Opioid receptor affinity and selectivity effects of second residue and carboxy terminal residue variation in a cyclic disulfide-containing opioid tetrapeptide

The previously described cyclic, delta opioid receptor-selective tetrapeptide H-Tyr-d -Cys-Phe-d -Pen-OH, where Pen, penicillamine, is β,β-dimethylcysteine, was modified at residues 2 and 4 by varying combinations of d - and l -Cys and d - and l -Pen, and effects on mu and delta opioid receptor binding affinities and on potency in the mouse vas deferens (MVD) smooth muscle assay were evaluated. A comparison was drawn between consequences of alterations in this series of analogs and those of analogous modifications in the related cyclic pentapeptide series which includes the highly delta receptor-selective [d -Pen²,d -Pen⁵]enke-phalin, DPDPE. Unlike effects observed in the cyclic pentapeptide series, the mu receptor binding affinities of the cyclic tetrapeptides are not dramatically influenced by substitution of Pen for Cys at residue 2. Conversely, while binding of the pentapeptides is only slightly affected by alteration of the chirality of the carboxy-terminal residue, modification of stereochemistry at the carboxy terminus in the tetrapeptides critically alters binding behavior at both mu and delta sites. In contrast with the pentapeptide series, the tetrapeptides appear to be highly dependent upon primary sequence for binding and activity, as only the lead compound binds with high affinity to the delta site. Results suggest that the less flexible cyclic tetrapeptides, lacking the Gly³ residue, display more stringent structural requirements for binding and activity than do the corresponding cyclic pentapeptides.     

Cyclic dermorphin-like tetrapeptides with delta-opioid receptor selectivity. 3. Effect of residue 3 modification on in vitro opioid activity

A series of residue 3-modified analogs of the cyclic, delta-opioid receptor-selective, dermorphin-like tetrapeptide Tyr-D-Cys-Phe-D-Pen and the corresponding residue 4-modified analog of the related delta receptor-selective cyclic pentapeptide [D-Pen2,D-Pen5] enkephalin were synthesized and evaluated in opioid receptor binding assays and in the in vitro mouse vas deferens (MVD) bioassay. In both series, substitutions that would be expected to alter the orientation of the phenylalanine-substituted aromatic side chain relative to the rest of the peptide, due to changes in the conformation of the peptide backbone, had deleterious effects on binding affinity and MVD potency. In general, these adverse effects were more pronounced in the pentapeptide series, owing, most likely, to the greater rigidity and, therefore, reduced susceptibility to conformational perturbation of the tetrapeptides. Substitution of phenylalanine by p-fluorophenylalanine enhances binding affinity in the pentapeptide series, consistent with previous observations in the enkephalins, but is without effect on binding in the tetrapeptide series. Substitution of phenylalanine by homophenylalanine, which alters the relationship of the aromatic phenyl ring to the remainder of the peptide by inserting an additional methylene group between the aromatic moiety and the backbone, greatly reduces binding affinity and MVD potency in the pentapeptide. The corresponding modification in the tetrapeptide series has little effect on delta receptor binding affinity and MVD potency and enhances binding to mu opioid receptors. Several possible interpretations of these results are discussed.     

Substitution on the Phe3 aromatic ring in cyclic delta opioid receptor-selective dermorphin/deltorphin tetrapeptide analogues: electronic and lipophilic requirements for receptor affinity.

In an effort to explore structural features affecting receptor recognition in a series of conformationally restricted tetrapeptides related to the cyclic, delta opioid receptor-selective analogue, [formula: see text] electronic, lipophilic, and steric effects at the Phe3 residue were assessed by substitution at different positions of the side-chain aromatic ring by halogens, alkyl, hydroxyl, and nitro groups. Effects on opioid receptor binding affinity and selectivity were determined. The results, which are generally consistent with reports of analogous modifications in linear and cyclic pentapeptide enkephalins, indicate that steric, lipophilic, and electronic properties are all important determinants of delta opioid receptor recognition. Specifically, modifications which increase lipophilicity or exert electron-withdrawing effects on the aromatic ring enhance binding affinity, while hydrophilic, bulky, or electron-releasing modifications are detrimental. These observations are in excellent agreement with quantitative structure-activity relationship (QSAR) results reported for Phe4 modifications in linear opioid pentapeptide enkephalin analogues, suggesting that the Phe3 tetrapeptide side chain and the Phe4 pentapeptide side chain interact with the same delta receptor binding subsite.     

