Characterization of the bioactive form and molecular determinants of recognition of cyclic enkephalin peptides at the δ-opioid receptor

An extensive and systematic search strategy to determine the conformational profile of 12 cyclic disulfide-bridged opioid peptides with varying affinities at the δ receptor has been carried out to identify the structure that is recognized by the δ receptor for each analogue. The methods and procedures used here for the conformational search have already been validated for [D -Pen2, D -Pen5] enkephalin (DPDPE), one member of this family. Use of these methods led to a low-energy solution conformation of DPDPE in excellent agreement with all the geometric properties deduced from its solution nmr spectra. Each of the analogue was subjected to the same procedure, involving a combination of molecular dynamics simulations at high and low temperature. The study was repeated in two environmental conditions, an apolar environment, simulated by using a distance-dependent dielectric constant, and a polar environment by embedding the peptides in a high constant dielectric ( ε = 80). An automated comparison of the different conformers based on their backbone rms and average distance between the key aromatic moieties was followed by graphic analysis using maximum structural overlap. The cross-comparison of the conformations for each analogue revealed a unique conformer that may be recognized by the δ receptor for each high-affinity analogue that permitted maintaining the critical elements required for recognition in a simple spatial orientation, while maximizing similarity in other regions. © 1993 John Wiley & Sons, Inc.     

Conformationally readdressed CCK-B/δ-opioid pepitide ligands

The sequence of a cholecystokinin (CCK) related peptide was modified to obtain analogues, which intereact selectively either with CCK-B, or with δ-opioid receptors. Two kinds of peptides were designed, namely, the cyclic peptides of the H-Tyr-cyclo(D -Pen-Gly-Trp-L -/D-3-transmecaptoproline)-Asp-Phe-NH₂ sequence (compounds 1a and 1b , respectively), and the linear peptides of the H-Tyr-D -Val-Gly-Trp-L /D -3-trans-methylmercaptoproline-Asp-Phe-NH₂ sequence (compounds 2a and 2b , respectively). The only difference between the chemical structures of the linear analogues compared to the cyclic ones is that one covalent bond has been eliminated and a sulfur atom is replaced by a methyl group. Molecular modeling showed that, among low-energy conformers of cyclic compounds 1 , there are three-dimensional structures compatible to the model for δ- receptor- bound conformer, suggested earlier[G. V. Nikiforovich. V. J. Hruby. O. Prakash, and C. A. Gehrig (1991) Biopolymers. vol. 31. pp. 941–955]. Results of binding assays fully supported the rationale for the design of compounds 1 and 2 . The cyclic analogue 1a has K_i values of 4.5 and > 5000 n M at δ- and μ-opioid receptors, respectively; IC₅₀ values of 3000 n M for both CCK-A and CCK-B receptors, whereas its linear counterpart 2a has k_i values of 462 and 229 nM at δ- and μ-opioid receptors, respectively; and IC₅₀ values of 1.6 and > 10.000 nM for CCK-A and CCK-B receptors, respectively. The results of this study demonstrate a possibility to redirect a peptide sequence that interacts with one type of receptors (CCK-B receptors) toward interaction with another type (δ-opioid receptors) belonging to a different physiological system. This redirection could be performed by changing the conformational properties of the peptide with very minimal changes in its chemical structure. © 1995 John Wiley & Sons, Inc.     

Chronic exposure to antibodies directed against anti-opiate peptides alter δ-opioid receptor levels

The development of addictive states in response to chronic opioid use may be regulated partially by the release of endogenous peptides. These anti-opiate peptides (AOP) are secreted or released into the CNS and produce diverse actions that counterbalance the effects of prolonged opiate exposure. Though the mechanism(s) by which these peptides exert their physiological properties remain largely unknown, there is some indication that AOP’s modulate opioid receptor levels. In this study, we investigated the effects of chronically infused α-melanocyte stimulating hormone (α-MSH), dynorphin_1-8 (DYN_1-8), dynorphin A (DYNA), and NPFF antibodies on δ-opioid receptor expression in rat brains. Quantitative autoradiographic experiments revealed that antibodies directed against α-MSH and DYNA produced significant increases in delta receptor levels in the caudate, claustrum, and cingulate cortex of the rat brain. Conversely, NPFF monoclonal antibodies caused significant decreases in the caudate, nucleus accumbens, olfactory tubercle, and cingulate cortex. These results suggest that the density of δ-opioid receptors is affected by changes in the levels of the anti-opioid peptides in the extracelluar fluid in the rat brain.     

