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.     

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

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

Structure-activity relationships of cyclic opioid peptide analogues containing a phenylalanine residue in the 3-position

Ten analogues of the highly mu-receptor selective cyclic opioid peptide H-Tyr-D-Orn-Phe-Asp-NH2 (1) were synthesized by the solid phase method and were characterized in vitro in mu- and delta-receptor representative binding assays and bioassays. These cyclic analogues are structurally related to the linear opioid peptides dermorphin and beta-casomorphin (morphiceptin), which also contain a phenylalanine residue in the 3-position of the peptide sequence. The obtained results indicate that analogous structural modifications (configurational inversion at positions 2, 3, and 4 or N alpha-methylation of Phe3) in cyclic peptide 1 and in dermorphin-related peptides had qualitatively the same effect on opioid activity, whereas the corresponding modifications in beta-casomorphins had the opposite effect. These findings can be interpreted to indicate that the mode of receptor binding of H-Tyr-D-Orn-Phe-Asp-NH2 is identical with that of dermorphin, but differs from that of beta-casomorphins. The side-chain length of the aromatic residue in position 3 of cyclic analogue 1 was shown to be critical for receptor affinity and selectivity, suggesting that mu- and delta-receptors differ from one another in the relative topographical disposition of the binding sites for the Tyr1 tyramine moiety and the Phe3 aromatic ring. Cyclic lactam analogue H-Tyr-D-Asp-Phe-A2bu-NH2, containing a reduced-size (12-membered) ring structure, showed increased mu-receptor selectivity, whereas the more flexible, cystine-containing analogue H-Tyr-D-Cys-Phe-Cys-NH2 (11-membered ring) was less selective. The latter results indicate that both ring size and ring flexibility affect receptor affinity and selectivity.     

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.     

Conformational restriction of the phenylalanine residue in a cyclic opioid peptide analogue: effects on receptor selectivity and stereospecificity.

In an effort to determine the effect of side chain conformational restriction on opioid receptor selectivity, the cyclic phenylalanine analogues 2-aminoindan-2-carboxylic acid (Aic), 2-aminotetralin-2-carboxylic acid (Atc), and tetrahydroisoquinoline-3-carboxylic acid (Tic) were substituted for Phe in the potent cyclic opioid peptide analogue H-Tyr-D-Orn-Phe-Glu-NH2, which lacks significant opioid receptor selectivity. Compounds were tested in mu- and delta-opioid receptor representative binding assays and bioassays in vitro. The analogue H-Tyr-D-Orn-Aic-Glu-NH2 was found to be a potent agonist with high preference of mu receptors over delta receptors. Opening of the five-membered ring of Aic in the latter peptide, as achieved through substitution of C alpha-methylphenylalanine or o-methylphenylalanine, resulted in only slightly selective compounds, indicating that the high mu selectivity of the Aic analogue is exclusively the consequence of the imposed side chain conformational restriction. Both diastereoisomers of H-Tyr-D-Orn-(D,L)-Atc-Glu-NH2 were highly mu-selective and, in contrast to the weak affinity observed with the D-Phe3 analogue as compared to the L-Phe3 analogue, both had similar potency. Thus, stereospecificity was lost as a consequence of side chain conformational restriction. Further structure-activity data obtained with analogues containing L- or D-homophenylalanine (Hfe) or 3-(1'-naphthyl)alanine (Nap) in place of Phe3 and consideration of geometric interrelationships between Nap and the L and D isomers of Atc, Hfe, and Phe led to the proposal that the D-Phe3 and the D-Atc3 analogue may have different modes of binding to the receptor. The very low potency observed with H-Tyr-D-Orn-N alpha MePhe-Glu-NH2 (N alpha MePhe = N alpha-methylphenylalanine) and H-Tyr-D-Orn-Tic-Glu-NH2 indicated that N alpha-alkylation at the 3-position is detrimental to activity.     

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

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

Solid phase synthesis and evaluation of Tyr-Tic-Phe-Phe(p-NHCOCH(2)Br) ([Phe(p-bromoacetamide)(4)]TIPP), a potent affinity label for delta opioid receptors

Derivatives of the delta opioid receptor selective peptide Tyr-Tic-Phe-Phe-OH (TIPP) containing a p-bromoacetamide moiety on the phenyl ring of Phe(3) or Phe(4) were prepared by solid phase synthesis. [Phe(p-NHCOCH(2)Br)(4)]TIPP exhibited high affinity for cloned delta receptors (IC(50) = 5.4 nM), and incubation with only 2.5 nM resulted in 85% wash resistant inhibition of radioligand binding to delta receptors. Therefore, this peptide is a potent affinity label for further study of delta opioid receptors.     