Modifications of the cyclic mu receptor selective tetrapeptide Tyr‐c[d‐Cys‐Phe‐d‐Pen]NH2 (Et): effects on opioid receptor binding and activation

Abstract: The previously described cyclic mu opioid receptor‐selective tetrapeptide Tyr‐c[d ‐Cys‐Phe‐d ‐Pen]NH₂ (Et) (JOM‐6) was modified at residues 1 and 3 by substitution with various natural and synthetic amino acids, and/or by alteration of the cyclic system. Effects on mu and delta opioid receptor binding affinities, and on potencies and efficacies as measured by the [³⁵S]‐GTPγS assay, were evaluated. Affinities at mu and delta receptors were not influenced dramatically by substitution of Tyr¹ with conformationally restricted phenolic amino acids. In the [³⁵S]‐GTPγS assay, all of the peptides tested exhibited a maximal response comparable with that of fentanyl at the mu opioid receptor, and all showed high potency, in the range0.4–9 nm . However, potency changes did not always correlate with affinity, suggesting that the conformation required for binding and the conformation required for activation of the opioid receptors are different. At the delta opioid receptor, none of the peptides were able to produce a response equivalent to that of the full delta agonist BW 373,U86 and only one had an EC₅₀ value of less than 100 nm . Lastly, we have identified a peptide, d ‐Hat‐c[d ‐Cys‐Phe‐d ‐Pen]NH₂ (Et), with high potency and > 1000‐fold functional selectivity for the mu over delta opioid receptor as measured by the [³⁵S]‐GTPγS assay.     

Roles of residues 3 and 4 in cyclic tetrapeptide ligand recognition by the κ‐opioid receptor

Abstract: A series of cyclic, disulfide‐ or dithioether‐containing tetrapeptides based on previously reported potent μ‐ and δ‐selective analogs has been explored with the aim of improving their poor affinity to the κ‐opioid receptor. Specifically targeted were modifications of tetrapeptide residues 3 and 4, as they presumably interact with residues from transmembrane helices 6 and 7 and extracellular loop 3 that differ among the three receptors. Accordingly, tetrapeptides were synthesized with Phe³ replaced by aliphatic (Gly, Ala, Aib, Cha), basic (Lys, Arg, homo‐Arg), or aromatic sides chains (Trp, Tyr, p‐NH₂Phe), and with d ‐Pen⁴ replaced by d ‐Cys⁴, and binding affinities to stably expressed μ‐, δ‐, and κ‐receptors were determined. In general, the resulting analogs failed to exhibit appreciable affinity for the κ‐receptor, with the exception of the tetrapeptide Tyr‐c[d ‐Cys‐Phe‐d ‐Cys]‐NH₂, cyclized via a disulfide bond, which demonstrated high binding affinity toward all opioid receptors (Ki^μ = 1.26 nm , Ki^δ = 16.1 nm , Ki^κ = 38.7 nm ). Modeling of the κ‐receptor/ligand complex in the active state reveals that the receptor‐binding pocket for residues 3 and 4 of the tetrapeptide ligands is smaller than that in the μ‐receptor and requires, for optimal fit, that the tripeptide cycle of the ligand assume a higher energy conformation. The magnitude of this energy penalty depends on the nature of the fourth residue of the peptide (d ‐Pen or d ‐Cys) and correlates well with the observed κ‐receptor binding affinity.     

