Dermorphin‐based potential affinity labels for µ‐opioid receptors*

Abstract: Dermorphin and [Lys⁷]dermorphin, selective µ‐opioid receptor ligands originating from amphibian skin, have been modified with various electrophiles in either the ‘message’ or ‘address’ sequences as potential peptide‐based affinity labels for µ‐receptors. Introduction of the electrophilic isothiocyanate and bromoacetamide groups on the para position of Phe³ and Phe⁵ was accomplished by incorporating Fmoc‐Phe(p‐NHAlloc) into the peptide followed by selective deprotection and modification. The corresponding amine‐containing peptides were also prepared. The pure peptides were evaluated in radioligand binding experiments using Chinese hamster ovary (CHO) cells expressing µ‐ and δ‐opioid receptors. In dermorphin, introduction of the electrophilic groups in the ‘message’ domain lowered the binding affinity by > 1000‐fold; only [Phe(p‐NH₂)³]dermorphin retained nanomolar affinity for µ‐receptors. Modifications in the ‘address’ region of both dermorphin and [Lys⁷]dermorphin were relatively well tolerated. In particular, [Phe(p‐NH₂)⁵,Lys⁷]dermorphin showed similar affinity to dermorphin, with almost 2‐fold higher selectivity for µ‐receptors. [Phe(p‐NHCOCH₂Br)⁵]‐ and [Phe(p‐NHCOCH₂Br)⁵,Lys⁷]dermorphin exhibited relatively high affinity (IC₅₀ = 27.7 and 15.1 nm , respectively) for µ‐receptors. However, neither of these peptides inhibited [³H]DAMGO binding in a wash‐resistant manner.     

An affinity label for δ‐opioid receptors derived from [d‐Ala2]deltorphin I*

Abstract: A series of potential affinity label derivatives of the amphibian opioid peptide [d ‐Ala²]deltorphin I were prepared by incorporation at the para position of Phe³ (in the ‘message’ sequence) or Phe⁵ (in the ‘address’ sequence) of an electrophilic group (i.e. isothiocyanate or bromoacetamide). The introduction of the electrophile was accomplished by incorporating Fmoc‐Phe(p‐NHAlloc) into the peptide, followed later in the synthesis by selective deprotection of the Alloc group and modification of the resulting amine. While para substitution decreased the δ‐opioid receptor affinity, selected analogs retained nanomolar affinity for δ receptors. [d ‐Ala²,Phe(p‐NCS)³]deltorphin I exhibited moderate affinity (IC₅₀ = 83 nm ) and high selectivity for δ receptors, while the corresponding amine and bromoacetamide derivatives showed pronounced decreases in δ‐receptor affinity (80‐ and >1200‐fold, respectively, compared with [d ‐Ala²]deltorphin I). In the ‘address’ sequence, the Phe(p‐NH₂)⁵ derivative showed the highest δ‐receptor affinity (IC₅₀ = 32 nm ), while the Phe(p‐NHCOCH₂Br)⁵ and Phe(p‐NCS)⁵ peptides displayed four‐ and tenfold lower δ‐receptor affinities, respectively. [d ‐Ala²,Phe(p‐NCS)³]deltorphin I exhibited wash‐resistant inhibition of [³H][d ‐Pen²,D‐Pen⁵]enkephalin (DPDPE) binding to δ receptors at a concentration of 80 nm . [d ‐Ala², Phe(p‐NCS)³]deltorphin I represents the first affinity label derivative of one of the potent and selective amphibian opioid peptides, and the first electrophilic affinity label derivative of an agonist containing the reactive functionality in the ‘message’ sequence of the peptide.     

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.     