Synthesis of highly mu and delta opioid receptor selective peptides containing a photoaffinity group

A series of cyclic, conformationally constrained photolabile peptides related to the enkephalins and to somatostatin were designed and synthesized in an effort to develop highly selective and potent peptides for the delta and mu opioid receptors. The following new peptides were prepared and tested for their delta opioid receptor potency and selectivity in the guinea pig ileum assay, the mouse vas deferens assay, and the rat brain binding assay: H-Tyr-D-Pen-Gly-p-NH2Phe-D-Pen-OH (1, [p-NH2Phe4]DPDPE) and H-Tyr-D-Pen-Gly-p-N3Phe-D-Pen-OH (2, [p-N3Phe4]-DPDPE). The following new peptides were prepared and tested for their mu opioid receptor potency and selectivity in the same assays: H-D-Phe-Cys-p-NH2Phe-D-Trp-Lys-Thr-Pen-Thr-NH2 (3, [p-NH2Phe3]CTP) and D-Phe-Cys-p-N3Phe-D-Trp-Lys-Thr-Pen-Thr-NH2 (4, [p-N3Phe3]CTP). The delta selective photoaffinity peptide 2 displayed both high affinity (IC50 = 9.5 nM) and good selectivity (IC50 mu/IC50 delta = 1053) as an agonist at delta opioid receptors in bioassays, and 2 also displayed moderate affinity (33 nM) and excellent selectivity (IC50 mu/IC50 delta = 110) for rat brain delta opioid receptors. The mu selective photoaffinity peptide 4 displayed very weak affinity (8% contraction at 300 nM) at mu opioid receptors in bioassays, but good affinity (IC50 = 48.6 nM) and excellent selectivity (IC50 delta/IC50 mu = 412) for the rat brain mu opioid receptors. These conformationally constrained cyclic photoaffinity peptides may be useful tools to investigate the pharmacology of delta and mu opioid receptors.     

Benzophenone dicarboxylic acid antagonists of leukotriene B4. 2. Structure-activity relationships of the lipophilic side chain

A series of lipophilic benzophenone dicarboxylic acid derivatives were found to inhibit the binding of the potent chemotaxin leukotriene B4 (LTB4) to its receptor on intact human neutrophils. Activity at the LTB4 receptor was determined by using a [3H]LTB4-binding assay. The structure-activity relationship for the lipophilic side chain was systematically investigated. Compounds with n-alkyl side chains of varying lengths were prepared and tested. Best inhibition of [3H]LTB4 binding was observed with the n-decyl derivative. Analogues with alkyl chains terminated with an aromatic ring showed improved activity. The 6-phenylhexyl side chain was optimal. Substitution on the terminal aromatic ring was also evaluated. Methoxyl, methylsulfinyl, and methyl substituents greatly enhanced the activity of the compound. For a given substituent, the para isomer had the best activity. Thus the nature of the lipophilic side chain can greatly influence the ability of the compounds to inhibit the binding of LTB4 to its receptor on intact human neutrophils. The most active compound from this series, 84 (LY223982), bound to the LTB4 receptor with an affinity approaching that of the agonist.     

Design of peptidomimetic delta opioid receptor antagonists using the message-address concept

Highly selective nonpeptide ligands with potent delta opioid receptor antagonist activity have been developed using the message-address concept. This approach envisaged the delta opioid receptor to contain two major recognition subsites; a message subsite which recognizes the pharmacophore, and an address subsite that is unique for the delta receptor type and confers selectivity. The message and address components of the delta-selective enkephalins were postulated to be Tyr1 and Phe4, respectively, with Gly2-Gly3 functioning as a spacer. The message component of the target compounds in this study was derived from naltrexone and related structures. An indole system was fused to the C ring of naltrexone as a mimic of the address component. The benzene moiety of indole was viewed as the delta address component, mimicking the phenyl group of Phe4, and the pyrrole portion was used as a rigid spacer. Members of the series (1-23) were evaluated for opioid antagonist activity on the guinea pig ileum (GPI) and mouse vas deferens (MVD) preparations. Naltrindole (NTI, 1) was the most potent member of the series, with Ke values of approximately 0.1 nM at delta receptors. The antagonism by NTI was approximately 220- and 350-fold greater at delta than at mu and kappa opioid receptors. The binding of NTI and selected members of the series to guinea pig brain membranes was qualitatively consistent with their pharmacologic antagonist activity profiles in the MVD and GPI, but the Ki values were not in the same rank order. The selectivity of NTI arises mainly as a consequence of increased affinity at delta receptors. Thus, the Ke and Ki values of NTI were 1/530 and 1/90 that of the delta antagonist enkephalin analogue, ICI 174864. In contrast to NTI, ICI174864 derives its selectivity through greatly decreased recognition at mu and kappa receptors. The implications of the high affinity and selectivity of NTI as a consequence of its conformational rigidity are discussed. It is suggested that any attempt to model a receptor-bound conformation of an opioid peptide should consider affinity and potency at multiple receptor sites rather than selectivity alone.     