Development and validation of opioid ligand–receptor interaction models: The structural basis of mu vs. delta selectivity

Abstract: Opioid receptor binding conformations for two structurally related, conformationally constrained tetrapeptides, JOM‐6 (µ receptor selective) and JOM‐13 (δ receptor selective), were deduced using conformational analysis of these ligands and analogs with additional conformational restrictions. Docking of these ligands in their binding conformations to opioid receptor structural models, based upon the published rhodopsin X‐ray structure, implicates specific structural features of the µ and δ receptor ligand binding sites as forming the basis for the µ selectivity of JOM‐6 and the δ selectivity of JOM‐13. In particular, the presence of E229 in the µ receptor (in place of the corresponding D210 of the δ receptor) causes an adverse electrostatic interaction with C‐terminal carboxylate‐containing ligands, resulting in the observed preference of ligands with an uncharged C‐terminus for the µ receptor. In addition, the requirement that the Phe³ side chain of JOM‐13 assume a gauche orientation for optimal δ binding, whereas the Phe³ side chain of JOM‐6 must be in a trans orientation for high‐affinity µ binding can be largely attributed to the steric effect of replacement of L300 of the δ receptor by W318 of the µ receptor. Testing this hypothesis by examining the binding of JOM‐6 and several of its key analogs with specific µ receptor mutants is described. Our initial results are consistent with the proposed ligand–receptor interaction models.     

Single residue modifications of the delta opioid receptor selective peptide, [d-Pen2,d-Pen5]-enkephalin (DPDPE)

Six analogs of the highly delta opioid receptor selective, conformationally restricted, cyclic peptide [d -Pen²,d -Pen⁵]enkephalin, Tyr-d -Pen-Gly-Phe-d -PenOH (DPDPE), were synthesized and evaluated for opioid activity in rat brain receptor binding and mouse vas deferens (MVD) smooth muscle assays. All analogs were single amino acid modifications of DPDPE and employed amino acid substitutions of known effects in linear enkephalin analogs. The effect on binding affinity and MVD potency of each modification within the DPDPE structural framework was consistent with the previous reports on similarly substituted linear analogs. Conformational features of four of the modified DPDPE analogs were examined by ¹H NMR spectroscopy and compared with DPDPE. From these studies it was concluded that the observed pharmacological differences with DPDPE displayed by diallyltyrosine¹-DPDPE ([DAT¹]DPDPE) and phenylglycine⁴-DPDPE ([Pgl⁴]DPDPE) are due to structural and/or conformational differences localized near the substituted amino acid. The observed enhanced μ receptor binding affinity of the carboxamide terminal DPDPE-NH₂ appears to be founded solely upon electronic differences, the NMR data suggesting indistinguishable conformations. The observation that the α-aminoisobutyric acid substituted analog [Aib³]DPDPE displays similar in vitro opioid behavior as DPDPE while apparently assuming a significantly different solution conformation suggests that further detailed conformational analysis of this analog will aid the elucidation of the key structural and conformational features required for action at the δ opioid receptor.     

Coupling of multiple opioid receptors to GTPase following selective receptor alkylation in brain membranes

