Synthesis and opioid activity of partial retro-inverso analogs of dermorphin

We studied the effect of partial retro-inverso modification of selected peptide bonds of dermorphin (H-Tyr-d -Ala-Phe-Gly-Tyr-Pro-Ser-NH₂. The modifications concern two consecutive peptide bonds (Phe³-Cly⁴-Tyr⁵, I) or a single one (Gly⁴-Tyr⁵-, II or Phe³-Gly⁴, III). All pseudoheptapeptides showed low opioid activity in the in vitro and in vivo tests. Compound III has a biological potency comparable to that of morphine but only 2–5% of original dermorphin when tested in guinea pig ileum preparation and in mice tail-flick assay after intra-cerebro or subcutaneous administration.     

Synthesis and receptor binding analysis of dermorphin hepta-, hexa- and pentapeptide analogues

Sixteen dermorphin analogues were synthesized and characterized for μ- and δ-opioid receptor binding properties using [³H]DAGO and [³H]DPDPE, respectively. The analogues included the following: substitutions at position 4 and/or the C-terminal residue; deletions of Gly⁴ or Pro⁶-Ser⁷; inclusion of Z or an acetyl group on the β-amino group of Lys⁷; and the presence of either a C-terminal amide or free acid group. Two peptides, [Lys7-OH]- and [Lys⁷-NH₂]dermorphin, had μ-affinities (K_iμ= 0.15–0.13 nm ) and μ-selectivities (K_iδ/K_iμ= 1158–1482) higher than dermorphin (K_iμ= 0.28 nm ; K_iδ/K_iμ= 295) and best fitted a one-site binding model similar to dermorphin. Significantly better (P <0.0001) fits to a two-site binding model vs. a one-site model were observed with four dermorphin analogues: [Lys(Z)⁷-OH]heptapeptide, [des-Gly⁴(Tyr⁴,Pro⁵,Asn⁶-OH)]hexapeptide and two pentapeptides, [Tyr⁵-NH₂] and [Trp⁴,Asn⁵-OH]. Our data revealed a complex binding pattern for dermorphin analogues to brain μ-receptors in which Hill coefficients less than 0.85 generally suggest heterogeneity of μ-receptors; however, only detailed analyses of the data derived from the non-linear regression fits for one- or two-components gave evidence for the possible existence of two separate [³H]DAGO binding sites. Eight of our dermorphin analogues had significantly better fits for a two-site model (P <0.05), but only four seemed to have two distinct Kⁱ, values (P <0.0001).     

Opioid peptides Synthesis and binding properties of dermorphin related heptapeptides

C-Terminal amino acid residues of dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH₂) were replaced by N^α-methyl- or D-amino acids in order to examine the effect on opioid activity. In binding studies based on displacement of μ, Δ, and κ opioid receptor selective radiolabels from guinea pig brain membranes, the 13 new analogues showed, like dermorphin, a negligible affinity for the κ binding site. The introduction of N^α-methyl- or D-amino acid residues at position 5, 6, or 7 of dermorphin, when matched with C-terminal amide function modifications, generally produced analogues with reversed μ/δ specificity.     

Synthesis and pharmacological activity of dermorphin and its N-terminal sequences

H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH₂ (demorphin), an opiate-like peptide, and tri-, tetra-, penta- and hexapeptide-amide analogs, were synthesized by conventional methods in solution, to determine the minimum peptide chain-length, required for analgesic activity.     

Synthesis and pharmacological activity of the N-terminal dermorphin tetrapeptide analogs with CH2-NH peptide bond isosteres

The synthesis of pseudotetrapeptides H-Tyr-D-Ala-Phe-NH-(CH₂)₂-NH₂ (1a), H-Tyr-D-Ala-Phe-ψ(CH₂-NH)-Gly-NH₂ (2a), H-Tyr-D-Ala-ψ(CH₂-NH)-Phe-Gly-NH₂ (3a), and H-Tyr-ψ(CH₂-NH)-D-Ala-Phe-Gly-NH₂ (4a), representing the N-terminal tetrapeptide sequence of dermorphin, in which amide bonds are replaced by CH₂-NH bond, is described. N-acetyl-Tyr and desamino-Tyr pseudopeptide analogs (1-4b), (1-3c) are also described. The analogs were assayed in binding studies based on displacement of μ and δ-receptor selective radiolabels from rat brain membrane and in a bioassay using guinea pig ileum (GPI). Pseudopeptides in which the C-terminal (1a) or D-Ala-Phe (3a) amide bond are substituted, exhibit higher μ-affinities and μ-receptor selectivity than the corresponding Phe-Gly or Tyr-D-Ala analogs (2a, 4a). Acetyl-and desamino-Tyr pseudopeptide analogs (1-4b) and (1-3c) did not exhibit μ and δ-opioid receptor affinity at nM concentration. The relevance of the single peptide replacement and of its association to acetylation or amino group elimination of Tyr, is discussed on the basis of a receptor model for μ and δ opioids.     