Highly selective kappa-opioid analgesics. 2. Synthesis and structure-activity relationships of novel N-[(2-aminocyclohexyl)aryl]acetamide derivatives

This paper describes the chemical synthesis and the development of structure-activity relationships (SAR) for the kappa opioid receptor affinity and mu/kappa opioid receptor selectivity of novel N-[(2-aminocyclohexyl)aryl]acetamide derivatives. The SAR of this series are investigated by consideration of structural modifications made to the aromatic moiety, the amide linkage, and cyclohexane and the pyrrolidine ring substituents of the prototype kappa selective agonist, PD117302 (trans-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzo[b]thiophene-4- acetamide) (1). The kappa and mu opioid receptor binding affinities of 23 novel compounds are reported. It is observed that optimal mu/kappa receptor selectivity is obtained with a benzo[b]thiophene aromatic system attached via the C-4 position, which is discussed in terms of steric and electronic parameters. The amide linkage has been replaced with the reversed amide, an ester, an aminomethylene, a thioamide, and a secondary amide. The best of these isosteres is the N-methyl amide. Substitution of the pyrrolidine ring of PD117302 in the 3-position with a hydroxymethylene group increases the mu/kappa selectivity compared to the unsubstituted compound, e.g. compound 14, trans-(+/-)-N-methyl-N-[2-[3-(hydroxymethyl)-1-pyrrolidinyl] cyclohexyl]-4-benzo[b]furanacetamide monohydrochloride, mu/kappa receptor selectivity = 244. The cis fused, 4,5 dimethyl ether substituted cyclohexane analogue trans-(+/-)-N-methyl-N-[4,5-dimethoxy-2-(1-pyrrolidinyl) cyclohexyl]-benzo[b]thiophene-4-acetamide monohydrochloride (32) has high in vitro kappa opioid receptor affinity (Ki = 16 nM) and equipotent analgesic activity to morphine after iv administration in rats.     

Synthesis and evaluation of N,N-dialkyl enkephalin-based affinity labels for delta opioid receptors

To develop affinity labels for delta opioid receptors based on peptide antagonists, the Phe(4) residues of N,N-dibenzylleucine enkephalin and N,N-diallyl[Aib(2),Aib(3)]leucine enkephalin (ICI-174, 864) were substituted with either Phe(p-NCS) or Phe(p-NHCOCH(2)Br). A general synthetic method was developed for the conversion of small peptide substrates into potential affinity labels. The target peptides were synthesized using Phe(p-NH(2)) and a Boc/Fmoc orthogonal protection strategy which allowed for late functional group conversion of a p-amine group in the peptides to the desired affinity labeling moieties. A key step in the synthesis was the selective deprotection of a Boc group in the presence of a tert-butyl ester using trimethylsilyl trifluoromethanesulfonate (TMS-OTf). The target peptides were evaluated in radioligand binding experiments in Chinese hamster ovary (CHO) cells expressing delta or mu opioid receptors. The delta receptor affinities of the N, N-dibenzylleucine enkephalin analogues were 2.5-10-fold higher than those for the corresponding ICI-174,864 analogues. In general, substitution at the para position of Phe(4) decreased binding affinity at both delta and mu receptors in standard radioligand binding assays; the one exception was N, N-dibenzyl[Phe(p-NCS)(4)]leucine enkephalin (2) which exhibited a 2-fold increase in affinity for delta receptors (IC(50) = 34.9 nM) compared to N,N-dibenzylleucine enkephalin (IC(50) = 78.2 nM). The decreases in mu receptor affinities were greater than in delta receptor affinities so that all of the analogues tested exhibited significantly greater delta receptor selectivity than the unsubstituted parent peptides. Of the target peptides tested, only N, N-dibenzyl[Phe(p-NCS)(4)]leucine enkephalin (2) exhibited wash-resistant inhibition of radioligand binding to delta receptors. To our knowledge, 2 represents the first peptide-based affinity label to utilize an isothiocyanate group as the electrophilic affinity labeling moiety. As a result of this study, enkephalin analogue 2 emerges as a potential affinity label useful for the further study of delta opioid receptors.     