β1‐adrenergic receptor antagonists signal via PDE4 translocation

It is generally assumed that antagonists of G_s‐coupled receptors do not activate cAMP signalling, because they do not stimulate cAMP production via G_s‐protein/adenylyl cyclase activation. Here, we report a new signalling pathway whereby antagonists of β₁‐adrenergic receptors (β₁ARs) increase cAMP levels locally without stimulating cAMP production directly. Binding of antagonists causes dissociation of a preformed complex between β₁ARs and Type‐4 cyclic nucleotide phosphodiesterases (PDE4s). This reduces the local concentration of cAMP‐hydrolytic activity, thereby increasing submembrane cAMP and PKA activity. Our study identifies receptor/PDE4 complex dissociation as a novel mechanism of antagonist action that contributes to the pharmacological properties of β₁AR antagonists and might be shared by other receptor subtypes.     

Quantitative two-dimensional nmr study of dermenkephalin,a highly potent and selective δ-opioid peptide

Dermenkephalin, H-Tyr-(D ) Met-Phe-His-Leu-Met-Asp-NH₂, a highly potent and selective δ-opioid peptide isolated from frog skin, was studied in DMSO-d₆ solution by two-dimensional nmr spectroscopy, including the determination of NH temperature coefficients, the evaluation of ³J coupling constants from phase-sensitive correlated spectroscopy (COSY) and the volumes of nuclear Overhauser effect (NOE) correlations. The two-dimensional NOE spectroscopy (NOESY) spectrum of dermenkephalin revealed sequential, medium-, and long-range effects. To put this information on a quantitative basis, special attention was devoted to J cross-peak suppression, quantification of the NOE volumes and analysis of the overlaps, normalization of the NOEs against diagonal peaks and H_ββ′ geminal interactions. Although most of the dihedral angles deduced from the ³J_Nα coupling constants together with several N_iα_i and α_iN_{i + 1} NOEs pointed to a partially extended peptide backbone, several N_i N_{i + 1} NOEs and β_i N_{i + 1} interactions argued in favor of a folded structure. Moreover, several long-range correlations of strong intensities were found that supported a close spatial proximity between the side chains of D -Met² and Met⁶, Tyr¹ and His⁴, Tyr¹ and Asp⁷, and His⁴ and the C-terminal amide group. In Phe, the g^? rotamer in the side chain is deduced from the ³J_αβ coupling constants and αβ and Nβ NOE correlations. Whereas the amide proton dependency was not indicative of stable hydrogen bonds, the nonuniform values of the temperature coefficient may reflect an equilibrium mixture of folded and extended conformers. The overall data should provide realistic starting models for energy minimization and modelization studies. © 1993 John Wiley & Sons, Inc.     

Development of a model for the δ-opioid receptor pharmacophore: 3. Comparison of the cyclic tetrapeptide Tyr-c[D-Cys-Phe-D-Pen] OH with other conformationally constrained δ-receptor selective ligands

