phe²⁸⁷-Phe²⁸⁸, Gln²⁹¹ in helix 6; and Phe³⁰⁷, Leu³¹⁰, Ala³¹⁴ and Asn³¹⁷ in helix 7 appeared to be the most important ones. Many of these residues are invariant for either the GPCR superfamily or the neurophyseal (vasopressin V2R, V1aR and V1bR and oxytocin OR) subfamily of receptors. Moreover, some of the equivalent residues in V1aR have already been found critical for the ligand affinity [Mouillac et al., J. Biol. Chem, 270 (1995) 25771].