Receptor selectivity of enkephalin analogs carrying artificial address peptides

The synthetic [Leu]enkephalin analogs YGGFLGP(KS'S'S')2-OMe (S' represents Sar residue, ENK-S'8), YGGFLGP(KPPP)2-OMe (ENK-P8), and YGGFLGP(KA'LA')2-OMe (A' represents 2-aminoisobutyric acid (Aib), ENK-A'8) were designed to investigate the role of membrane affinity in opioid receptor binding. CD measurement in ethanol revealed that ENK-S'8 took a random coil conformation, while ENK-P8 and ENK-A'8 partly adopted a 3(1)- and alpha-helical conformation, respectively. ENK-A'8 was shown to be distributed to lipid bilayer membrane due to the amphiphilic alpha-helical structure, while ENK-S'8 and ENK-P8 stayed in aqueous phase. Opioid receptor affinities ENK-S'8 and ENK-P8 were similar to [Leu]enkephalinamide, indicating that the connection of hydrophilic peptide segments to the C-terminal of [Leu]enkephalinamide did not affect the receptor affinity. On the other hand, delta- and mu-receptor affinities of ENK-A'8 were about 1/20 and 1/6 times, respectively, those of [Leu]enkephalinamide. Therefore, ENK-A'8 has higher selectivity for mu-receptor than [Leu]enkephalinamide. These results are explained in terms of the membrane compartment concept proposed by Schwyzer.     

Photoaffinity inactivation of the enkephalin receptor.

An arylazide enkephalin derivative, [D-Ala2,Met5]enkephalin-Tyr-N-(2-nitro-4-azidophenyl) ethylenediamine (ETN), has been synthesized. In the dark, it inhibited the binding of [3H]enkephalinamide to enkephalin receptor-rich NG-108 cell membranes with an I50 = 2.2 X 10(-8) M or KI = 7 X 10(-9) M, assuming competitive inhibition. Photolysis of membranes in the presence of ETN caused irreversible inactivation of the enkephalin receptor, but inactivation was prevented by the addition of enkephalin, the half-effective concentration being 3 x 10(-9) M. ETN appears to be an effective photoaffinity label for the enkephalin receptor.     

Dimeric dermorphin analogues as mu-receptor probes on rat brain membranes. Correlation between central mu-receptor potency and suppression of gastric acid secretion

The opioid receptor preference for dermorphin and several dimerized structural analogues was investigated using rat brain synaptosomes and correlated with the potencies of intracerebroventricularly administered dimeric dermorphin peptides to inhibit gastric acid secretion. The carboxyl terminus of dermorphin or amino-terminal dermorphin analogues was bridged by dihydrazide or (poly)ethylenediamine structures. Synaptosomal membranes were prepared for radioligand binding assay in the presence of soybean trypsin inhibitor and preincubated to remove endogenously bound opioid peptides before storage at -70 degrees C. Specific radiolabeled agonists used in the radioligand binding assays were [D-Ala2,N-methyl-Phe4,Gly-ol5] [3H] enkephalin for mu-receptors and [D-Ala2,D-Leu5] [3H]enkephalin for delta-receptors. delta-Receptor binding assays were conducted in the presence of 2.6 microM [N-Me-Phe3,D-Pro4]morphiceptin to suppress peptide binding to mu-receptors. [D-Ala2,N-methyl-Phe4,Gly-ol5]enkephalin and dermorphin had affinities of 1.39 and 1.22 nM for mu-receptors and 355.8 and 178.6 nM for delta-receptors, respectively. Affinities of dimeric-dermorphin0 for mu- and delta-receptors, and the mu-selectivity ratio, exceeded values characteristic of dermorphin. The dimerized amino-terminal dermorphin analogues are peptides whose receptor binding differed from the parent molecule; e.g. the affinity of dimeric tetrapeptides toward mu-receptors was reduced but was increased for delta-receptors relative to monomeric dermorphin-(1-4)-amide. Dimeric tetradermorphin linked by a bridge containing 12 methylene units (di-tetra-dermorphin12), exhibited a dramatic loss in the mu-selectivity ratio as a result of diminished mu-affinity. On the other hand, substitution of Gly4 by Sar in di-tetra-dermorphin2 enhanced binding to mu-receptors: substitution of D-Arg2 for D-Ala resulted in an increased binding to mu-receptors while decreasing binding to delta-receptors, yielding a peptide with the highest mu-selectivity ratio. These substitutions of D-Arg2 and Sar4 in dimeric amino-terminal dermorphin pentapeptides enhanced binding to both mu- and delta-receptors relative to dermorphin-(1-5)-amide, but led to a decrease in its mu-selectivity ratio. Several dimeric dermorphin analogues exhibited an enhanced mu-selectivity ratio relative to their monomeric analogues. Dimeric peptides, which had a relatively high affinity for mu-receptors, were effective in the suppression of gastric acid secretion.     