Opioid agonists of the mu, kappa and delta types stimulated low-K_m guanosine triphosphatase (GTPase) in membranes, from the brain of the rat by up to 34%, with potencies the rank order of which corresponded to the respective binding affinities to opioid receptor. In general, kappa ligands stimulated GTPase to a lesser degree than mu or delta opiates. The coupling of a given type of opioid receptor to GTPase was resolved by direct or protective alkylation of the other receptors. Treatment of the membranes with β-funaltrexamine abolished the stimulation of GTPase by sufentanil and levorphanol (mu), but not by bremazocine (kappa) or DSLET (delta). On the other hand, prior incubation with Superfit, an alkylating agent with selectivity for the delta opioid receptor, specifically eliminated the effect of DSLET. Partial alkylation by increasing concentrations of Superfit gradually reduced the extent of stimulation of GTPase by DSLET. The successive treatment of membranes with Superfit and β-funaltrex-amine blocked the actions of DSLET, sufentanil and levorphanol, but had no effect on the stimulation of the GTPase by bremazocine. Selective coupling of an opioid receptor to GTPase was also obtained after incubation of membranes with β-chlornaltrexamine in the presence of protective concentrations of mu, kappa or delta opioid ligands. Alkylation resolved the coupling of the non-selective opiate etorphine: the sum of stimulation of GTPase in the receptor-selective membranes equalled maximal stimulation of enzyme in untreated membranes. Naloxone blocked the stimulation of GTPase by mu, kappa or delta agonists, but ICI-174,864 specifically inhibited the effect of DSLET. The results describe the use of receptor-selective membranes from brain to characterize the coupling of multiple opioid receptors to high-affinity GTPase, the inhibitory binding protein for GTP of the adenylate cyclase complex.     

Substitution of aromatic and nonaromatic amino acids for the Phe3 residue in the δ-selective opioid peptide deltorphin I: Effects on binding affinity and selectivity

Deltorphins I and II (Tyr-D-Ala-Phe-Asp-Val-Val-Gly NH₂ and Tyr-D-Ala-Phe-Glu-Val-Val-Gly NH₂) display a high degree of 6-opioid receptor selectivity. Since they lack the intervening Gly3 residue found between the Tyr and Phe aromatic moieties in pentapeptide enkephalins, deltorphins I and II resemble a previously described series of cyclic tetrapep-tides based on Tyr-c[D-Cys-Phe-D-Pen] (JOM-13). With the goal of development of structure-activity relationships for deltorphins and comparison with that of the cyclic tetrapep-tides, ten analogs of deltorphin I were synthesized in which Phe³ was replaced with specific aromatic and nonaromatic amino acids with varying physicochemical properties. Results indicated that analogs containing the bicyclic aromatic amino acids 3-(l-naphthyl)-L-alanine [1-Nal; K_i(μ) = 767 nM, K_i(§) = 7.70 nM], 3-(2-naphthyl)-L-alanine [2-Nal; K_i(μ)= 1910 nM, K_i(§) = 49.2 nM], tryptophan [K_i(μ)= 1250 nM, K_i(§) = 23.9nM], and 3-(3-benzothienyl)-L-alanine [Bth; K_i(μ)= 112nM, K_i(§) = 3.36 nM] were fairly well tolerated at μ- and §-receptors, though affinity was compromised to varying degrees relative to deltorphin I. Shortening the Phe side chain by incorporation of phenylglycine (Pgl) was detrimental to both μ (K_i= 4710 nM) and § (K_i= 15.6 nM) binding, while extension of the side chain with homophenylalanine (Hfe) enhanced μ binding (K_i= 67.8 nM), leaving § affinity unaffected (K_i= 2.64 nM). Substitution with nonaromatic amino acids valine and isoleucine led expectedly to poor opioid binding [K_i(μ) =≥ 10000 nM for each, K_i(§) = 160 and 94.7 nM, respectively], while peptides containing cyclohexylalanine (Cha) and leucine surprisingly retained affinity at both μ (K_i= 322 and 1240 nM, respectively) and § (K_i= 10.5 and 12.4 nM, respectively) sites. In general, these trends mirror those observed for similar modification in Tyr-c[D-Cys-Phe-D-Pen].     

