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.     

Synthesis and activity of the melanocortin Xaa‐d‐Phe‐Arg‐Trp‐NH2 tetrapeptides with amide bond modifications

Abstract: The melanocortin receptor (MCR) pathway has been identified as participating in several physiologically important pathways including pigmentation, energy homeostasis, inflammation, obesity, hypertension, and sexual function. All the endogenous MCR agonists contain a core His‐Phe‐Arg‐Trp sequence identified as important for receptor molecular recognition and stimulation. Several structure–activity studies using the Ac‐His‐d ‐Phe‐Arg‐Trp‐NH₂ tetrapeptide template have been performed in the context of modifying N‐terminal ‘capping’ groups and amino acid constituents. Herein, we report the synthesis and pharmacologic characterization of modified Xaa‐d ‐Phe‐Arg‐Trp‐NH₂ (Xaa = His or Phe) melanocortin tetrapeptides (N‐site selective methylation, permethylation, or amide bond reduction) at the mouse MC1, MC3, MC4 and MC5 receptors. The modified peptides generated in this study resulted in equipotent or reduced MCR potency when compared with control ligands. The reduced amide bond analog of the Phe‐d ‐Phe‐Arg‐Trp‐NH₂ peptide converted its agonist activity into an antagonistic at the central mMC3 and mMC4 receptors involved in the regulation of energy homeostasis, while retaining full agonist activity at the peripheral MC1 and MC5 receptors.     

Modified melanocortin tetrapeptide Ac‐His‐dPhe‐Arg‐Trp‐NH2 at the arginine side chain with ureas and thioureas

Abstract: The Ac‐His‐d Phe‐Arg‐Trp‐NH₂ tetrapeptide is a nonselective melanocortin agonist and replacement of Arg in the tetrapeptide with acidic, basic or neutral amino acids results in reduced potency at the melanocortin receptor (MCR) isoforms (MC1R and MC3–5R). To determine the importance of the positive charge and the guanidine moiety for melanocortin activity, a series of urea‐ and thiourea‐substituted tetrapeptides were designed. Replacement of Arg with Lys or ornithine reduced agonist activity at the mouse mMC1 and mMC3–5 receptors, thus supporting the hypothesis that the guanidine moiety is important for receptor potency, particularly at the MC3–5 receptors. The Arg side chain‐modified tetrapeptides examined in this study include substituted phenyl, naphthyl, and aliphatic urea and thiourea residues using a Lys side‐chain template. These ligands elicit full‐agonist pharmacology at the mouse MCRs examined in this study.     

Radiochemical and radiopharmacological characterization of a 64Cu‐labeled α‐MSH analog conjugated with different chelators

Radiolabeled α‐melanocyte‐stimulating hormone (α‐MSH) derivatives have a high potential for diagnosis and treatment of melanoma, because of high specificity and binding affinity to the melanocortin‐1 receptor (MC1R). Hence, the α‐MSH‐derived peptide NAP‐NS1 with a β‐Ala linker (ε‐Ahx‐β‐Ala‐Nle‐Asp‐His‐D‐Phe‐Arg‐Trp‐Gly‐NH₂) was conjugated to different chelators: either to NOTA (p‐SCN‐Bn‐1,4,7‐triazacyclononane‐1,4,7‐triacetic acid), to a hexadentate bispidine carbonate derivative (dimethyl‐9‐(((4‐nitrophenoxy)carbonyl)oxy)‐2,4‐di(pyridin‐2‐yl)‐3,7‐bis(pyridin‐2‐ylmethyl)‐3,7‐diazabicyclo[3.3.1]nonane‐1,5‐dicarboxylate), or to DMPTACN (p‐SCN‐Ph‐bis(2‐pyridyl‐methyl)‐1,4,7‐triaza‐cyclononane), labeled with ⁶⁴Cu, and investigated in terms of radiochemical and radiopharmacological properties. For the three ⁶⁴Cu‐labeled conjugates negligible transchelation, suitable buffer and serum stability, as well as appropriate water solubility, was determined. The three conjugates exhibited high binding affinity (low nanomolar range) in murine B16F10, human MeWo, and human TXM13 cells. The B_max values of [⁶⁴Cu]Cu‐bispidine‐NAP‐NS1 ([⁶⁴Cu]Cu‐ 2 ) and [⁶⁴Cu]Cu‐DMPTACN‐NAP‐NS1 ([⁶⁴Cu]Cu‐ 3 ) were higher than those of [⁶⁴Cu]Cu‐NOTA‐NAP‐NS1 ([⁶⁴Cu]Cu‐ 1 ), implying that different charged chelate units might have an impact on binding capacity. Preliminary in vivo biodistribution studies suggested the main excretion pathway of [⁶⁴Cu]Cu‐ 1 and [⁶⁴Cu]Cu‐ 3 to be renal, while that of [⁶⁴Cu]Cu‐ 2 seemed to be both renal and hepatobiliary. An initial moderate uptake in the kidney decreased clearly after 60 minutes. All three ⁶⁴Cu‐labeled conjugates should be considered for further in vivo investigations using a suitable xenograft mouse model.     