Antinociceptive potencies of beta-casomorphin analogs as compared to their affinities towards mu and delta opiate receptor sites in brain and periphery

beta-Casomorphins and their analogs were tested for their opioid activities in the myenteric plexus longitudinal muscle preparation of the guinea pig ileum (GPI), the isolated mouse vas deferens (MVD), and for their affinities to mu- delta- and kappa- binding sites in rat brain membranes. C-terminal amidation of beta-casomorphin-4 and (-5) increased opioid potency in both organ preparations (GPI, MVD) and affinity to mu-binding sites in brain whereas binding to delta-sites was diminished. These beta-casomorphin-amides displayed a 2-3 times greater naloxone reversible antinociceptive effect than natural beta-casomorphins. Introduction of D-alanine at position 2 in the beta-casomorphin-amides increased potency in the GPI whereas activity in the MVD was only slightly changed. These compounds, however, showed a remarkable increase in binding to delta-sites in brain with an unaffected or slightly increased binding to mu-sites and decreased binding to kappa-sites. D-Ala2-beta-casomorphin-4 and (-5) amides were 10 times more potent antinociceptive agents than corresponding beta-casomorphin-amides. These results suggest firstly, that peripheral delta-receptors in the MVD are not as closely related to delta-binding sites at rat brain membranes as is the case with mu-receptors in the GPI and mu-binding sites, and secondly, in addition to mu-receptors, delta-receptors may be of importance in mediating antinociception.     

Possible participation of membrane thiol groups on the mechanism of NAD(P)+-stimulated Ca2+ efflux from mitochondria

The in vitro opioid activities of a series of leucine enkephalin analogs containing a thioamide linkage in place of the peptide bond at various positions of the backbone were determined in mu- and delta-receptor-selective bio- and binding-assays. Thioamide substitution in the 1-2 position resulted in an inactive compound, whereas the same modification in the 2-3 and 4-5 position produced potency enhancement. Most interestingly, the 2-3 modified analog showed a 3 to 5 times higher preference for delta- over mu-receptors than natural leucine enkephalin. These results suggest that subtle backbone modifications can have a profound effect on receptor affinity and selectivity of biologically active peptides.     

Multivalent Ligand System Carrying Enkephalin and Neurotensin Coimmobilized on Liposomes

A multivalent ligand system was constructed by coimmobilization of two kinds of peptide ligands, enkephalin and neurotensin derivatives having a dioctadecyl group, on dimyristoylphosphatidylcholine (DMPC) liposomes. The enkephalin derivatives are Tyr-D -Ala-Gly-Trp-Leu- (Sar-Sar-Pro)_n-[N(C₁₈H₃₇)₂] (Enk3nD, n=0, 1, 2), where a dioctadecyl group was connected to the C-terminal side of enkephalin directly or through a hydrophilic and flexible spacer chain of different lengths. The neurotensin derivatives are Ac-Glu[N(C₁₈H₃₇)₂]-(Sar-Sar-Pro)_n-Arg-Arg-Pro-Tyr-Ile-Leu-OH (D3nNT, n=0, 1, 2, 3). The derivatives were spontaneously immobilized on DMPC liposomes by overnight incubation. The receptor affinity of the enkephalin derivatives became significantly higher upon immobilization on liposomes. The highest affinity was obtained for the δ receptor by Enk6D immobilized on DMPC liposomes. This affinity is higher than that of enkephalinamide. Neurotensin derivatives coimmobilized with large amounts of Enk3D on DMPC liposomes show higher affinity than the neurotensin derivatives immobilized alone. The effect of Enk3D on the receptor affinity of the coimmobilized neurotensin derivative disappeared by the addition of [Ala², MePhe⁴, Gly-ol⁵]enkephalin (DAGO). Therefore, the receptor affinity of a peptide hormone is altered by immobilization on DMPC liposomes and by coimmobilization with other peptide hormones. It was confirmed by fluorescent microscopy that the multivalent ligand system binds to receptors without release of the bound ligands from DMPC liposomes.     