Investigation of the selectivity of oxymorphone- and naltrexone-derived ligands via site-directed mutagenesis of opioid receptors: exploring the "address" recognition locus

The delta-selective opioid antagonist naltrindole (NTI), as well as the kappa-selective opioid antagonists norbinaltorphimine (norBNI) and 5'-guanidinonaltrindole (GNTI), are derived from naltrexone, a universal opioid antagonist. Previous studies have indicated that extracellular loop III is the key region for discrimination by naltrexone-derived selective ligands between the delta, mu, and kappa opioid receptor types. It has been proposed that selective ligands could bind to all three receptor types if the appropriate portions of the extracellular loops were eliminated. To investigate this possibility, several single-point mutant opioid receptors have been generated with the aim of conferring enhanced affinity of selective ligands for their nonpreferred receptor types. Mutations were made in all three types of opioid receptors with the focus on two positions at the extracellular end of transmembrane regions (TM) VI and VII. It was found that the delta-selective NTI could bind both mu and kappa receptors with significantly enhanced affinity when an aromatic residue in TM VII was replaced with alanine (mu[W318A] and kappa[Y312A]). Similarly, kappa-selective antagonists, norBNI and GNTI, showed enhanced affinity for the mu[W318A] mutant and for both mu and delta receptors when a glutamate residue was incorporated into the extracellular end of TM VI (mu[K303E] and delta[W284E]). These results demonstrate that naltrexone-derived selective ligands achieve their selectivity via a combination of enhanced affinity of the address for a particular subsite along with loss of affinity due to steric interference at nonpreferred types. The results reveal key residues in the "address" recognition locus that contribute to the selectivity of opioid ligands and support the hypothesis that recognition of the naltrexone moiety is essentially the same for all three receptor types.     

A Naloxone–Steroid Hybrid Azine with Selective and Long-Acting Opioid Antagonism at Delta Receptors In Vitro

The interaction of naloxone estrone azine (N-EH) with various opioid receptor types was studied in vitro. Its potency as an antagonist of opioid effects was compared to that of naloxone on the electrically evoked contractions of mouse vas deferens (Mvd) and guinea pig ileum myenteric plexus longitudinal muscle (Gpi) preparations. N-EH was found to be 9-fold more potent than naloxone in antagonizing the effects of D-Ala²-Leu⁵-enkephalin in the Mvd and 22-fold less potent in antagonizing nor-morphine in the Gpi. In the Mvd, the recovery half-time for N-EH was longer than 1000 min. Neither compound showed agonism. The two compounds were also compared for their capacity to displace the binding of ³H-D-Ala²-Leu⁵-enkephalin, ³H-dihydromorphine, and ³H-ethylketocyclazocine to rat brain membranes under conditions where delta, mu, and kappa sites were labeled. The relative affinities were 0.70, 0.16, and 0.14 for N-EH and 0.05, 0.87, and 0.08 for naloxone, respectively. Thus, compared to naloxone, which is mu selective, N-EH is a delta-selective antagonist.     

Characterization of the receptor binding profile of (3H)-dermorphin in the rat brain

Amphibian skin synthesizes a variety of biologically active peptides. Of these, dermorphin (Tyr-d -Ala-Phe-Gly-Tyr-Pro-Ser-NH₂) is an extraordinarily potent opioid peptide up to 1000 times more active than morphine in inducing analgesia after intracerebroventricular administration. Dermorphin has little in common with the sequence of all hitherto known mammalian opioid peptides and is unique in having a d -amino acid residue in position 2. Specific binding properties of tritium labeled dermorphin were characterized in the rat brain. Scatchard or Hill analysis of equilibrium measurements performed over a large range of concentrations revealed a single population of dermorphin binding sites with a Kd value of 0.46 nM. Dermorphin and the selective μ-receptor ligand (d -Ala², MePhe⁴, Gly⁵-ol)- enkephalin (DAGO) had similar high potencies in competing with (³H)-dermorphin binding, whereas the inverse holds for the prototypical delta receptor ligand (d -Pen²,d -Pen⁵)-enkephalin (DPDPE), which exhibited a potency three orders of magnitude lower. Dermorphin was tested for its relative affinity to μ and delta binding sites by determining its potency in displacing (³H)-DAGO and (³H)-DPDPE from rat brain membrane preparations. Based on these comparisons, dermorphin exhibited a selectivity ratio Ki(DPDPE)/Ki(DA-GO) = 100, a value almost identical to that of DAGO, this ligand being considered as the protypical μ-receptor probe. The high affinity and selectivity of (³H)-dermorphin together with its very low nonspecific binding make this peptide a useful tool for dissecting the role(s) of the μ-receptor(s).     