Novel endomorphin‐2 analogs with μ‐opioid receptor antagonist activity

Abstract: A series of position 4‐substituted endomorphin‐2 (Tyr‐Pro‐Phe‐Phe‐NH₂) analogs containing 3‐(1‐naphthyl)‐alanine (1‐Nal) or 3‐(2‐naphthyl)‐alanine (2‐Nal) in l ‐ or d ‐configuration, was synthesized. The opioid activity profiles of these peptides were determined in the μ‐opioid receptor representative binding assay and in the Guinea‐Pig Ileum assay/Mouse Vas Deferens assay (GPI/MVD) bioassays in vitro, as well as in the mouse hot‐plate test of analgesia in vivo. In the binding assay the affinity of all new analogs for the μ‐opioid receptor was reduced compared with endomorphin‐2. The two most potent analogs were [d ‐1‐Nal⁴]‐ and [d ‐2‐Nal⁴]endomorphin‐2, with IC₅₀ values 14 ± 1.25 and 19 ± 2.1 nm , respectively, compared with 1.9 ± 0.21 nm for endomorphin‐2. In the GPI assay these analogs were found to be weak antagonists and they were inactive in the MVD assay. The in vitro GPI assay results were in agreement with those obtained in the in vivo hot‐plate test. Antinociception induced by endomorphin‐2 was reversed by concomitant intracerebroventricula (i.c.v.) administration of [d ‐1‐Nal⁴]‐ and [d ‐2‐Nal⁴]‐endomorphin‐2, indicating that these analogs were μ‐opioid antagonists. Their antagonist activity was compared with that of naloxone. At a dose 5 μg per animal naloxone almost completely inhibited antinociceptive action of endomorphin‐2, while [d ‐1‐Nal⁴]endomorphin‐2 in about 46%.     

Binding of endomorphin‐2 to μ‐opioid receptors in experimental mouse mammary adenocarcinoma

Abstract: Endomorphin‐2 (Tyr‐Pro‐Phe‐Phe‐NH₂) binds with high affinity and selectivity to the μ‐opioid receptor. In the present study, [¹²⁵I]endomorphin‐2 has been used to characterize μ‐opioid‐binding sites on transplantable mouse mammary adenocarcinoma cells. Cold saturation experiments performed with [¹²⁵I]endomorphin‐2 (1 nm ) show biphasic binding curves in Scatchard coordinates. One component represents high affinity and low capacity (K_d = 18.79 ± 1.13 nm , B_max = 635 ± 24 fmol/mg protein) and the other shows low affinity and higher capacity (K_d = 7.67 ± 0.81 μm , B_max = 157 ± 13 pmol/mg protein) binding sites. The rank order of agonists competing for the [¹²⁵I]endomorphin‐2 binding site was [d ‐1‐Nal³]morphiceptin > endomorphin‐2 ? [d ‐Phe³]morphiceptin > morphiceptin > [d ‐1‐Nal³]endomorphin‐2, indicating binding of these peptides to μ‐opioid receptors. The uptake of ¹³¹I‐labeled peptides administered intraperitoneally to tumor‐bearing mice was also investigated. The highest accumulation in the tumor was observed for [d ‐1‐Nal³]morphiceptin, which reached the value of 8.19 ± 1.14% dose/g tissue.     

Cyclic enkephalin analogs containing various para‐substituted phenylalanine derivatives in place of Tyr1 are potent opioid agonists*

Abstract: The cyclic enkephalin analog H‐Tyr‐c[d ‐Cys‐Gly‐Phe(pNO₂)‐d ‐Cys]NH₂ is a highly potent opioid agonist with IC₅₀s of 35 pm and 19 pm in the guinea‐pig ileum (GPI) and mouse vas deferens (MVD) assays, respectively. The Phe¹‐analog of this peptide showed 370‐fold and 6790‐fold lower agonist potency in the GPI and MVD assays, respectively, indicating the importance of the Tyr¹ hydroxyl‐group in the interaction with μ and δ opioid receptors. In the present study, the effect of various substituents (‐NH₂, ‐NO₂, ‐CN, ‐CH₃, ‐COOH, ‐COCH₃, ‐CONH₂) introduced in the para‐position of the Phe¹‐residue of H‐Phe‐c[d ‐Cys‐Gly‐Phe(pNO₂)‐d ‐Cys]NH₂ on the in vitro opioid activity profile was examined. Most analogs showed enhanced μ and δ agonist potencies in the two bioassays, except for the Phe(pCOOH)¹‐analog, which was weakly active, probably as a consequence of the negative charge. The most potent compounds were the Phe(pCOH₃)¹‐ and the Phe(pCONH₂)¹‐analogs. The latter compound showed subnanomolar μ and δ agonist potencies and represents the most potent enkephalin analog lacking the Tyr¹ hydroxyl‐group reported to date. Taken together, these results indicate that various substituents introduced in the para‐position of Phe¹ enhance opioid activity via hydrogen bonding or hydrophobic interactions with the receptor. Comparison with existing structure‐activity relationship on phenolic hydroxyl replacements in morphinans indicates that these nonpeptide opiates and some of the cyclic enkephalin analogs described here may have different modes of binding to the receptor.     