The effect of modification of the carboxyl group in short D-arginine 2-containing enkephalin analogs on their affinity and selectivity for opiate receptors

Radioreceptor binding assay using a membrane fraction from the rat brain was applied to study [D-Arg2, Leu5] enkephalin and two series of its analogues truncated at the C-terminus with a free or modified carboxyl group: tetra- and tripeptide amides and ethyl esters. The affinity to mu-specific opiate receptor subtype of the N-terminal [D-Arg2] tetrapeptide ethyl ester was 44 times as high as that of the tripeptide with a free carboxyl, and thus the ester retained up to 10% of leucine-enkephalin binding potency. However, a comparable esterification of the carboxyl group in the N-terminal [D-Arg2] tripeptide led to a 6-fold reduction in its affinity to mu-receptors. Consequently, identical modifications of the C-terminal carboxyl group in enkephalin analogues of various length can have completely different effects. Substitution of the natural glycine residue by D-arginine residue in position 2 of the enkephalin molecule truncated at the C-terminus increased the mu-receptor binding potency of the tetrapeptide, whereas its delta receptor binding potency declined by more than one order of magnitude. Simultaneous replacement of glycine2 by D-arginine2 and carboxyl amidation resulted in the short enkephalin analogue Tyr--D--Arg--Gly--Phe--NH2, whose affinity to mu receptors was four times as high as that of leucine--enkephalin, the tetrapeptide being 284 times more selective for the mu vs. delta opiate receptors.     

Opiate receptor binding affinities of some D-amino acid substituted beta-casomorphin analogs

beta-Casomorphin-(5) and some analogs modified by the introduction of some D-amino acids and D-pipecolic acid as well as by C-terminal amidation were tested for their affinities to mu- and delta-binding sites in rat brain membranes. The binding affinities of these compounds are compared with the known activities in the guinea pig ileum (GPI) and mouse vas deferens (MVD) test and their antinociceptive potencies in rats. The substitution of D-proline for proline in position 4 in beta-casomorphin-(5) and beta-casomorphin-(4)amide (morphiceptin) results in derivatives with very high mu-binding affinity and mu-selectivity. These affinities correspond to the respective analgesic potencies. Both binding to mu-receptors and analgesic potency are also enhanced by the introduction of D-Phe in position 3. Testing D-Ala2 substituted derivatives with respect to their ability to compete for 3H-naloxone, we observed apparent differences between the pentapeptide amides (biphasic displacement curves) and the tetrapeptide amides (monophasic displacement curves). The substitution of L-Pro2 by D-pipecolic acid yields an analog with preferential delta-receptor affinity in the organ preparations (MVD) but preferential mu-receptor affinity in brain membranes. This finding suggests a possible difference between peripheral and central mu-binding sites.     

Affinity labeling of delta-opiate receptors using [D-Ala2,Leu5,Cys6]enkephalin. Covalent attachment via thiol-disulfide exchange

[D-Ala2,Leu5,Cys6]Enkephalin (DALCE) is a synthetic enkephalin analog which contains a sulfhydryl group. DALCE binds with high affinity to delta-receptors, with moderate affinity to mu-receptors, and with negligible affinity to kappa-receptors. Pretreatment of rat brain membranes with DALCE resulted in concentration-dependent loss of delta-binding sites. Using 2 nM [3H][D-Pen2,D-Pen5]enkephalin (where Pen represents penicillamine) to label delta-sites, 50% loss of sites occurred at about 3 microM DALCE. Loss of sites was not reversed by subsequent incubation in buffer containing 250 mM NaCl and 100 microM guanyl-5'-yl imidodiphosphate (Gpp(NH)p), conditions which cause dissociation of opiate agonists. By contrast, the enkephalin analogs [D-Ala2,D-Leu5]enkephalin, [D-Ser2,Leu5,Thr6]enkephalin, [D-Pen2,D-Pen5]enkephalin, and [D-Ala2,D-Leu5,Lys6]enkephalin were readily dissociated by NaCl and Gpp(NH)p, producing negligible loss at 3 microM. This suggests that DALCE binds covalently to the receptors. Pretreatment of membranes with the reducing agents dithiothreitol and beta-mercaptoethanol had no effect on opiate binding. Thus, loss of sites required both specific recognition by opiate receptors and a thiol group. The irreversible effect of DALCE was completely selective for delta-receptors. Pretreatment with DALCE had no effect on binding of ligands to mu- or kappa-receptors. The effect of DALCE on delta-binding was: 1) markedly attenuated by inclusion of dithiothreitol in the preincubation buffer, 2) partially reversed by subsequent incubation with dithiothreitol, 3) slightly enhanced when converted to the disulfide-linked dimer, and 4) prevented by blocking the DALCE sulfhydryl group with N-ethylmaleimide or iodoacetamide. These results indicate that DALCE binds covalently to delta-receptors by forming a disulfide bond with a sulfhydryl group in the binding site. The mechanism may involve a thiol-disulfide exchange reaction.     