We have previously proposed a model of the δ-opioid receptor bound conformation for the cyclic tetrapeptide, Tyr-c[D -Cys-Phe-D -Pen]OH (JOM-13) based on its conformational analysis and from conformation-affinity relationships observed for its analogues with modified first and third residues. To further verify the model, it is compared here with results of conformational and structure-activity studies for other known conformationally constrained δ-selective ligands: the cyclic pentapeptide agonist, Tyr-c[D -Pen-Gly-Phe-D -Phe]OH (DPDPE); the peptide antagonist, Tyr-Tic-Phe-PheOH (TIPP); the alkaloid agonist, 7-spiroindanyloxymorphone (SIOM); and the related alkaloid antagonist, oxymorphindole (OMI). A candidate δ-bound conformer is identified for DPDPE that provides spatial overlap of the functionally important N-terminal N⁺₃ and C-terminal COO⁻ groups and the aromatic rings of the Tyr and Phe residues in both cyclic peptides. It is shown that all δ-selective ligands considered have similar arrangements of their pharmacophoric elements, i.e., the tyramine moiety and a second aromatic ring (i.e., the rings of Phe³, Phe⁴, and Tic² residues in JOM-13, DPDPE, and TIPP, respectively; the indole ring system in OMI, and the indanyl ring system in SIOM). The second aromatic rings, while occupying similar regions of space throughout the analogues considered, have different orientations in agonists and antagonists, but identical orientations in peptide and alkaloid ligands with the same agonistic or antagonistic properties. These results agree with the previously proposed binding model for JOM-13, are consistent with the view that δ-opioid agonists and antagonists share the same binding site, and support the hypothesis of a similar mode of binding for opioid peptides and alkaloids. © 1996 John Wiley & Sons, Inc.     

Concentration‐dependent and surface‐assisted self‐assembly properties of a bioactive estrogen receptor α‐derived peptide

We have synthesized a 17‐mer peptide (ERα17p) that is issued from the hinge region of the estrogen receptor α and which activates the proliferation of breast carcinoma cells in steroid‐deprived conditions. In the present paper, we show that at a concentration of ~50 μM, it rapidly forms amyloid‐like fibrils with the assistance of electrostatic interactions and that at higher concentrations, it spontaneously forms a hydrogel. By using biophysical, spectral and rheological techniques, we have explored the structural, biophysical and mechanical characteristics of ERα17p with respect to fibril formation and gelation. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Interaction of linear and cyclic peptide antagonists at the human B(2) kinin receptor

The ligand receptor interactions involving the C-terminal moiety of kinin B(2) receptor antagonists Icatibant (H-DArg-Arg-Pro-Hyp-Gly-Thi-Ser-Dtic-Oic-Arg-OH), MEN 11270 (H-DArg-Arg-Pro-Hyp-Gly-Thi-c(Dab-Dtic-Oic-Arg)c(7gamma-10alpha)) and a series of analogs modified in position 10 were investigated by radioligand-binding experiments at the wild type (WT) and at the Ser(111)Ala and Ser(111)Lys mutant human kinin B(2) receptors. Icatibant and [Lys(10)]-Icatibant maintained the same high affinity towards the three receptors. For Icatibant-NH(2), [Ala(10)]-Icatibant, MEN 11270 and [Glu(10)]-MEN 11270, the changes in affinity at the WT and Ser(111)Lys receptors indicated that the presence of a net positive or negative charge at the C-terminal moiety of these peptides caused a decrease in affinity to the WT receptor and that Ser(111) residue is in proximity of the side chain of residue 10. The changes in affinity measured with [desArg(10)]-Icatibant and [desArg(10)]-Icatibant-NH(2), moreover, confirmed that a C-terminal charge compensation between the positive charge of Arg(10) side chain and the C-terminal free carboxylic function favours a high affinity interaction.     

Design and characterization of a model αβ peptide

CD and nmr spectroscopy were used to compare the conformational properties of two related peptides. One of the peptides, Model AB, was designed to adopt a helix-turn-extended strand (αβ) tertiary structure in water that might be stabilized by hydrophobic interactions between two leucine residues in the amino-terminal segment and two methionine residues in the carboxyl terminal segment. The other peptide, AB Helix, has the same amino acid sequence as Model AB except that it lacks the-Pro-Met-Thr-Met-Thr-Gly segment at the carboxyl-terminus. Although the carboxyl-terminal segment of Model AB was found to be unstructured, its presence increases the number of residues in a helical conformation, shifts the pKas of three ionizable side chains by 1 pH unit or more compared to an unstructured peptide, stabilizes the peptide as a monomer in high concentrations of ammonium sulfate, increases the conformational stability of residues at the terminal ends of the helix, and results in many slowly exchanging amide protons throughout the entire backbone of the peptide. These results suggest that interactions between adjacent segments in a small peptide can have significant structure organizing effects. Similar kinds of interactions may be important in determining the structure of early intermediates in protein folding and may be useful in the de novo design of independently folding peptides. © 1995 John Wiley & Sons, Inc.     