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.     

Extensive structure–activity studies of lactam derivatives of MT‐II and SHU‐9119: their activity and selectivity at human melanocortin receptors 3, 4, and 5

Abstract: The melanocortin system is involved in the regulation of several diverse physiologic pathways. Recently we have demonstrated that replacing His⁶ by Pro⁶ in the well‐known antagonist SHU‐9119 resulted in a potent agonist at the hMC5R (EC₅₀ = 0.072 nm ) with full antagonist activity at the hMC3R and the hMC4R. We have designed, synthesized, and pharmacologically characterized a series of peptide analogs of MT‐II and SHU‐9119 at the human melanocortin receptors MC3R, MC4R and MC5R. All these peptides were modified at position 6 with a Pro instead of a His residue. In this study, we have identified new scaffolds which are antagonists at the hMC4R and hMC3R. Additionally, we have discovered a new selective agonist at the hMC4R, Ac‐Nle‐c[Asp‐Pro‐D‐Phe‐Arg‐Trp‐Lys]‐Pro‐Val‐NH₂ ( 6 , PG‐931) which will be useful in further biologic investigations of the hMC4R. PG‐931 was about 100‐fold more selective for the hMC4R vs. the hMC3R (IC₅₀ = 0.58 and 55 nm , respectively). Some of these new analogs have exceptional biologic potencies at the hMC5R and will be useful in further efforts to differentiate the substructural features responsible for selectivity at the hMC3R, hMC4R, and hMC5R.     

Contribution of the transmembrane domain 6 of melanocortin-4 receptor to peptide [Pro, dNal (2′)]-γ-MSH selectivity

The melanocortin receptor (MCR) subtype family is a member of the GPCR superfamily and each of them has a different pharmacological profile regarding the relative potency of the endogenous and synthetic melanocortin peptides. Substitution of Trp with dNal (2′) in γ-MSH resulted in the loss of binding affinity and potency at hMC4R. However, the molecular mechanism of this ligand selectivity is unclear. In this study, we utilized chimeric receptors and site-directed mutagenesis approaches to investigate the molecular basis of MC4R responsible for peptide [Pro⁵, dNal (2′)⁸]-γ-MSH selectivity. Cassette substitutions of the second, third, fourth, fifth, and sixth TM of the human MC4R (hMC4R) with the homologous regions of hMC1R were constructed and the binding affinity of peptide [Pro⁵, dNal (2′)⁸]-γ-MSH at these chimeric receptors was evaluated. Our results indicate that the cassette substitutions of TM2, TM3, TM4 and TM5 of hMC4R with homologous regions of the hMC1R did not significantly increase peptide [Pro⁵, dNal (2′)⁸]-γ-MSH binding affinity and potency but substitution of the TM6 of the hMC4R with the same region of the hMC1R significantly enhances [Pro⁵, dNal (2′)⁸]-γ-MSH binding affinity and potency. Further site-directed mutagenesis study indicates that four amino acid residues, Phe267, Tyr268, Ile269 and Ser270, in TM6 of the hMC4R may play an important role in [Pro5, dNal (2′)-γ-MSH selective activity at MC4R.     