Probing receptor binding activity of interleukin-8 dimer using a disulfide trap

Interleukin-8 (IL-8), a member of the chemokine superfamily, exists as both monomers and dimers, and mediates its function by binding to neutrophil CXCR1 and CXCR2 receptors that belong to the G protein-coupled receptor class. It is now well established that the monomer functions as a high-affinity ligand, but the binding affinity of the dimer remains controversial. The approximately 1000-fold difference between monomer-dimer equilibrium constant (microM) and receptor binding constant (nM) of IL-8 does not allow receptor-binding affinity measurements of the native IL-8 dimer. In this study, we overcame this roadblock by creating a "trapped" nondissociating dimer that contains a disulfide bond across the dimer interface at the 2-fold symmetry point. The NMR studies show that the structure of this trapped dimer is indistinguishable from the native dimer. The trapped dimer, compared to a trapped monomer, bound CXCR1 with approximately 70-fold and CXCR2 with approximately 20-fold lower affinities. Receptor binding involves two interactions, between the IL-8 N-loop and receptor N-domain residues, and between IL-8 N-terminal and receptor extracellular loop residues. In contrast to a trapped monomer that bound an isolated CXCR1 N-domain peptide with microM affinity, the trapped dimer failed to show any binding, indicating that dimerization predominantly perturbs the binding of only the N-loop residues. These results demonstrate that only the monomer is a high-affinity ligand for both receptors, and also provide a structural basis for the lower binding affinity of the dimer.     

Receptor selectivity of Met-enkephalin-Arg6-Phe7, an endogenous opioid peptide, in cerebral cortex of human and rat

This study was undertaken to examine the receptor selectivity of Met-enkephalin-Arg6-Phe7 (MERF) employing radioreceptor binding assays in human cerebral cortex membranes, and to elucidate the responsible receptors that mediate the regulatory action of MERF on high (20 mM) K+-stimulated release of [3H]norepinephrine ([3H]-NE) in rat cortex slices. Specific binding of [3H]MERF was inhibited by DAMGO, Tyr-D-Arg-Phe-Sar(TAPS), bremazocine and ethylketocyclazocine (EKC), but not by U69,593 (U69) and DPDPE. MERF showed high affinity for specific binding sites of [3H]DAMGO. However, MERF had little influence on the specific binding of [3H]DPDPE, [3H]U69 and [3H]diprenorphine ([3H]DIP) in the presence of 1 microM each of DAMGO, DPDPE and U69. In [3H]NE release experiments using rat cortex slices, DAMGO, MERF and EKC, in order of their potency, inhibited K+-stimulated release of [3H]NE. The inhibitory effects of MERF and DAMGO were more sensitive than that of EKC to antagonism by CTAP, nor-binaltorphimine (nor-BNI) and naloxone. These results suggested that MERF possesses high affinity for mu-receptors, but not for delta-, kappa1-, and very low affinity for kappa2-receptors in human cerebral cortex membranes. Also, the inhibitory effect of MERF on the K+-stimulated release of [3H]NE appears to be mediated by mu-receptors in rat cerebral cortex slices.     