[d-Pen2,d-Pen5]enkephalin,the standard delta opioid agonist,induces morphine-like behaviors in mice

[d-Pen²,d-Pen⁵]enkephalin (DPDPE; 3–30 µg) and morphine (10 µg) both caused Straub tails, increased locomotion, and circling after ICV administration to ICR mice. DPDPE-induced tail stiffening was reduced when mice were pretreated with naloxone (0.5 mg/kg SC) or-funaltrexamine (10 µg ICV), but not with ICI 174864 (2 mg/kg SC), the selective antagonist at delta opioid receptors. These results point to (a) mu receptors mediating the tail stiffening and (b) the loss of delta receptor selectivity after 10 and 30 µg DPDPE.     

Solution conformations of the peptide backbone for DPDPE and its β-MePhe4-substituted analogs

The solution structures of DPDPE, a conformationally restricted pentapeptide with the sequence H-Tyr¹-d -Pen²-Gly³-Phe⁴-d -Pen⁵-OH, and its four β-MePhe⁴-substituted analogs were examined by a combined approach including the NMR measurements in DMSO and water as well as independent energy calculations. It was concluded that several low energy conformers of DPDPE backbone satisfy the NMR data obtained in this study as well as in previous studies by other authors. These possible solution conformers of DPDPE in both DMSO and water share virtually the same type of cyclic backbone structure, with the Gly³ residue in a conformation close to a γ-turn, and the Phe⁴ residue in a conformation close to α-helical torsion angles. They differ in the space arrangements of the flexible Tyr¹ moiety. The solution structures of the β-MePhe⁴-substituted analogs of DPDPE are interesting. For analogs with an S-configuration at the C^α atom in the Phe⁴ residue, the cyclic backbone conformations resemble those of DPDPE itself, whereas for analogs with an R-configuration at the C^α atom, the backbone conformation is somewhat different. This observation is in line with the high biological potencies and selectivities displayed by the former compounds but not by the latter ones. It was noted also that as far as the peptide backbone conformers are concerned, some of the possible DPDPE conformers in water are similar to the previously suggested model for the δ-receptor-bound conformation of DPDPE, becoming virtually identical to this conformation by rotating the side chains of the Tyr¹ and the Phe⁴ residues.     

Proton NMR Conformational Analysis of Cyclic β-Casomorphin Analogues of the Type Tyr-cyclo[-Nω-D-Orn-Xaa-Yaa-Gly-]

A conformational study of the cyclic β-casomorphin-5 analogues H-Tyr-cyclo[-D-Orn-2-Nal-Pro-Gly-] ( 1 ) (μ-selective agonist; 2-Nal = 2-naphthylalanine), H-Tyr-cyclo[-D-Orn-2-Nal-D-Pro-Gly-] ( 2 ) (mixed μ agonist/δ antagonist) and H-Tyr-cyclo[-D-Orn-Phe-D-Pro-Gly-] ( 3 ) (highly potent μ and δ agonist) has been carried out using ¹H NMR spectroscopy. A complete assignment of the proton resonances of the three pentapeptides has been achieved. Compound 1 was shown to exist in two conformations, a major one (90%) characterized by a cis amide bond between 2-Nal³ and Pro⁴, and a minor one (10%) showing cis amide bonds both between D-Orn² and 2-Nal³ and between 2-Nal³ and Pro⁴. Peptides 2 and 3 each showed only one conformer with all-trans peptide bonds in both cases. Temperature dependence studies of the amide proton chemical shifts indicated the existence of several intramolecular hydrogen bonds in the case of compounds 2 and 3 but not in the case of peptide 1. The backbone conformations of 2 and 3 were found to be similar, both being characterized by two consecutive γ turns around the D-Pro⁴ and D-Orn² residues, respectively, and by a D-Orn²-CO←HN^δ-D-Orn² hydrogen bond. Altogether, the overall backbone conformation and the preferred side chain conformation were found to be roughly similar for the three title peptides. For all three compounds a close proximity between the aromatic moiety of the 3-position residue (2-Nal or Phe) and the D(or L)-Pro⁴ residue was established on the basis of ROESY experiments. The examination of low energy conformations obtained in molecular modelling studies by taking into account the various experimentally found NMR parameters (NOEs, vicinal H,H coupling constants, torsion angles, H-bonds) led to proposals of the solution conformation for each peptide. These conformations are in close agreement with a pharmacophore model for μ opioid receptor binding compounds.     