Modification of the Phe3 aromatic moiety in delta receptor-selective dermorphin/deltorphin-related tetrapeptides Effects on opioid receptor binding

The previously described cyclic delta opioid receptor-selective tetrapeptide H-Tyr-d -Cys-Phe-d -Pen-OH (JOM-13) was modified at residue 3 by incorporation of both natural and unnatural amino acids with varying steric, electronic, and lipophilic properties. Effects on mu and delta opioid receptor binding affinities were evaluated by testing the compounds for displacement of radiolabeled receptor-selective ligands in a guinea pig brain receptor binding assay. Results obtained with the bulky aromatic 1-Nal³ and 2-Nal³ substitutions suggest that the shape of the receptor subsite with which the side chain of the internal aromatic residue interacts differs for delta and mu receptors. This subsite of either receptor can accommodate the transverse steric bulk of the 1-Nal³ side chain but only the delta receptor can readily accept the more elongated 2-Nal³ side chain. Several analogs with pi-excessive heteroaromatic side chains in residue 3 were examined. In general, these analogs display diminished binding to mu and delta receptors, consistent with previous findings for analogs with residue 3 substitutions of modified electronic character. Several analogs with alkyl side chains in residue 3 were also examined. While delta receptor binding affinity is severely diminished with Val³, Ile³, and Leu³ substitutions, Cha³ substitution is very well tolerated, indicating that, contrary to the widely held belief, an aromatic side chain in this portion of the ligand is not required for delta receptor binding. Where possible, comparison of results in this delta-selective tetrapeptide series with those reported for analogous modification in the cyclic delta-selective pentapeptide [d -Pen², d -Pen⁵]enkephalin (DPDPE) and linear pentapeptide enkephalins reveals similar trends.     

Synthesis and preliminary biological investigations of O-sulphated dermorphin

Synthesis by a classical solution method of a sulphated analogue of dermorphin is reported. The product has proved to be devoid of any peripheral and central opiate- or CCK-like activity in the tests assayed.     

Structural requirements for delta opioid receptor binding

Structural features influencing opioid activity of enkephalin analogs were investigated through the synthesis and evaluation of opioid receptor binding affinities of a series of cyclic dithioether-containing analogs and structurally related linear analogs of the cyclic, disulfide-containing peptides, [D-Pen2, D-Pen5]enkephalin and [D-Pen2, L-Pen5]enkephalin, where Pen (penicillamine) is beta, beta-dimethylcysteine. The major effect of increasing the ring size of the cyclic moiety from disulfide to dithioether analogs was a large decrease in delta opioid receptor binding affinity which suggests that relatively compact conformations of the peptide ligand are necessary for optimal binding to this receptor. The effect of bulky, hydrophobic residues at position 2 in the peptide chain was evaluated by preparing the linear analogs, [D-t-Leu2, D-t-Leu5]enkephalin (t-Leu, 2-amino-3,3-dimethylbutanoic acid) and [D-Abu2, D-t-Leu5]enkephalin (Abu, 2-aminobutanoic acid). The former analog was found to be 36- and 450-fold less potent at delta and mu receptor sites, respectively, than was the latter, suggesting that bulky side chain substituents in position 2 of enkephalin analogs lead to a deleterious steric interaction at delta and particularly at mu receptors.     