Evidence for a mu-delta opioid receptor complex in CHO cells co-expressing mu and delta opioid peptide receptors

Based on non-competitive binding interactions we suggested that mu and delta receptors associate as a mu/delta receptor complex in rat brain. We hypothesized that the same non-competitive binding interactions observed in rat brain will be seen in CHO cells that co-express mu and delta receptors, but not in cells that express just mu or delta receptors. We used CHO cells expressing the cloned human mu receptor, cloned human delta receptor, or cloned mouse delta/human mu ("dimer cell"). Cell membranes were prepared from intact cells pretreated with 100nM SUPERFIT. [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding assays followed published procedures. SUPERFIT, a delta-selective irreversible ligand, decreased [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding to delta receptors by approximately 75% and to mu receptors by approximately 50% in dimer cells. SUPERFIT treatment did not decrease [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding to mu cells. The IC(50) values observed in SUPERFIT-treated dimer cells were: [d-Pen(2),d-Pen(5)]enkephalin (1820nM) and morphine (171nM). Saturation binding experiments with SUPERFIT-treated dimer cells showed that [d-Pen(2),d-Pen(5)]enkephalin (5000nM) was a competitive inhibitor. In contrast, morphine (1000nM) lowered the B(max) from 1944fmol/mg to 1276fmol/mg protein (35% decrease). Both [d-Pen(2),d-Pen(5)]enkephalin and morphine competitively inhibited [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding to SUPERFIT-treated mu cells. The results indicate that the mu-delta opioid receptor complex defined on the basis of non-competitive binding interactions in rat brain over 20 years ago likely occurs as a consequence of the formation of mu-delta heterodimers. SUPERFIT-treated dimer cells may provide a useful model to study the properties of mu-delta heterodimers.     

Identification of an opioid receptor subunit carrying the mu binding site

The enkephalin affinity reagent [3H]Tyr-D-Ala-Gly-Phe-Leu-CH2Cl [( 3H]DALECK) was synthesized. It exhibited high-affinity reversible binding, at pH 7.4, to both mu and delta opioid receptor sites in rat brain membranes. At pH 8.1, nanomolar levels of [3H]DALECK produced an irreversible labeling in synaptic membranes, essentially only in one subunit of 58 000 daltons. The irreversible phase of the reaction reduced the subsequent binding of a mu-selective enkephalin derivative but not that of a delta-selective one. It is concluded that a mu subunit of the opioid receptor exists, can be alkylated specifically, and is of Mr 58 000.     

Design of biologically active peptides with non-peptidic structural elements. Biological and physical properties of a synthetic analogue of beta-endorphin with unnatural amino acids in the region 6-12

Modelling studies with beta-endorphin have clearly demonstrated that an amphiphilic secondary structural segment is a salient feature of the biologically active conformation of this 31-residue opioid peptide hormone. Here, we have initiated the synthesis of peptide models using unnatural building blocks by designing a beta-endorphin analogue (peptide 6) in which the hydrophilic linker region between the NH2-terminal enkephalin (residues 1-5) and the COOH-terminal helix (residues 10-28, sequence identical to that of peptide 3 in region 13-31, Fig. 1) consists of four units of gamma-amino-gamma-hydroxymethylbutyric acid connected by isopeptidic linkages. Peptide 6 has physical properties similar to that of peptide 3, as shown by surface monolayer and circular dichroism studies. The binding affinities of the two peptides to delta- and mu-receptors are also similar. In rat vas deferens assays, the present model is equipotent to peptide 3. The most striking result of all is the potent analgesic activity displayed by peptide 6 when injected intracerebroventricularly into mice. The potencies of peptides 6 and 3 are comparable in these assays. These studies clearly illustrate that one can use unusual building blocks to construct structural regions of synthetic analogues and still preserve the biological activity of peptide hormones.     

Mutation W284L of the human delta opioid receptor reveals agonist specific receptor conformations for G protein activation