Theoretical conformational analysis of six arginine vasopressin analogs with the l‐naphthylalanine in position 3

Abstract: Introduction of the naphthylalanine residue into either position 3 of arginine vasopressin (AVP), or its analogs results in peptides with interesting pharmacological properties. The single substituted analog of AVP with l ‐2‐Nal in position 3 causes moderate antiduretic activity, whereas [Mpa¹, (l ‐1‐Nal)³, (d ‐Arg)⁸] VP and [Mpa¹, (l ‐2‐Nal)³, (d ‐Arg)⁸] VP are potent and selective V₂ agonists. Moreover [(l ‐2‐Nal)³, (d ‐Arg)⁸] VP is among the most potent and selective antagonists of V_1a receptors. In this study we carried out conformational calculations on [(l ‐1‐Nal)³] AVP, [(l ‐2‐Nal)³] AVP, [(l ‐1‐Nal)³, (d ‐Arg)⁸] VP, [(l ‐2‐Nal)³, (d ‐Arg)⁸] VP, [Mpa¹, (l ‐1‐Nal)³, (d ‐Arg)⁸] VP, [Mpa¹, (l ‐2‐Nal)³, (d ‐Arg)⁸] VP, using the ECEPP/3 force field with and without including hydration to simulate aqueous and nonpolar environments. It was found that in all six compound studied, the low‐energy conformations have common geometry and relative energies. Therefore, the modifications of the Phe in position 3 influence the binding to the receptor by changing the size of the third residue, rather than by changing the conformational space. The lowest‐energy conformations in the presence and absence of water had β‐turns at residues Phe³‐Gln⁴ and Gln⁴‐Asn⁵ and Gln⁴‐Asn⁵, respectively. The conformation at the Gln⁴‐Asn⁵ turn was most similar to the crystal structure of the pressinoic acid (the cyclic moiety of vasopressin).     

In vivo structure–activity studies on the dark‐color‐inducing neurohormone of locusts

Abstract: In the 11‐residue long dark‐color‐inducing neurohormone (DCIN = [His⁷]‐corazonin), of locusts, from residue 2 to residue 11, one amino acid at each time was substituted by d ‐phenylalanine (d ‐Phe). The dark‐color‐inducing effect of these peptides was investigated in comparison with unaltered DCIN by a bioassay based on nymphs of a DCIN‐deficient albino mutant of the migratory locust, Locusta migratoria. Substitution of any single amino acid by d ‐Phe always reduced the activity, but did not abolish it completely. Maximum inactivation was obtained after substitution of Gln4, Ser6, or Trp9. The latter two residues are within the partial sequence ‐Ser‐Xxx‐Gly‐Trp‐ (Xxx = His in the DCIN) that seems to be important for the dark‐color‐inducing activity, as found also in another study (Insect Biochem. Mol. Biol. 32, 2002, 909). Gln4, however, is outside of this partial sequence. Minimal, although still considerable, inactivation occurred after substitution of Gly8, Phe3, or Asn11, despite the fact that Gly8 is within the ‐Ser‐Xxx‐Gly‐Trp‐ partial sequence. In conclusion, no single active core was found, indicating that the whole sequence of the DCIN is necessary to induce maximum darkening effect. No difference was found in the activity of the peptides in which Gly8 was substituted by d ‐Phe or by l ‐Phe. Therefore the ‐Ser‐Xxx‐Gly‐Trp‐ partial sequence does not seem to be stabilized by a type II β‐turn. Nevertheless, existence of another kind of turn that includes this partial sequence is feasible. A single unsuccessful attempt was made to discover an antagonist to the DCIN.     

Chemical synthesis and receptor binding of catfish somatostatin: a disulfide‐bridged β‐d‐Galp‐(1→3)‐α‐d‐GalpNAc O‐glycopeptide*

Abstract: The glycopeptide hormone catfish somatostatin (somatostatin‐22) has the amino acid sequence H‐Asp‐Asn‐Thr‐Val‐Thr‐Ser‐Lys‐Pro‐Leu‐Asn‐Cys‐Met‐Asn‐Tyr‐Phe‐Trp‐Lys‐Ser‐Arg‐Thr‐Ala‐Cys‐OH; it includes a cyclic disulfide connecting the two Cys residues, and the major naturally occurring glycoform contains d ‐GalNAc and d ‐Gal O‐glycosidically linked to Thr⁵. The linear sequence was assembled smoothly starting with an Fmoc‐Cys(Trt)‐PAC‐PEG‐PS support, using stepwise Fmoc solid‐phase chemistry. In addition to the nonglycosylated peptide, two glycosylated forms of somatostatin‐22 were accessed by incorporating as building blocks, respectively, N^α‐Fmoc‐Thr(Ac₃‐α‐D‐GalNAc)‐OH and N^α‐Fmoc‐Thr(Ac₄‐β‐D‐Gal‐(1→3)‐Ac₂‐α‐D‐GalNAc)‐OH. Acidolytic deprotection/cleavage of these peptidyl‐resins with trifluoroacetic acid/scavenger cocktails gave the corresponding acetyl‐protected glycopeptides with free sulfhydryl functions. Deacetylation, by methanolysis in the presence of catalytic sodium methoxide, was followed by mild oxidation at pH 7, mediated by N^α‐dithiasuccinoyl (Dts)‐glycine, to provide the desired monomeric cyclic disulfides. The purified peptides were tested for binding affinities to a panel of cloned human somatostatin receptor subtypes; in several cases, presence of the disaccharide moiety resulted in 2‐fold tighter binding.     