Direct electrophilic fluorination of tyrosine in dermorphin analogues and its effect on biological activity,receptor affinity and selectivity

In a preliminary communication we reported [(Tetrahedron Lett. 31, 619 (1990)] that acetyl hypofluorite can be used efficiently to introduce fluorine regiospecifically (ortho to OH) into the phenolic ring of tyrosine-containing peptides. This procedure has been applied to the fluorination of a number of μ-selective opioid peptides derived from dermorphin. While the procedure can be used even when the side chains of Arg, Lys, and Tyr are left unprotected, the sulfoxide of a Met(O)-containing analogue was oxidized to sulfone faster than fluorination of the phenolic ring. This method can also be used when the peptide is attached to Merrifield resin. Thus, Tyr(3-F)-d -Ala-Phe-Gly-NH₂ and Tyr(3-F)-d -Arg-Phe-Lys-NH₂ (F-DALDA) have been prepared, purified, and characterized. Affinities of these fluorinated peptides for both μ-and δ-opioid receptors are reduced (two- to nine-fold) relative to their nonfluorinated analogues, but their selectivity for μ-opioid receptors is not significantly altered. Similarly, the in vitro biological potencies (GPI and MVD assays) of the fluorinated analogues are reduced (two- to seven-fold) relative to their nonfluorinated parent peptides. Thus, F-DALDA, which has high affinity (K δ= 15.2nM) and selectivity (K δ/K δ= 5390) for μ-opioid receptors, has potential use in biochemical studies which utilize ¹⁹F or ¹⁸F- labeled compounds.     

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.     

Synthesis and biological studies of dermorphin and its analogs substituted at positions 5 and 7

Dermorphin and seven of its analogs substituted at positions 5 and/or 7, have been synthesized by the solid phase method employing mainly 9-fluorenylmethyloxycarbonylamino acid trichlorophenyl esters in presence of 1-hydroxybenzotriazole, the solid support being the Merrifield resin. Among the analogs synthesized, the most interesting is [Tyr⁷]dermorphin. It is one of the most potent dermorphin analogs reported so far. Compared to the natural peptide, it is about two times more potent in the GPI (in vitro) and nearly 1.4 times more potent in its analgesic activity in mice by the hot plate test (in vivo). Further, its antidiarrhoeal activity in mice (in vivo) is comparable to that of dermorphin. On the other hand, [Thr⁷]dermorphin is almost as potent as dermorphin.     

μ阿片受体功能调节

μ阿片受体识别选择性配基的区域还有争论 ,受体的TM内氨基酸的空间取向影响选择性配基的亲和性。阿片受体活性的调节可能并没有涉及到它们将GTP和G蛋白相联系的能力 ,以及相继的异源三聚体的解离能力。激动剂诱导的 μ受体的磷酸化与受体的脱敏是否相关尚无定论。μ受体细胞内过程需要形成多蛋白复合物 ,形成受体信号复合物的细胞蛋白募集很重要。通用转录因子之间的相互作用决定受体基因的转录是很明确的。     

Synthesis and biological activity of carboxyl terminally extended dermorphins

Dermorphinoyl(DMR)-glycine, DMR-sarcosine and DMR-glycyl-arginine have been prepared in order to examine the effect of C-terminal extension of dermorphin (H-Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH₂) on opioid activity. On GPI preparation the addition of Gly, Sar, or Gly-Arg to the carboxyl terminus of dermorphinoic acid was detrimental to μ activity: dermorphinoyl-derivatives, in fact, retain only 5–20% of dermorphin potency. Following intracerebroventricular administration (tail-flick test), whereas the analgesic activities of compounds showed the trend dermorphin >DMR-Sar> DMR-Gly-Arg>DMR-Gly>morphine, the nonapeptide displayed highest activity after subcutaneous injection in mice: DMR-Gly-Arg was 2.5 and 10 times more potent than dermorphin and morphine, respectively.     

阿片依赖病理过程中NMDA受体与阿片受体的相互作用及分子机制

阿片依赖的病理生理学基础与机体长期接触阿片类物质后产生的代偿性适应相关。近年来研究发现 ,NMDA受体拮抗剂、抗体和反义核酸能抑制阿片躯体和精神依赖。该文从神经生化和分子生物学角度 ,综述了NMDA受体作用系统与阿片受体作用系统的相互作用以及NMDA受体参与阿片依赖的分子机制 ,并评价了NMDA受体拮抗剂及其他相关物质治疗阿片依赖的应用前景     

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).     