Intrinsic activities of different delta opioid agonists were determined in a [35S]GTPgammaS binding assay using cell membranes from Chinese hamster ovary (CHO) cells stably expressing the wild type (hDOR/CHO) or W284L mutant human delta opioid receptor (W284L/CHO). Agonist binding affinities were regulated more robustly by sodium and guanine nucleotide in W284L/CHO than in hDOR/ CHO cell membranes. The W284L mutation selectively reduced the affinity of SNC 80 while having moderate effect ((-) TAN 67) or no effect (DPDPE) on the affinities of other delta selective agonists. The mutation had opposite effects on the intrinsic activities of agonists belonging to different chemical classes. The effects of the mutation on agonist affinities and potencies were independent from its effects on the intrinsic activities of the agonists. Maximal stimulation of [35S]GTPgammaS binding by SNC 80 was 2-fold higher in W284L mutant cell membranes than in wild type hDOR/CHO cell membranes, despite lower receptor expression levels in the W284L/CHO cells. The binding affinity of SNC 80 however, was significantly reduced (15-fold and 30-fold in the absence or presence of sodium+GDP respectively) in W284L/CHO cell membranes relative to wild type hDOR/CHO membranes. Conversely, the Emax of (-)TAN 67 in the [35S]GTPgammaS binding assay was markedly reduced (0.6-fold of that of the wild type) with only a slight (6-fold) reduction in its binding affinity. The affinity and intrinsic activity of DPDPE on the other hand remained unchanged at the W284L mutant hDOR. The mutation had similar effects on the affinities potencies and intrinsic activities of (-)TAN 67 and SB 219825. The results indicate that delta opioid agonists of different chemical classes use specific conformations for G protein activation.     

Dimeric pentapeptide enkephalin: a novel probe of delta opiate receptors

A dimeric pentapeptide enkephalin (DPE2) consisting of two molecules of [D-Ala 2, Leu 5] enkephalin linked at C-terminal leucine with ethylenediamine, (H-Tyr-D-Ala-Gly-Phe-Leu-NH-Ch2)2 is a bivalent ligand for the delta enkephalin receptors of rat brain and neuroblastoma-glioma hybrid (NG108-15) cells. This new enkephalin analog shows dramatically increased affinity in radioligand assays using whole brain membranes when delta but not mu specific radioligands are employed. When membranes from NG108-15 cells are used, the dimer shows greatly increased activity irrespective of the mu or delta specificity of the tracer. The dimer DPE2 shows a four-fold, "sodium shift" in its IC50 for competition with [3H]naloxone, suggestive of agonist behavior. Agonist activity was confirmed by demonstrating that DPE2 inhibits cyclic AMP production in prostaglandin E1 stimulated NG108-15 cells, and by demonstrating very high potency in the mouse vas deferens bioassay. DPE2 binds to the same delta sites as the delta-selective monomer [D-Ala2, D-Leu5] enkephalin, since the two ligands show complete crossdisplacement. Radiolabeled 3H-DPE2 shows a five-fold higher affinity constant, a 2.5-fold higher association rate constant, and a two-fold lower dissociation rate than the monomer. These results are consistent with the hypothesis that the dimeric pentapeptide enkephalin can bridge two delta receptors. This enkephalin dimer provides a valuable new probe of opiate receptors and their organization in cell membranes.     

Enkephalin and alpha-adrenergic receptors: evidence for both common and differentiable regulatory pathways and down-regulation of the enkephalin receptor

Several clones of neuroblastoma-glioma NG108-15 hybrid cells were used to reveal whether the regulation of opiate receptor density interacts with the regulation of alpha-adrenergic or acetyl-choline receptors. Low density of alpha-adrenergic receptors in 3 selected clones was accompanied with similar reduction in the density of enkephalin receptors but not in muscarinic acetyl-choline receptors. Yet opiate antagonists that increased the number of opiate receptors in the parent NG108-15 cells in a stereospecific manner had no similar effect on the number of alpha-adrenergic receptors. Moreover, the stable enkephalin analogue D-ala-2-methionine enkephalinamide, but not the opiate alkaloid morphine, decreased the binding of 3H-DAMEA to the membranes and induced down-regulation of enkephalin receptors. Yet DAMEA had no effect on the binding of the alpha-adrenergic antagonist 3H-yohimbine. The study suggests that alpha-adrenergic and enkephalin receptors may share some common regulatory pathways but opiate peptides and antagonists selectively decrease or increase the density of enkephalin receptors, respectively, with no effect on alpha-adrenergic receptor density.     