Conformational comparison of µ‐selective endomorphin‐2 with its C‐terminal free acid in DMSO solution,by 1H NMR spectroscopy and molecular modeling calculation

Abstract: In order to make clear the structural role of the C‐terminal amide group of endomorphin‐2 (EM2, H‐Tyr‐Pro‐Phe‐Phe‐NH₂), an endogenous µ‐receptor ligand, in the biological function, the solution conformations of endomorphin‐2 and its C‐terminal free acid (EM2OH, H‐Tyr‐Pro‐Phe‐Phe‐OH), studied using two‐dimensional ¹H NMR measurements and molecular modeling calculations, were compared. Both peptides were in equilibrium between the cis and trans isomers around the Tyr‐Pro ω bond in a population ratio of ≈ 1 : 2. The lack of significant temperature and concentration dependence of NH protons suggested that the NMR spectra reflected the conformational features of the respective molecules themselves. Fifty possible 3D structures for the each isomer were generated by the dynamical simulated annealing method under the proton?proton distance constraints derived from the ROE cross‐peaks. These energy‐minimized conformers, which were all in the φ torsion angles estimated from J_NHCαH coupling constants within ± 30°, were then classified in groups one or two according to the folding backbone structures. All trans and cis EM2 conformers adopt an open conformation in which their extended backbone structures are twisted at the Pro²–Phe³ moiety. In contrast, the trans and cis conformers of EM2OH show conformational variation between the ‘bow’‐shaped extended and folded backbone structures, although the cis conformers of its zwitterionic form are refined into the folded structure of the close disposition of C‐ and N‐terminal groups. These results indicate clearly that the substitution of carboxyl group for C‐terminal amide group makes the peptide flexible. The conformational requirement for µ‐receptor activation has been discussed based on the active form proposed for endomorphin‐1 and by comparing conformational features of EM2 and EM2OH.     

A solid‐phase synthetic strategy for the preparation of peptide‐based affinity labels: synthesis of dynorphin A analogs

Abstract: Solid‐phase synthetic methodology was developed for the preparation of peptide‐based affinity labels. The initial peptides synthesized were dynorphin A (Dyn A) analogs [Phe(p‐X)⁴,d ‐Pro¹⁰]Dyn A(1–11)NH₂ containing isothiocyanate (X = –N=C=S) and bromoacetamide (X = –NHCOCH₂Br) groups. The peptides were assembled on solid supports using Fmoc‐protected amino acids, and the side chain amine to be functionalized, Phe(p‐NH₂), was protected by the Alloc (allyloxycarbonyl) group. Following removal of the Alloc group by palladium(0), the reactive isothiocyanate and bromoacetamide functionalities were successfully introduced while the peptides were still attached to the resin. Synthesis of these peptides was carried out on polystyrene (PS) and polyethylene glycol–polystyrene (PEG–PS) resins containing the PAL [peptide amide linker, 5‐(4‐Fmoc‐aminomethyl‐3,5‐dimethoxyphenoxy)valeric acid] linker. Both the rate of Alloc deprotection and the purity of the crude affinity‐labeled peptides obtained were found to be dependent on the resin used for peptide assembly.     

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.     

Cyclic Opioid Peptide Agonists and Antagonists Obtained Via Ring‐Closing Metathesis

The opioid peptide H‐Tyr‐c[D‐Cys‐Phe‐Phe‐Cys]NH₂ cyclized via a methylene dithiother is a potent and selective μ opioid agonist (Przydial M.J. et al., J Peptide Res, 66, 2005, 255). Dicarba analogues of this peptide with Tyr, 2′,6′‐dimethyltyrosine (Dmt), 3‐[2,6‐dimethyl‐4‐hydroxyphenyl)propanoic acid (Dhp) or (2S)‐2‐methyl‐3‐(2,6‐dimethyl‐4‐hydroxyphenyl)propanoic acid [(2S)‐Mdp] in the 1‐position were prepared. The peptides were synthesized on solid‐phase by substituting d ‐allylglycine and (2S)‐2‐amino‐5‐hexenoic acid in position 2 and 5, respectively, followed by ring‐closing metathesis. Mixtures of cis and trans isomers of the resulting olefinic peptides were obtained, and catalytic hydrogenation yielded the saturated –CH₂–CH₂– bridged peptides. All six Tyr¹‐ and Dmt¹‐dicarba analogues retained high μ and δ opioid agonist potency and showed only slight or no preference for μ over δ receptors. As expected, the six Dhp¹‐ and (2S)‐Mdp¹‐dicarba analogues turned out to be μ opioid antagonists but, surprisingly, displayed a range of different efficacies (agonism, partial agonism or antagonism) at the δ receptor. The obtained results indicate that the μ versus δ receptor selectivity and the efficacy at the δ receptor of these cyclic peptides depend on distinct conformational characteristics of the 15‐membered peptide ring structure, which may affect the spatial positioning of the exocyclic residue and of the Phe³ and Phe⁴ side chains.     