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

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

Effects of the substitution of Phe4 in the opioid peptide [D-Ala8]dynorphin A-(1-11)NH2

Phenylalanine at position 4 of the peptide dynorphin A (Dyn A) is an important residue for opioid receptor affinity and activity, but there is very little information available on the structure-activity relationships or conformational preference of this residue for interaction with kappa-opioid receptors. Based on the hypothesis that the spatial orientation of the aromatic ring at position 4 of Dyn A is important for opioid receptor affinity and selectivity, a series of Dyn A analogues with various Phe derivatives substituted at position 4 were synthesized and evaluated for their opioid receptor affinity and activity. The L- and D-Homophe4 (homophenylalanine) analogues of [D-Ala8]Dyn A-(1-11)NH2 were compared to the (R)- and (S)-Atc4 (2-aminotetralin-2-carboxylic acid) derivatives (Aldrich et al. Chirality 2001, 13, 125-129). [l-Homophe4,D-Ala8]Dyn A-(1-11)NH2 exhibited higher kappa-opioid receptor affinity than the D-Homophe4 isomer, while [(R)-Atc4,D-Ala8]Dyn A-(1-11)NH2 exhibited higher kappa-opioid receptor affinity than the (S)-Atc4 isomer. Comparing the structure of Atc to those of Phe and Homophe, these results suggest that the Atc isomers are functioning more as constrained Homophe rather than Phe analogues in these Dyn A derivatives. The higher kappa-opioid receptor affinity of the (R)-Atc4 analogue suggests that Phe4 of Dyn A most likely adopts a gauche (-) or trans conformation in the kappa-opioid receptor binding site. Comparison of [D-Ala8]Dyn A-(1-11)NH2 derivatives containing Aic4 (2-aminoindan-2-carboxylic acid) and Tic4 (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) with the peptides containing their acyclic counterparts alpha-MePhe4 and N-MePhe4, respectively, suggest that the loss in opioid receptor affinity seen for the Aic4 and Tic4 analogues is probably due to an improper orientation of the aromatic ring in these residues. Most of the analogues in this series showed much lower affinity for delta-opioid receptors than the parent peptide, suggesting that kappa- and delta-opioid receptors have distinct binding pockets for the residue at position 4 of Dyn A. All of the analogues with high affinity for kappa-opioid receptors exhibited full agonist activity in the adenylyl cyclase assay using cloned kappa-opioid receptors, indicating that changes in the position or orientation of the phenyl ring in this residue did not alter the ability of the peptides to activate the receptor.     

2',6'-dimethylphenylalanine (Dmp) can mimic the N-terminal Tyr in opioid peptides

Substitution of 2',6'-dimethyltyrosine (Dmt) for the N-terminal Tyr in opioid peptides has recently been shown to be a promising tool for improving opioid receptor affinity and biological activity. We have also demonstrated that another unnatural amino acid, 2',6'-dimethylphenylalanine (Dmp), is not only an excellent substitute for Phe at position 3 but also can mimic the aromatic N-terminal Tyr residue in a micro opioid receptor-selective dermorphin analogue (YRFB: Tyr-D-Arg-Phe-betaAla-NH(2)). To further evaluate the value of Dmp in opioid peptides, we investigated Dmp(1)-substituted analogues of the delta receptor ligands, deltorphin II (DLT: Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH(2)) and enkephalin (ENK: Tyr-Gly-Gly-Phe-Leu). In the receptor binding assay, both [Dmp(1)]DLT and [Dmp(1)]ENK bound to the delta-receptor with high affinity and selectivity, and were nearly as effective as the parent peptides. The potency of the Dmp(1)-peptides on the MVD and GPI assays correlated well with the receptor binding affinity data. These results are in contrast to the tendency of corresponding Dmt(1)-analogues to have poor receptor selectivity. Taken together with the results with YRFB, we conclude that the Dmp(1)-peptide is superior to the corresponding Dmt(1)-peptide in its receptor selectivity. [Dmp(1)]DLT and [Dmp(1)]YRFB may serve as pharmacological tools for the studies of ligand recognition and opioid receptor signal transduction.     

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

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

Stereospecificity of amino acid side chains in deltorphin defines binding to opioid receptors.