Role of steric interactions in the delta opioid receptor selectivity of [D-Pen2, D-Pen5]enkephalin

In order to assess the individual effects of each of the 3-methyl groups in residue 2 of [D-Pen2, D-Pen5]enkephalin on binding affinity to mu and delta opioid receptors, (2S,3S)methylcysteine ((3S)Me-D-Cys) and (2S,3R)methylcysteine ((3R)Me-D-Cys) were synthesized and incorporated into the analogs, [(3S)Me-D-Cys2, D-Pen5] enkephalin and [(3R)Me-D-Cys2, D-Pen5]enkephalin. Of these analogs, [(3S)Me-D-Cys2, D-Pen5]enkephalin appears from 1H n.m.r. spectra to assume a conformation similar to those of [D-Pen2, D-Pen5]enkephalin and the less delta receptor-selective, but more potent, [D-Cys2, D-Pen5]enkephalin. Assessment of binding affinity to mu and delta receptors revealed that [(3S)Me-D-Cys2, D-Pen5]enkephalin exhibits delta receptor affinity intermediate between [D-Pen2, D-Pen5]enkephalin and [D-Cys2, D-Pen5]enkephalin while its mu receptor affinity is similar to that of [D-Cys2, D-Pen5]enkephalin. These results suggest that, for [D-Pen2, D-Pen5]enkephalin, adverse steric interactions between the D-Pen2 pro-R methyl group and the mu receptor binding site lead to the low mu receptor binding affinity observed for this analog. By contrast, both the pro-R and pro-S D-Pen2 methyl groups lead to minor steric interactions which contribute to the somewhat lower delta receptor affinity of this compound.     

Gbetagamma affinity for bovine rhodopsin is determined by the carboxyl-terminal sequences of the gamma subunit

Two native betagamma dimers, beta(1)gamma(1) and beta(1)gamma(2), display very different affinities for receptors. Since these gamma subunits differ in both primary structure and isoprenoid modification, we examined the relative contributions of each to Gbetagamma interaction with receptors. We constructed baculoviruses encoding gamma(1) and gamma(2) subunits with altered CAAX (where A is an aliphatic amino acid) motifs to direct alternate or no prenylation of the gamma chains and a set of gamma(1) and gamma(2) chimeras with the gamma(2) CAAX motif at the carboxyl terminus. All the gamma constructs coexpressed with beta(1) in Sf9 cells yielded beta(1)gamma dimers, which were purified to near homogeneity, and their affinities for receptors and Galpha were quantitatively determined. Whereas alteration of the isoprenoid of gamma(1) from farnesyl to geranylgeranyl and of gamma(2) from geranylgeranyl to farnesyl had no impact on the affinities of beta(1)gamma dimers for Galpha(t), the non-prenylated beta(1)gamma(2) dimer had significantly diminished affinity. Altered prenylation resulted in a <2-fold decrease in affinity of the beta(1)gamma(2) dimer for rhodopsin and a <3-fold change for the beta(1)gamma(1) dimer. In each case with identical isoprenylation, the beta(1)gamma(2) dimer displayed significantly greater affinity for rhodopsin compared with the beta(1)gamma(1) dimer. Furthermore, dimers containing chimeric Ggamma chains with identical geranylgeranyl modification displayed rhodopsin affinities largely determined by the carboxyl-terminal one-third of the protein. These results indicate that isoprenoid modification of the Ggamma subunit is essential for binding to both Galpha and receptors. The isoprenoid type influences the binding affinity for receptors, but not for Galpha. Finally, the primary structure of the Ggamma subunit provides a major contribution to receptor binding of Gbetagamma, with the carboxyl-terminal sequence conferring receptor selectivity.     

AT4 receptor structure-binding relationship: N-terminal-modified angiotensin IV analogues

The effect of structural changes in the N-terminal amino acid of AIV, with respect to AT₄ receptor binding, was examined by competition with [¹²⁵I]AIV in bovine adrenal membranes. Analogues with modifications of the first residue -amino group possessed lower affinities than the primary amine-containing parent compound. Peptides with a residue 1 -carbon in the conformation exhibited poor affinity for the AT₄ receptor. Modifications of the residue 1 R-group demonstrate that a straight chain aliphatic moiety containing four carbons is optimal for receptor-ligand binding, as evidenced by the extremely high affinity of [Nle¹]AIV (K_i = 3.59±0.51 pM). Replacement of the 1–2 peptide bond of AIV with the methylene bond isostere Ψ (CH₂-NH), increased the K_i approximately fivefold, indicating that the peptide bond may be replaced wihle maintaining relatively high-affinity receptor binding.