Characterization of a soluble form of the C3b/C4b receptor (CR1) in human plasma

A radioimmunoassay with the use of soluble 125I-Fab monoclonal anti-CR1 and rabbit IgG anti-CR1 bound to Staphylococcus aureus particles was employed to detect and quantitate CR1 antigen in human plasma. Among 16 normal individuals the concentration of soluble CR1 in plasma ranged from 13 to 81 ng/ml, and a similar range of concentration was found in plasma from 15 patients having systemic lupus erythematosus (SLE). The amount of plasma CR1 in normal donors, but not in SLE patients, significantly correlated with the number of CR1 sites on erythrocytes (r = 0.90, p less than 0.001), and was 7.1% of the amount of receptor that was present on erythrocytes in blood. The concentration of soluble CR1 was not diminished by ultracentrifugation or ultrafiltration of plasma, was not affected by various modes of anti-coagulation or even by clotting of blood, and did not change during incubation of blood at 4 degrees C for up to 4 hr. On sucrose density gradient ultracentrifugation of plasma the CR1 was distributed as a broad peak that overlapped the plasma protein profile. The Mr of plasma CR1 was identical to that of erythrocyte CR1 when assessed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and immunoblotting. In addition, the plasma form of CR1 exhibited the same structural phenotype as did receptor from erythrocytes of the same individual. CR1 antigen purified from plasma was as active as CR1 from erythrocytes in promoting the cleavage by factor I of C3b to iC3b, C3c, and C3dg. Therefore, a functionally and structurally intact form of soluble CR1 resides in plasma.     

Comparative response of δ13C, δ18O and δ15N in durum wheat exposed to salinity at the vegetative and reproductive stages

This study compared the performance of the stable isotope composition of carbon (δ¹³C), oxygen (δ¹⁸O) and nitrogen (δ¹⁵N) by tracking plant response and genotypic variability of durum wheat to different salinity conditions. To that end, δ¹³C, δ¹⁸O and δ¹⁵N were analysed in dry matter (dm) and the water‐soluble fraction (wsf) of leaves from plants exposed to salinity, either soon after plant emergence or at anthesis. The δ¹³C and δ¹⁸O of the wsf recorded the recent growing conditions, including changes in evaporative conditions. Regardless of the plant part (dm or wsf), δ¹³C and δ¹⁸O increased and δ¹⁵N decreased in response to stress. When the stress conditions were established just after emergence, δ¹⁵N and δ¹³C correlated positively with genotypic differences in biomass, whereas δ¹⁸O correlated negatively in the most severe treatment. When the stress conditions were imposed at anthesis, relationships between the three isotope signatures and biomass were only significant and positive within the most severe treatments. The results show that nitrogen metabolism, together with stomatal limitation, is involved in the genotypic response to salinity, with the relative importance of each factor depending on the severity and duration of the stress as well as the phenological stage that the stress occurs.     

Structural basis of the interaction between integrin α6β4 and plectin at the hemidesmosomes

The interaction between the integrin α6β4 and plectin is essential for the assembly and stability of hemidesmosomes, which are junctional adhesion complexes that anchor epithelial cells to the basement membrane. We describe the crystal structure at 2.75 Å resolution of the primary α6β4–plectin complex, formed by the first pair of fibronectin type III domains and the N‐terminal region of the connecting segment of β4 and the actin‐binding domain of plectin. Two missense mutations in β4 (R1225H and R1281W) linked to nonlethal forms of epidermolysis bullosa prevent essential intermolecular contacts. We also present two structures at 1.75 and 2.05 Å resolution of the β4 moiety in the absence of plectin, which reveal a major rearrangement of the connecting segment of β4 on binding to plectin. This conformational switch is correlated with the way α6β4 promotes stable adhesion or cell migration and suggests an allosteric control of the integrin.     