Synthesis,biology, NMR and conformation studies of the topographically constrained δ‐opioid selective peptide analogs of [β‐iPrPhe3]deltorphin I

Abstract: Replacement of Phe³ in the endogenous δ‐opioid selective peptide deltorphin I with four optically pure stereoisomers of the topographically constrained, highly hydrophobic novel amino acid β‐isopropylphenylalanine (β‐iPrPhe) produced four pharmacologically different deltorphin I peptidomimetics. Radiolabeled ligand‐binding assays and in vitro biological evaluation indicate that the stereoconfiguration of the iPrPhe residue plays a crucial role in determining the binding affinity, bioactivity and selectivity of [β‐iPrPhe³]deltorphin I analogs: a (2S,3R) configuration of the iPrPhe³ residue in [β‐iPrPhe³]deltorphin I provided the most desirable biological properties with binding affinity (IC₅₀ = 2 n m ), bioassay potency (IC₅₀ = 1.23 n m in MVD assay) and exceptional selectivity for the δ‐opioid receptor over the µ‐opioid receptor (30 000). Further conformational studies based on two‐dimensional NMR and computer‐assisted molecular modeling suggested a model for the possible bioactive conformation in which the Tyr¹ and (2S,3R)‐β‐iPrPhe³ residues adopt trans side‐chain conformations, and the linear peptide backbone favors a distorted β‐turn conformation.     

‘Tethered ligand’derived pentapeptide agonists of thrombin receptor: a study of side chain requirements for human platelet activation and GTPase stimulation

Proteolytic action of α-thrombin on human thrombin receptor results in cleavage of a portion of the N-terminus, thereby generating a‘tethered ligand’at the newly exposed N-terminus, which then activates the receptor in an intramolecular fashion. Agonist peptides incorporating the amino acid sequence of the newly exposed N-terminal portion of the cleaved receptor cause receptor activation without requiring prior cleavage of the receptor by thrombin. The pentapeptide amide Ser-Phe-Leu-Leu-Arg-NH₂, which retains the N-terminal sequence of the‘tethered ligand’of the receptor, has been shown to be the minimum sequence to cause receptor activation. To understand the importance of the side chains of various residues within the pentapeptide amide, we carried out an extensive structure-activity study of the ability of peptides to stimulate gel-filtered platelet aggregation. In this study 106 pentapeptide amides were synthesized, utilizing naturally occurring l -amino acids, unnatural amino acids, D-amino acids and N-methyl amino acids for replacements. At position-1, charged residues (acidic or basic) were not tolerated, and the size and shape of the residue were important. Position-2 tolerated only aromatic residues. Position-3 accommodated various residues. A significant finding of this study was that two very different residues, [3-(2-naphthyl)]-l -alanine and l -arginine, when substituted for leucine residue at position-3, resulted in more active agonists. At position-4 aromatic and aliphatic residues were well tolerated, whereas basic and acidic residues were less tolerated. Position-5 mimicked position-3 in its ability to tolerate a wide range of residues. In general, an acidic residue was not preferred in any position. In all positions a -Phe-residue was tolerated. Positions 1 and 3 tolerated -Pro-residue, whereas positions 2, 4 and 5 did not. d -Amino acid substitution was not tolerated in any position. While N-methylation of residues at positions-3 and -4 led to poor agonists, at position-2 it resulted in an inactive analog. These studies define the side chain requirements for the‘tethered ligand’derived pentapeptide agonists of thrombin receptor for human platelet activation. Selected peptides were tested for ability to stimulate platelet membrane GTPase. A good correlation was observed between peptides that stimulate GTPase and those that stimulate platelet aggregation, with the general finding that 3-10-fold higher concentrations were required to half maximally activate GTPase when compared with the concentrations needed for activation of platelet aggregation. © Munksgaard 1995.     