Involvement of endogenous opioid peptides in the antinociception induced by the novel dermorphin tetrapeptide analog amidino-TAPA

The antinociceptive effect of i.t. administered N-amidino-Tyr-d-Arg-Phe-β-Ala (amidino-TAPA), an N-terminal tetrapeptide analog of dermorphin, was characterized in ddY mice. In the opioid receptor ligand-binding assays using mouse brain membranes, amidino-TAPA showed a very high affinity for μ-opioid receptors, a low affinity to δ-opioid receptors and no affinity for κ-opioid receptors. In the mouse tail-flick test, i.t. treatment with amidino-TAPA produced a potent antinociception. The antinociception induced by amidino-TAPA was significantly attenuated by i.t. pretreatment with the μ-opioid receptor antagonist β-funaltrexamine, the κ-opioid receptor antagonist nor-binaltorphimine and the δ-opioid receptor antagonist naltrindole. Moreover, the antinociception induced by amidino-TAPA was significantly attenuated by i.t. pretreatment with antisera against the endogenous κ-opioid peptides dynorphin A, dynorphin B and -neo-endorphin; and the endogenous δ-opioid peptide [Leu⁵]enkephalin. In mice lacking prodynorphin, the precursor of the endogenous κ-opioid peptides, the antinociceptive effect of amidino-TAPA was significantly attenuated compared to that in wild-type C57BL/6J mice. However, there was no difference in G-protein activation by amidino-TAPA in the spinal cord membranes from prodynorphin knockout mice and C57BL/6J mice. The present results suggest that the spinal antinociception induced by the μ-opioid receptor selective peptide amidino-TAPA is mediated in part by the release of endogenous opioid peptides in the spinal cord, which is caused by the direct stimulation of μ-opioid receptors.     

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.     

Cyclic morphiceptin analogs: cyclization studies and opioid activities in vitro

Attempts were undertaken to develop cyclic β-casomorphin-5 analogs with improved opioid activity profiles by deletion of the glycine residue in position 5, leading to analogs structurally related to the opioid peptide morphiceptin (H-Tyr-Pro-Phe-Pro-NH₂). The tetrapeptide sequence Boc-Tyr(tBu)-D-Xaa-Phe-Yaa-OH (Xaa = Lys, Orn, A₂bu; Yaa = Pro in L- or D-configuration) was used to study the influence of ring size and chirality on the yield of cyclization between the ω-amino group of Xaa and the C-terminal carboxyl group. In all cases the cyclization reaction was performed under identical experimental conditions to allow a direct comparison with regard to yield and homogeneity. The reaction products were purified by crystallization and liquid chromatography, and were characterized by HPLC, TLC, electrospray mass spectrometry and ¹H-NMR spectroscopy. In none of the reactions performed with the cyclization precursors containing proline in the L-configuration could a cyclic monomer be detected, and the cyclodimer ( 7–9 ) was the exclusive product in each case. Cyclodimerization was also the favored reaction in the attempted formation of the 11-membered ring of the cyclic [D-A₂bu², D-Pro⁴]-morphiceptin analog 12 , since only traces of the monomer were found. In the case of both the [D-Lys², D-Pro⁴]-analog 10 and the [D-Orn², D-Pro⁴]- analog 11 , the cyclomonomer/cyclodimer ratio was about 80: 20. The cyclic monomers 10 and 11 showed high opioid activity in the μ-receptor-representative guinea pig ileum assay (IC₅₀= 2–5 nM) and in the δ-receptor representative mouse vas deferens assay (IC₅₀= 50–60 nM), whereas the potency of the cyclodimers was 2–3 orders of magnitude lower in both in vitro bioassays.     

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.     

Synthesis and binding properties of specific photoaffinity ligands for μ and δ opioid receptor subtypes

We report the synthesis and binding properties of specific photoaffinity ligands for μ and δ opioid receptor subtypes. These ligands are derived from DAGO: Tyr-D-Ala-Gly-NMePhe-Gly-ol, a μ selective probe and DTLET: Tyr-D-Thr-Gly-Phe-Leu-Thr, a δ selective probe by modifying the Phe 4 residue. These modifications are: i) a nitro group on the para position of Phe ring as Phe(4 NO₂) or Nip, ii) an azido group as Phe(4 N₃) or AZ. Pharmacological responses on mouse vas deferens (δ sites) and guinea pig ileum (μ sites), as well as competition experiments with [³H] DAGO and [³H] DTLET on crude rat brain membranes have been performed. The nitro group on the phenyl ring of the Phe residue preserves the affinity and selectivity of each probe: NipDAGO for the μ sites, NipDTLET for the δ ones. However the nitro probes do not appear to be photo-activable by u.v. irradiation. Likewise, azidation of the phenyl ring of the Phe residue does not change the receptor selectivity of each probe, but AZDAGO has less affinity than its parent molecule DAGO, while AZDTLET has more affinity than DTLET. These compounds are photoactivable and provide an efficient tool to characterize and isolate the different receptor subtypes, especially the δ site.