Contribution of the transmembrane domain 6 of melanocortin-4 receptor to peptide [Pro5, DNal (2')8]-gamma-MSH selectivity

The melanocortin receptor (MCR) subtype family is a member of the GPCR superfamily and each of them has a different pharmacological profile regarding the relative potency of the endogenous and synthetic melanocortin peptides. Substitution of Trp with DNal (2') in gamma-MSH resulted in the loss of binding affinity and potency at hMC4R. However, the molecular mechanism of this ligand selectivity is unclear. In this study, we utilized chimeric receptors and site-directed mutagenesis approaches to investigate the molecular basis of MC4R responsible for peptide [Pro5, DNal (2')8]-gamma-MSH selectivity. Cassette substitutions of the second, third, fourth, fifth, and sixth TM of the human MC4R (hMC4R) with the homologous regions of hMC1R were constructed and the binding affinity of peptide [Pro5, DNal (2')8]-gamma-MSH at these chimeric receptors was evaluated. Our results indicate that the cassette substitutions of TM2, TM3, TM4 and TM5 of hMC4R with homologous regions of the hMC1R did not significantly increase peptide [Pro5, DNal (2')8]-gamma-MSH binding affinity and potency but substitution of the TM6 of the hMC4R with the same region of the hMC1R significantly enhances [Pro5, DNal (2')8]-gamma-MSH binding affinity and potency. Further site-directed mutagenesis study indicates that four amino acid residues, Phe267, Tyr268, Ile269 and Ser270, in TM6 of the hMC4R may play an important role in [Pro5, DNal (2')-gamma-MSH selective activity at MC4R.     

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.     

Conformational investigation of α,β‐dehydropeptides. X. Molecular and crystal structure of Ac‐ΔAla‐NMe2 compared with those of Ac‐l‐Ala‐NMe2, Ac‐dl‐Ala‐NMe2 and other dimethylamidesa

Abstract: A series of three homologous dimethyldiamides Ac‐ΔAla‐NMe₂, Ac‐l ‐Ala‐NMe₂ and Ac‐dl ‐Ala‐NMe₂ has been synthesized and the structures of these amides determined from single‐crystal X‐ray diffraction data. To learn more about the conformational preferences of compounds studied, the fully relaxed (φ–ψ) conformational energy maps in vacuo (AM1) of Ac‐ΔAla‐NMe₂ and Ac‐l ‐Ala‐NMe₂ were obtained, and the calculated minima reoptimized with the DFT/B3LYP/6–31G** method. The crystal‐state results have been compared with the literature data. Ac‐ΔAla‐NMe₂ and other α,β‐dehydroamino acid dimethyldiamides, Ac‐ΔXaa‐NMe₂ adopt the conservative conformation of the torsion angles φ, ψ = ～ ?45°, ～130°, which are located in the high‐energy region (region H) of Ramachandran diagram. Ac‐l ‐Ala‐NMe₂ and Ac‐dl ‐Ala‐NMe₂, as well as other saturated amino acid dimethylamides Ac‐l /dl ‐Xaa‐NMe₂, present common peptide structures, and no conformational preferences are observed. Molecular packing of the amides analysed reveals two general hydrogen‐bonded motifs. Dehydro and dl ‐species are paired into centrosymmetric dimers, and l ‐compounds form catemers. However, Ac‐ΔAla‐NMe₂ and Ac‐DL ‐Ala‐NMe₂ constitute exceptions: their molecules also link into catemers.     