Potent Opioid Peptide Agonists Containing 4′‐[N‐((4′‐phenyl)‐phenethyl)carboxamido]phenylalanine (Bcp) in Place of Tyr

Analogues of the opioid peptides H‐Tyr‐c[d ‐Cys‐Gly‐Phe(pNO₂)‐d ‐Cys]NH₂ (non‐selective), H‐Tyr‐d ‐Arg‐Phe‐Lys‐NH₂ (μ‐selective) and dynorphin A(1‐11)‐NH₂ (κ‐selective) containing 4′‐[N‐((4′‐phenyl)‐phenethyl)carboxamido]phenylanine (Bcp) in place of Tyr¹ were synthesized. All three Bcp¹‐opioid peptides retained high μ opioid receptor binding affinity, but showed very significant differences in the opioid receptor selectivity profiles as compared with the corresponding Tyr¹‐containing parent peptides. The cyclic peptide H‐Bcp‐c[d ‐Cys‐Gly‐Phe(pNO₂)‐d ‐Cys]NH₂ turned out to be an extraordinarily potent, μ‐selective opioid agonist, whereas the Bcp¹‐analogue of dynorphin A(1‐11)‐NH₂ displayed partial agonism at the μ receptor. The obtained results suggest that the large biphenylethyl substituent contained in these compounds may engage in a hydrophobic interaction with a receptor subsite and thereby may play a role in the ligand’s ability to induce a specific receptor conformation or to bind to a distinct receptor conformation in a situation of conformational receptor heterogeneity.     

Biological and conformational study of β‐substituted prolines in MT‐II template: steric effects leading to human MC5 receptor selectivity*

Abstract: To investigate the molecular basis for the interaction of the χ‐constrained conformation of melanotropin peptide with the human melanocortin receptors, a series of β‐substituted proline analogs were synthesized and incorporated into the Ac‐Nle‐c[Asp‐His‐d ‐Phe‐Arg‐Trp‐Lys]‐NH₂ (MT‐II) template at the His⁶ and d ‐Phe⁷ positions. It was found that the binding affinities generally diminished as the steric bulk of the p‐substituents of the 3‐phenylproline residues increased. From (2S, 3R)‐3‐phenyl‐Pro⁶ to (2S, 3R)‐3‐(p‐methoxyphenyl)‐Pro⁶ analogs the binding affinity decreased 23‐fold at the human melanocortin‐3 receptor (hMC3R), 17‐fold at the hMC4R, and eight‐fold at the hMC5R, but selectivity for the hMC5R increased. In addition, the substitution of the d ‐Phe⁷ residue with a (2R, 3S)‐3‐phenyl‐Pro resulted in greatly reduced binding affinity (10³–10⁵) at these melanocortin receptors. Macromodel's Large Scale Low Mode (LLMOD) with OPLS‐AA force field simulations revealed that both MT‐II and SHU‐9119 share a similar backbone conformation and topography with the exception of the orientation of the side chains of d ‐Phe⁷/d ‐Nal (2′)⁷ in χ space. Introduction of the dihedrally constrained phenylproline analogs into the His⁶ position (analogs 2 – 6 ) caused topographical changes that might be responsible for the lower binding affinities. Our findings indicate that hMC3 and hMC4 receptors are more sensitive to steric effects and conformational constraints than the hMC5 receptor. This is the first example for melanocortin receptor selectivity where the propensity of steric interactions in χ space of β‐modified Pro⁶ analogs of MT‐II has been shown to play a critical role for binding as well as bioefficacy of melanotropins at hMC3 and hMC4 receptors, but not at the hMC5 receptor.     

