Dynorphin A analogs containing a conformationally constrained phenylalanine derivative in position 4: reversal of preferred stereochemistry for opioid receptor affinity and discrimination of kappa vs. delta receptors

Analogs of the opioid peptide [D-Ala8]dynorphin A-(1-11)NH2 containing optically pure (R)- and (S)-2-aminotetralin-2-carboxylic acid (Atc) in position 4 were synthesized and evaluated for opioid receptor affinity. These peptides are the first reported dynorphin A analogs containing a conformationally constrained amino acid in place of the important aromatic residue Phe4. By incorporating resolved Atc isomers, the opioid receptor affinity and the stereochemistry of the constrained residue could be unambiguously correlated. Both Dyn A analogs containing Atc in position 4 retained nanomolar affinity for kappa and mu opioid receptors. Unexpectedly the peptide containing (R)-Atc, corresponding to a conformationally constrained D-Phe analog, displaying higher affinity for both kappa and mu receptors than the peptide containing (S)-Atc. In contrast [D-Phe4,D-Ala8]Dyn A-(1-11)NH2 exhibited significantly lower affinity for kappa and mu receptors than the parent peptide, as expected. Conformational restriction of the Phe4 sidechain or incorporation of D-Phe in position 4 had the largest effect on delta receptor affinity, yielding compounds with negligible affinity for these receptors. Thus, there appear to be distinctly different structural requirements for this residue for kappa vs. delta receptors, and it is possible to completely distinguish between these two receptors by changing a single residue in Dyn A.     

In-vitro investigation of oxazol and urea analogues of morphinan at opioid receptors

A series of 2-amino-oxazole (7 and 8) analogs and 2-one-oxazole analogs (9 and 10) were synthesized from cyclorphan (1) or butorphan (2) and evaluated in-vitro by their binding affinity at mu, delta, and kappa opioid receptors and compared with their 2-aminothiozole analogs 5 and 6. Ligands 7-10 showed decreased affinities at kappa and mu receptors. Urea analogs (11-14) were also prepared from 2-aminocyclorphan (3) or 2-aminobutorphan (4) and evaluated in-vitro by their binding affinity at mu, delta, and kappa opioid receptors. The urea derived opioids retained their affinities at mu receptors while showing increased affinities at delta receptors and decreased affinities at kappa receptors. Functional activities of these compounds were measured in the [35S]GTPgammaS binding assay, illustrating that all of these ligands were kappa agonists. At the mu receptor, compounds 11 and 12 were mu agonist/antagonists.     

Use of hydrophilic diamines for bridging of two opioid peptide pharmacophores. Synthesis and receptor binding of two new analogues.

The bivalent ligand approach, which assumes that two pharmacophores are connected by a spacer, was used to design receptor type-selective ligands for opioid receptors. The first two opioid peptide bivalent ligands with different spacer lengths containing different numbers of hydroxyl groups, (Tyr-D-Ala-Gly-Phe-NH-CH2-CHOH-)2 (Tyr-D-Ala-Gly-Phe-NH-CH2-CHOH-CHOH-)2, were synthesized and their binding to mu, delta, and kappa opioid receptors was characterized. Both analogues were found to possess high opioid in vitro activities. The length of the hydrophilic spacer does not affect the affinity for delta receptors, whereas shorter spacer length increases affinity for mu and even more so for kappa receptors. Thus receptor type-selective peptides for opioid receptors can be designed using the bivalent approach.     

8-acenaphthen-1-yl-1-phenyl-1,3,8-triaza-spiro[4.5]decan-4-one derivatives as orphanin FQ receptor agonists.

A series of 8-acenaphthen-1-yl-1-phenyl-1,3,8-triaza-spiro[4.5]decan+ ++-4-one derivatives 1 was studied with respect to the binding affinity for the orphanin FQ (OFQ) and opioid (mu, kappa, delta) receptors. The influence of stereochemistry as well as the substitution pattern of the phenyl-ring in position 1 on the affinity for the orphanin FQ receptor and selectivity to opioid (mu, kappa, delta) receptors is discussed. The most interesting compound 1c was tested for its anxiolytic-like properties in vivo.     

The biological consequences of replacing hydrophobic amino acids in deltorphin I with amphiphilic alpha-hydroxymethylamino acids.

New analogues of deltorphin I (DT I), in which the Phe residue in position 3, and the Val residue in position 5 or 6 are replaced with respective amphiphilic alpha-hydroxymethylamino acid residues (HmAA), were synthesized and tested for receptor affinity and selectivity to mu and delta opioid receptors. The analogue with (R)-HmPhe at position 3 lost receptor selectivity, as a result of a partial decrease of affinity to delta and a significant increase of affinity to mu receptors. In contrast, an analogue with (S)-HmPhe in the same position, was very potent and more specific to delta receptors than parent DT I. The analogue with (R)-HmVal at position 5 expressed higher delta affinity and selectivity than parent DT I. The analogue with other possible isomer (S)-HmVal was less selective for delta opioid receptors, as a result of decreasing affinity to delta and increasing affinity to mu receptors. The analogues with (R)- or (S)-HmVal in position 6 expressed equally low receptor affinity and selectivity. The data obtained support a previously proposed model of active conformation of deltorphins.     

5'-halogenated analogs of oxymorphindole

The presence of a 6,7-fused indole group in the indolomorphinans was considered to be responsible for the delta opioid selectivity for this class of ligands. Herein is shown that 5'-halogenated analogs of oxymorphindole are opioids with little selectivity for delta receptors over mu opioid receptors.     