A series of individual D-amino acid replacement analogues of deltorphin A, several of which were in combination with a His4 deletion, were used to probe alterations of side-chain orientation on peptide binding parameters with rat brain opioid receptors. Peptides with D-amino acids in residues 1, 3, and 5 exhibited diminished affinities primarily for delta receptors (88-1200-fold) with selectivity decreasing by factors of 13-64-fold relative to deltorphin A (Ki delta = 0.45 nM; Ki mu/Ki delta = 764): the aromatic side chains Tyr1 and Phe3, which lie in the N-terminal "message" domain and the aryl side chain of Leu5 in the C-terminal "address" domain, appear to play essential roles in conferring high delta affinity and selectivity. Although D-His4 only decreased delta affinity by 6-fold and selectivity by a factor of 4, His appears to be involved as an integral component of both domains: [des-His4]deltorphin A and [des-His4] analogues containing consecutive D-amino acid replacements in the remaining residues exhibited weak binding to delta receptors and poor delta selectivity. Substitution of D-Met2 in deltorphin A by D-Ala or D-Nle decreased delta selectivities 3-6-fold through an elevation in mu affinities; however, the converse replacement, D-Met for D-Ala2 in deltorphin B, diminished beta selectivity by an order of magnitude only through the loss in delta affinity. The data show that the high delta affinity and selectivity of deltorphins correlate with and require a strict stereospecificity of the amino acid residue side chains.     

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

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

Synthesis and structural requirements of N-substituted norapomorphines for affinity and activity at dopamine D-1, D-2, and agonist receptor sites in rat brain

A series of N-substituted analogues of (R)-(-)-norapomorphine were synthesized to study the optimal structural requirements of the N-alkyl side chain to interact with D-1 and D-2 dopaminergic receptors as well as dopamine (DA) agonist binding sites. Evaluations included testing the affinity of these compounds for DA receptor sites in rat striatal tissue and assessing stereotypy as a behavioral index of dopaminergic activity. The electronic, steric, and lipophilic properties of the N-alkyl side chain were found to be related to affinity, D-2 selectivity, and dopaminergic activity. All 11 compounds evaluated had relatively low affinity at D-1 sites. Optimum D-2 and agonist-site affinity as well as agonist activity were exhibited by N-cyclopropylmethyl (7) greater than or equal to N-allyl (8) greater than or equal to N-propyl (4) or N-ethyl (3) substituted compounds. Branching of the N-alkyl side chain as in N-isopropyl (5) and N-isobutyl (6) markedly reduced the D-2 affinity and activity, presumably due to steric effects. The N-trifluoroethyl (10) and N-pentafluoropropyl (11) derivatives had low affinity for all their dopamine receptor sites and no agonistic activity; evidently, the highly electronegative F atoms decrease basicity of the N atom and therefore decrease the ability of the N atom to be cationic at physiological pH, a proposed requirement for high-affinity binding to DA receptors.     

Design and biological evaluation of linear and cyclic phosphopeptide ligands of the N-terminal SH2 domain of protein tyrosine phosphatase SHP-1

In an effort to gain further insight into the conformational and topographical requirements for recognition by the N-terminal SH2 domain of protein tyrosine phosphatase SHP-1, we synthesized a series of linear and cyclic peptides derived from the sequence surrounding phosphotyrosine 2267 in the receptor tyrosine kinase Ros (EGLNpYMVL). A molecular modeling approach was used to suggest peptide modifications sterically compatible with the N-SH2-peptide binding groove and possibly enhanced binding affinities compared to the parent peptide. The potencies of the synthesized compounds were evaluated by assaying their ability to stimulate phosphatase activity as well as by their binding affinities to the GST-fused N-SH2 domain of SHP-1. In the series of linear peptides, structural modifications of Ros pY2267 in positions pY + 1 to pY + 3 by amino acid residues structurally related to Phe, for example l-erythro/threo-Abu(betaPh) (5a, 5b), yielded ligands with increased binding affinity. The incorporation of d-amino acid residues at pY + 1 and pY + 3 led to inactive peptides. The replacement of Phe in both pY + 1 and pY + 3 by Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) was also not tolerated due to steric hindrance. Cyclic peptides (13, 14) that were linked via residues in positions pY - 1 (Lys) and pY + 2 (Asp/Glu) and contained a Gly residue in the bridging unit displayed much lower potencies for the stimulation of SHP-1 activity but increased binding affinities compared to Ros pY2267. They partially competed with Ros pY2267 in the activation assay. Such cyclic structures may serve as scaffolds for competitive SHP-1 inhibitor design targeting N-SH2 domain-protein interactions that block SHP-1 activation.