Synthesis and receptor binding of opioid peptide analogues containing β3‐homo‐amino acids

β‐Amino acids containing hybrid peptides and β‐peptides show great potential as peptidomimetics. In this paper we describe the synthesis and affinity toward the µ‐ and δ‐opioid receptors of β‐peptides, analogues of Leu‐enkephalin, deltorphin I, dermorphin and α,β‐hybrides, analogues of deltorphin I. Substitution of α‐amino acid residues with β³‐homo‐amino acid residues, in general resulted in decrease of affinity to opioid receptors. However, the incorporation β³h‐D ‐Ala in position 2 or β³hPhe in position 3 of deltorphin I resulted in potent and selective ligand for δ‐opioid receptor. The NMR studies of β‐deltorphin I analogue suggest that conformational motions in the central part of the peptide backbone are partially restricted and some conformational preferences can be expected. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

The versatility of the αβ T‐cell antigen receptor

The T‐cell antigen receptor is a heterodimeric αβ protein (TCR) expressed on the surface of T‐lymphocytes, with each chain of the TCR comprising three complementarity‐determining regions (CDRs) that collectively form the antigen‐binding site. Unlike antibodies, which are closely related proteins that recognize intact protein antigens, TCRs classically bind, via their CDR loops, to peptides (p) that are presented by molecules of the major histocompatibility complex (MHC). This TCR‐pMHC interaction is crucially important in cell‐mediated immunity, with the specificity in the cellular immune response being attributable to MHC polymorphism, an extensive TCR repertoire and a variable peptide cargo. The ensuing structural and biophysical studies within the TCR‐pMHC axis have been highly informative in understanding the fundamental events that underpin protective immunity and dysfunctional T‐cell responses that occur during autoimmunity. In addition, TCRs can recognize the CD1 family, a family of MHC‐related molecules that instead of presenting peptides are ideally suited to bind lipid‐based antigens. Structural studies within the CD1‐lipid antigen system are beginning to inform us how lipid antigens are specifically presented by CD1, and how such CD1‐lipid antigen complexes are recognized by the TCR. Moreover, it has recently been shown that certain TCRs can bind to vitamin B based metabolites that are bound to an MHC‐like molecule termed MR1. Thus, TCRs can recognize peptides, lipids, and small molecule metabolites, and here we review the basic principles underpinning this versatile and fascinating receptor recognition system that is vital to a host's survival.     

Structure-function studies of linear and cyclized peptide antagonists of the GnRH receptor.

Structurally new analogs of the peptidic GnRH receptor antagonist Cetrorelix as well as conformationally constrained cyclized deca- or pentapeptides were synthesized and selected peptides evaluated comprehensively. To understand how structural variations of the antagonistic peptide effect pharmacodynamic properties, binding affinities and antagonistic potencies toward the human and rat GnRH receptor were determined. Whereas large substituents in position 6 of linear peptides are compatible with high binding affinity (K(D) < 0.5 nM), all cyclized peptides except the cyclo[3-10] analog D-52391 depicted low binding affinity (K(D) > 10 nM). Binding affinity and antagonistic potency in vitro correlated for all peptides and surprisingly no discrimination between human and rat receptor proteins was observed. Since receptor residues W(101) and N(102) are involved in agonist and antagonist binding, equally potent but structurally different antagonists were tested for binding to the respective W(101)A and N(102)A mutants. In contrast to linear decapeptides, residues N(102) and W(101) are not involved in binding of D-23938 and W(101) is the critical residue for D-52391 binding. We conclude that although equally potent, peptidic GnRH receptor antagonists do have distinct interactions within the ligand binding pocket. Finally, selected antagonists were tested for testosterone suppression in male rats. The duration of testosterone suppression below castration levels differed largely from 1 day for Ganirelix to 27 days for D-23487. Systemic availability became evident as the most important parameter for in vivo efficacy.