Dehydro-enkephalins

Two kinds of dehydropeptide analogs of enkephalin containing a ΔTyr unit at the N-terminus have been synthesized by coupling Boc-ΔTyr-(Cl₂Bzl)-OH with amino acid amides and tetrapeptide esters using the water soluble carbodiimide-HOBt method. Pentapeptides consisting of ΔTyr¹, and ΔPhe⁴ or ΔLeu⁵ were also prepared. Ultraviolet difference spectroscopy was important in the characterization of the dehydro moieties, ΔTyr, ΔPhe and ΔLeu. Attempts to liberate ΔTyr¹-enkephalins have been unsuccessful because of the instability of an N-terminal ΔTyr residue having p-phenolic group in the side chain.     

Structure–activity studies at the rat tachykinin NK2 receptor: effect of substitution at position 5 of neurokinin A

A series of analogues of neurokinin A(4–10) was synthesized using solid phase techniques with Chiron pins, and purified by HPLC. The potencies of 10 peptides with substitution at Ser⁵ were assessed at rat fundus NK2 receptors. In membrane binding studies with [¹²⁵I]-[Lys⁵,Tyr(I₂)⁷,MeLeu⁹,Nle¹⁰]-NKA(4–10), all compounds except [Asp⁵]NKA(4–10) showed reasonable affinity, and analogues with Lys and Arg substitutions were five-fold more potent than NKA(4–10). In functional studies, all peptides were able to contract the rat isolated fundus strips. Analogues with Phe, His and Asn substitutions were substantially weaker in functional than in binding studies, whereas there was an excellent correlation (r = 0.95) between binding and functional potency for the remaining seven peptides. [Phe⁵]NKA(4–10) is in fact neurokinin B(4–10) and this residue may be critical in determining selectivity between NK2 and NK3 receptors. Analogues with a basic residue (Lys, Arg) at position 5 showed both increased affinity and functional potency, whereas the neutral [Asn⁵]NKA(4–10) was equally as weak in contractile studies as the acidic [Asp⁵]NKA(4–10). However, [Glu⁵]NKA(4–10) and [Gln⁵]NKA(4–10) were no different from NKA(4–10). Our results could indicate the presence of a negative charge on the NK2 receptor, close to position 5 of NKA. This would facilitate interaction with positively charged side chains and impede interaction with negatively charged side chains, particularly the inflexible side chain of aspartic acid. Thus, not only the charge, but also the length of the side chain of the residue at position 5, seems to be important for interaction with the rat NK2 receptor.     

Synthesis and receptor binding characteristics of [d-Ala2, cysteamine5]enkephalin,a thiol-containing probe for structural elements of opiate receptors

For the elucidation of structural elements in the opiate receptors, a thiol-containing enkephalin analog, [d -Ala², cysteamine⁵]enkephalin, and its dimeric analog were synthesized and evaluated in the radio-ligand receptor binding assays using rat brain membranes. The dimeric analog was very potent in both delta and mu assays. Comparison of receptor affinities of the thiol-containing enkephalin with those of standard mu or delta receptor specific ligands suggested that the mu receptor contains an essential thiol group which may interact with the thiol group at the C-terminus of the enkephalin analog. It also appears that no metal-ion site, postulated for the delta receptors, is present in the delta binding site.