[Nle3,d‐Phe6]‐γ2‐melanocyte‐stimulating hormone possesses the renal excretory but not the cardiovascular actions of the native γ2‐melanocyte‐stimulating hormone in anaesthetized rats

相似文献   

In vivo structure–activity studies on the dark‐color‐inducing neurohormone of locusts

In the 11‐residue long dark‐color‐inducing neurohormone (DCIN = [His⁷]‐corazonin), of locusts, from residue 2 to residue 11, one amino acid at each time was substituted by d ‐phenylalanine (d ‐Phe). The dark‐color‐inducing effect of these peptides was investigated in comparison with unaltered DCIN by a bioassay based on nymphs of a DCIN‐deficient albino mutant of the migratory locust, Locusta migratoria. Substitution of any single amino acid by d ‐Phe always reduced the activity, but did not abolish it completely. Maximum inactivation was obtained after substitution of Gln4, Ser6, or Trp9. The latter two residues are within the partial sequence ‐Ser‐Xxx‐Gly‐Trp‐ (Xxx = His in the DCIN) that seems to be important for the dark‐color‐inducing activity, as found also in another study (Insect Biochem. Mol. Biol. 32, 2002, 909). Gln4, however, is outside of this partial sequence. Minimal, although still considerable, inactivation occurred after substitution of Gly8, Phe3, or Asn11, despite the fact that Gly8 is within the ‐Ser‐Xxx‐Gly‐Trp‐ partial sequence. In conclusion, no single active core was found, indicating that the whole sequence of the DCIN is necessary to induce maximum darkening effect. No difference was found in the activity of the peptides in which Gly8 was substituted by d ‐Phe or by L ‐Phe. Therefore the ‐Ser‐Xxx‐Gly‐Trp‐ partial sequence does not seem to be stabilized by a type II β‐turn. Nevertheless, existence of another kind of turn that includes this partial sequence is feasible. A single unsuccessful attempt was made to discover an antagonist to the DCIN.     

Synthesis and biologic activity of conformationally constrained analogs of L‐363,301

Abstract: We report the synthesis, biological activity and conformational analysis of analogs of the cyclic hexapeptide L‐363,301, c[Pro⁶‐Phe⁷‐d ‐Trp⁸‐Lys⁹‐Thr¹⁰‐Phe¹¹] (numbering as in the native hormone somatostatin‐14). The d ‐Trp in position 8 was replaced with (2R,3S)‐ and (2R,3R)‐β‐MeTrp respectively, with an added methyl group in the beta position of Trp. The objective of our study was to determine the potency and selectivity generated by the added constraint in the beta position of the d ‐Trp upon binding to human somatostatin receptors hsst1‐5. We synthesized the building blocks enantioselectively and incorporated them into the peptides by SPPS. Competition binding assays revealed that both compounds 2 and 3 were selective for hsst2 over hsst5. The (2R,3S) analog 2 was approximately 30 times more potent at hsst2 than the (2R,3R) analog 3 . Interestingly, the (2R,3R) compound showed no binding affinity at hsst5.     

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.     

Synthesis and biological evaluation of constrained analogues of the opioid peptide H‐Tyr‐d‐Ala‐Phe‐Gly‐NH2 using the 4‐amino‐2‐benzazepin‐3‐one scaffold

Abstract: The synthesis of conformationally restricted dipeptidic moieties 4‐amino‐1,2,4,5‐tetrahydro‐2‐benzazepin‐3‐one (Aba)‐Gly ([(4S)‐amino‐3‐oxo‐1,2,4,5‐tetrahydro‐1H‐2‐benzazepin‐2‐yl]‐acetic acid) and 8‐hydroxy‐4‐amino‐1,2,4,5‐tetrahydro‐2‐benzazepin‐3‐one (Hba)‐d ‐Ala ([(4S)‐amino‐8‐hydroxy‐3‐oxo‐1,2,4,5‐tetrahydro‐benzo[c]azepin‐2‐yl]‐propionic acid) was based on a synthetic strategy that uses an oxazolidinone as an N‐acyliminium precursor. Introducing these Aba scaffolds into the N‐terminal tetrapeptide of dermorphin (H‐Tyr‐d ‐Ala‐Phe‐Gly‐Tyr‐Pro‐Ser‐NH₂)‐induced remarkable shifts in affinity and selectivity towards the opioid μ‐ and δ‐receptors. This paper provides the synthesis and biological in vitro and in vivo evaluation of constricted analogues of the N‐terminal tetrapeptide H‐Tyr‐d ‐Ala‐Phe‐Gly‐NH₂, which is the minimal subunit of dermorphin needed for dermorphin‐like opiate activity.     

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.     

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.