Structure–activity relationships of arodyn,a novel acetylated kappa opioid receptor antagonist*,†

Abstract: We previously reported that the novel dynorphin A (Dyn A, Tyr‐Gly‐Gly‐Phe‐Leu‐Arg‐Arg‐Ile‐Arg‐Pro‐Lys‐Leu‐Lys‐Trp‐Asp‐Asn‐Gln) analog arodyn (Ac[Phe^1,2,3,Arg⁴,d ‐Ala⁸]Dyn A‐(1–11)NH₂, Bennett, M.A., Murray, T.F. & Aldrich, J.V. (2002) J. Med. Chem. vol. 45, pp. 5617–5619) is a κ opioid receptor‐selective peptide [K_i(κ) = 10 nm , K_i ratio (κ/μ/δ) = 1/174/583] which exhibits antagonist activity at κ opioid receptors. In this study, a series of arodyn analogs was prepared and evaluated to explore the structure–activity relationships (SAR) of this peptide; this included an alanine scan of the entire arodyn sequence, sequential isomeric d ‐amino acid substitution in the N‐terminal ‘message’ sequence, NMePhe substitution individually in positions 1–3, and modifications in position 1. The results for the Ala‐substituted derivatives indicated that Arg⁶ and Arg⁷ are the most important residues for arodyn's nanomolar binding affinity for κ opioid receptors. Ala substitution of the other basic residues (Arg⁴, Arg⁹ and Lys¹¹) resulted in lower decreases in affinity for κ opioid receptors (three‐ to fivefold compared with arodyn). Of particular interest, while [Ala¹⁰]arodyn exhibits similar κ opioid receptor binding as arodyn, it displays higher κ vs. μ opioid receptor selectivity [K_i ratio (κ/μ) = 1/350] than arodyn because of a twofold loss in affinity at μ opioid receptors. Surprisingly, the Tyr¹ analog exhibits a sevenfold decrease in κ opioid receptor affinity, indicating that arodyn displays significantly different SAR than Dyn A; [Tyr¹]arodyn also unexpectedly exhibits inverse agonist activity in the adenylyl cyclase assay using Chinese hamster ovary cells stably expressing κ opioid receptors. Substitution of NMePhe in position 1 gave [NMePhe¹]arodyn which exhibits high affinity [K_i(κ) = 4.56 nm ] and exceptional selectivity for κ opioid receptors [K_i ratio (κ/μ/δ) = 1/1100/>2170]. This peptide exhibits antagonistic activity in the adenylyl cyclase assay, reversing the agonism of 10 nm Dyn A‐(1–13)NH₂. Thus [NMePhe¹]arodyn is a highly κ opioid receptor‐selective antagonist that could be a useful pharmacological tool to study κ opioid receptor‐mediated activities.     

Importance of the C‐terminal phenylalanine of gastrin for binding to the human CCK2 receptor

Abstract: The importance of the C‐terminal Phe of gastrin and structural requirements at position 17 for binding to the human CCK₂ receptor were assessed using analogs of [Leu¹⁵]G(11?17). The following peptides were synthesized, Ac[Leu¹⁵]G(11?17), Ac[Leu¹⁵]G(11?16)NH₂, [Leu¹⁵]G(11?17), [Leu¹⁵,Ala¹⁷]G(11?17), [Leu¹⁵,Abu¹⁷]G(11?17), [Leu¹⁵,Val¹⁷]G(11?17), [Leu¹⁵,Leu¹⁷]G(11?17), [Leu¹⁵,Cha¹⁷]G(11?17), [Leu¹⁵,Trp¹⁷]G(11?17), [Leu¹⁵,Tic¹⁷]G(11?17), [Leu¹⁵, d ‐Phe¹⁷]G(11?17) and [Leu¹⁵,p‐X‐Phe¹⁷]G(11?17), where X = F, Cl, Br, I, OH, CH₃, NH₂ and NO_2. Competition binding experiments with [³H]CCK‐8 were performed using human CCK₂ receptors stably expressed in CHO cells. Phe¹⁷ was shown to be important for binding. A hydrophobic side‐chain larger than Leu is required at position 17 but aromaticity does not appear to be essential. Constraint of the aromatic side‐chain either in the g(+) or g(–) conformation, as in the case of Tic, results in a significant decrease in affinity. In addition, the peptide conformation induced by incorporation of d ‐Phe decreases binding. The size and electron withdrawing/donating properties of the para substituent are not important for interaction with the receptor. The current study shows that the use of des‐Phe analogs of gastrin is not a viable strategy for development of antagonists for the human CCK₂ receptor.     

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.     

Development of highly potent and ective dynorphin A analogues as new medicines

Abstract: Dynorphin A (Dyn A), a 17 amino acid peptide H‐Tyr‐Gly‐Gly‐Phe‐Leu‐Arg‐Arg‐Ile‐Arg‐Pro‐Lys‐Leu‐Lys‐Trp‐Asp‐Asn‐Gln‐OH, is a potent opioid peptide which interacts preferentially with κ‐opioid receptors. Research in the development of selective and potent opioid peptide ligands for the κ‐receptor is important in mediating analgesia. Several cyclic disulphide bridge‐containing peptide analogues of Dyn A, which were conformationally constrained in the putative message or address segment of the opioid ligand, were designed, synthesized and assayed. To further investigate the conformational and topographical requirements for the residues in positions 5 and 11 of these analogues, a systematic series of Dyn A_1?11‐NH₂ cyclic analogues incorporating the sulphydryl‐containing amino acids l ‐ and d ‐Cys and l ‐ and d ‐Pen in positions 5 and 11 were synthesized and assayed. Cyclic lactam peptide analogues were also synthesized and assayed. Several of these cyclic analogues, retained the same affinity and selectivity (vs. the μ‐ and δ‐receptors) as the parent Dyn A_1?11‐NH₂ peptide in the guinea‐pig brain (GPB), but exhibited a much lower activity in the guinea‐pig ileum (GPI), thus leading to centrally vs. peripherally selective peptides. Studies of the structure–activity relationship of Dyn A peptide provide new insights into the importance of each amino acid residue (and their configurations) in Dyn A analogues for high potency and good selectivity at κ‐opioid receptors. We report herein the progress towards the development of Dyn A peptide ligands, which can act as agonists or antagonists at cell surface receptors that modulate cell function and animal behaviour using various approaches to rational peptide ligand‐based drug design.     

Design of high affinity cyclic pentapeptide ligands for κ‐opioid receptors

Abstract: Using results from our previously reported cyclic opioid peptide series and reliable models for μ‐, δ‐, and κ‐opioid receptors (MOR, DOR, and KOR, respectively) and their complexes with peptide ligands, we have designed and synthesized a series of cyclic pentapeptides of structure Tyr‐c[d ‐Cys‐Phe‐Phe‐X]‐NH₂, cyclized via disulfide, methylene, or ethylene dithioethers, and where X = d ‐ or l ‐Cys; or d ‐ or l ‐penicillamine (Pen; β,β‐dimethylcysteine). Determination of binding affinities to MOR, DOR, and KOR revealed that members of this series with X = d ‐ or l ‐Cys display KOR affinities in the low nanomolar range, demonstrating that a ‘DPDPE‐like’ tetrapeptide scaffold is suitable not only for DOR and MOR ligands, but also for KOR ligands. The cyclic pentapeptides reported here are not, however, selective for KOR, rather they display significant selectivity and high affinity for MOR. Indeed, peptide 8 , Tyr‐c[d ‐Cys‐Phe‐Phe‐Cys]‐NH₂‐cyclized via a methylene dithioether, shows picomolar binding affinity for MOR ( = 16 pm ) with more than 100‐fold selectivity for MOR vs. DOR or KOR, and may be of interest as a high affinity, high selectivity MOR ligand. Nonetheless, the high affinity KOR peptides in this series represent excellent leads for the development of structurally related, selective KOR ligands designed to exploit structurally specific features of KOR, MOR, and DOR.     

Design of chimeric peptide ligands to galanin receptors and substance P receptors

Several chimeric peptides were synthesized and found to be high-affinity ligands for both galanin and substance P receptors in membranes from the rat hypothalamus. The peptide galantide, composed of the N-terminal part of galanin and C-terminal part of substance P (SP), galanin-(1-12)-Pro-SP-(5-11) amide, which is the first galanin antagonist to be reported, recognizes two classes of galanin binding sites (K_D(1)<0.1 nM and K_D(2)∼ 6 nM) in the rat hypothalamus, while it appears to bind to a single population of SP receptors (K_D∼ 40 nM). The chimeric peptide has higher affinity towards galanin receptors than the endogenous peptide galanin-(1-29) (KD ∼ 1 nm ) or its N-terminal fragment galanin-(1-13) (K_D∼ 1,nm ), which constitutes the A′-terminus of the chimeric peptide. Galantide has also higher affinity for the SP receptors than the C-terminal SP fragment-(4-11) amide (K_D= 0.4μm ), which constitutes its C-terminal portion. Substitution of amino acid residues, which is of importance for recognition of galanin by galanin receptors, such as [Trp²], in the galanin portion of the chimeric peptide or substitution of ([Phe⁷] or [Met¹¹]-amide) in the SP portion of chimeric peptide both cause significant loss in affinity of the analogs of galantide for both the galanin- and the SP-receptors. These results suggest that the high affinity of the chimeric peptide, galantide, may in part be accounted for by simultaneous recognition/binding to both receptors. In line with this suggestion is the finding that the binding of the chimeric ligands to the galanin receptor is strongly influenced by the presence of SP (1 μm ) or spantide (1 μm ). We have performed the synthesis and binding studies with 11 chimeric peptides, all composed of the N-terminal galanin-(1-13) fragment or of its analogs, linked to the C-terminal portion of SP or its peptide antagonist, spantide. Our results, similar to earlier reports on chimeric peptides, suggest that high-affinity ligands to peptide receptors can be produced by linking biologically active N-terminal and C-terminal portions of peptides via linkers, enabling a) independent recognition of the chimeric peptide by the relevant receptors and b) intramolecular interactions between the joined N- and C-terminal peptide fragments. These two phenomena may also explain why some of the chimeric peptides have higher affinity than the endogenous peptide(s) and why galantide, and some of its analogs presented here, behave(s) as a galanin receptor antagonist(s).     

The chemical syntheses and bioactivities of novel peptide‐based endothelin antagonists

Abstract: Endothelin antagonists, novel tripeptides containing a series of unnatural amino acids, were synthesized and characterized. A linear peptide BQ‐485, perhydroazepin‐1‐yl‐l ‐leucyl (1)‐d ‐tryptophanyl (2)‐d ‐tryptophan (3), was selected as the parent compound. The introduction of d ‐Phe derivatives into these peptidic ET antagonists resulted in potent activity against the contraction of rat aortic smooth muscles induced by ET‐1 (10 nm ) which activated the ET receptors. Among these compounds, 15 tripeptides had high enough antagonistic activity at the level of 10^?7 mol/L (IC₅₀). The activity of three compounds was 10^?6 mol/L (IC₅₀). These HIM‐CO‐Leu‐d ‐Trp‐d ‐Phe(‐R)‐OH compounds as ET_A antagonists may provide a tool for the development of therapeutic agents in the treatment of putative ET‐1‐related disorders.     

Analogs of Substance P modified at the C‐terminus which are both agonist and antagonist of the NK‐1 receptor depending on the second messenger pathway

Abstract: The initial goal of this study was to analyze, using photolabeling, the interactions between Substance P and its tachykinin NK‐1 receptor. Therefore, the photoreactive amino acid para‐benzoyl‐phenylalanine (pBzl)Phe was incorporated into the Substance P sequence from position 4 to 11 leading to Bapa⁰[(pBzl)Phe^x]SP analogs. Biotinyl sulfone‐5‐aminopentanoic acid (Bapa) was introduced in order to purify the covalent complex. These photoreactive SP analogs were first assayed for their affinity for the two binding sites associated with the NK‐1 receptor, as well as for their potency in activating the phospholipase C and adenylate cyclase pathways. All analogs photoreactive from position 4 to 11 have moderate to high affinity for the two NK‐1 receptor‐binding sites, except for the analog modified at position 7. This affinity could be correlated to their potency to activate the phospholipase C and adenylate cyclase pathways, except for the analog photoreactive at position 11. Bapa⁰[(pBzl)Phe¹¹]SP was found to be an agonist in the phospholipase C pathway and an antagonist in the adenylate cyclase pathway, other analogs modified at position 11 were therefore analyzed. Among these, Bapa⁰[Pro⁹, (pBzl)Hcy(O₂)¹¹]SP is a partial agonist, whereas Bapa⁰[Hcy(ethylaminodansyl)¹¹]SP is a full agonist in the phospholipase C pathway, the two analogs being antagonist in the adenylate cyclase pathway. These results show that analogs of SP can be simultaneously agonist at one binding site and antagonist at the other binding site associated with the NK‐1 receptor.