Synthesis and binding properties of deltorphin I analogues containing (R) and (S)-alpha-hydroxymethylnaphtylalanine

New analogues of deltorphin I (DT I), in which the phenylalanine residue in position 3 is substituted with amphiphilic alpha,alpha-disubstituted amino acid enantiomers, (R) and (S)-alpha-hydroxymethylnaphtylalanine, were synthesized and tested for mu and delta opioid receptor affinity and selectivity. Although both analogues have lower affinity to delta receptors than DT I, they both expressed specificity to delta receptors.     

Chemistry of opium alkaloids. Part 44: synthesis and opioid receptor binding profile of substituted ethenoisomorphinans and ethenomorphinans

7- and 8-substituted 6alpha,14alpha-ethenoisomorphinans were synthesized by reaction of properly substituted morphinan-6,8-dienes (analogues of thebaine) with methyl vinyl ketone or ethyl acrylate. Reaction with the appropriate Grignard reagent gave the 7- and 8-dialkylmethanols, respectively. Cleavage of the 3-methyl ether with KOH/glycol or boron tribromide afforded the 3-hydroxyl derivatives. In general, the compounds with the ethoxycarbonyl or dimethylmethanol substituent at the 8alpha-position showed lower affinity for the mu, kappa, and delta opioid receptor subtypes than the corresponding 7alpha- and 7beta-substituted compounds. Introduction of a chloro substituent in position 18 increased the potency significantly. The 7-substituent could be connected to the 18-position without loss of affinity. 5Beta-alkyl substitution of 6alpha,14alpha-ethenoisomorphinans led to a decrease in affinity for the three opioid receptor subtypes. In the 5beta-methyl series the affinity for the mu and delta receptors increased from 7alpha-dimethylmethanol to 7alpha-methylhexylmethanol. In the 5beta-alkyl series, the affinity for the mu-receptor could be increased by connecting the 5- and 7-substituents, yielding a compound with high mu-selectivity. The new 6beta,14beta-ethenomorphinans did not show affinity for any of the opioid receptors, in accordance with the inactivity earlier found in in vivo experiments.     

Synthesis and evaluation of derivatives of leucine enkephalin as potential affinity labels for delta opioid receptors

As part of an effort to develop peptide-based affinity labels for opioid receptors, [Leu(5)]enkephalin (LeuEnk) and DTLET (Tyr-D-Thr-Gly-Phe-Leu-Thr), potent agonists for delta receptors, were selected as the parent peptides for further modification. The affinity label derivatives were prepared using standard Fmoc solid-phase peptide synthesis in conjunction with Fmoc-Phe(p-NHAlloc) (Fmoc: 9-flourenylmethoxycarbonyl;) and selective modification of the p-amino group on this residue. The electrophilic isothiocyanate and bromoacetamide groups were introduced into the para position of Phe(4); the corresponding free amine-containing peptides were also prepared for comparison. The pure peptides were evaluated in radioligand binding assays using Chinese hamster ovary (CHO) cells expressing delta and micro opioid receptors. Modification of Phe(4) in LeuEnk and DTLET significantly decreased delta-receptor binding affinity (40 to >2,000-fold). Among the synthesized analogues, [Phe(p-NH(2))(4)]DTLET showed the highest delta-receptor binding affinity (IC(50) = 39 nM) and enhanced selectivity for delta receptors compared to DTLET while other derivatives exhibited much lower delta receptor affinity. The differences in affinities between the two series of analogues and between the derivatives of LeuEnk and N,N-dibenzyl[Leu(5)]Enk reported previously suggest subtle differences in interactions of Phe(4) with delta receptors depending on other modifications in the sequences.     

(4-Carboxamido)phenylalanine is a surrogate for tyrosine in opioid receptor peptide ligands

(S)-4-(Carboxamido)phenylalanine (Cpa) is examined as a bioisosteric replacement for the terminal tyrosine (Tyr) residue in a variety of known peptide ligands for the mu, delta and kappa opioid receptors. The Cpa-containing peptides, assayed against cloned human opioid receptors, display comparable binding affinity (Ki), and agonist potency (EC50) to the parent ligands at the three receptors. Cpa analogs of delta selective peptides show an increase in delta selectivity relative to the mu receptor. Cpa is the first example of an amino acid that acts as a surrogate for Tyr in opioid peptide ligands, challenging the long-standing belief that a phenolic residue is required for high affinity binding.     

Dimerization of morphine and orphanin FQ/nociceptin receptors: generation of a novel opioid receptor subtype

Although orphanin FQ/nociceptin (OFQ/N) receptors are a member of the opioid receptor family of receptors, they bind traditional opioids with very poor affinity. We now demonstrate that mu opioid receptors can physically associate with OFQ/N receptors, resulting in a complex with a unique binding selectivity profile. Immunoprecipitation of epitope-tagged OFQ/N receptors co-precipitates mu receptors. When the two receptors were co-expressed in CHO cells, [3H]OFQ/N retained its high binding affinity for its receptor. However, co-expression of the two receptors increased by up to 250-fold the affinity of a series of opioids in [3H]OFQ/N binding assays. This enhanced affinity was limited to agonists with high affinity for mu receptors. Selective kappa(1) and delta opioids did not lower binding. Despite the dramatic increase in affinity for the opioid agonists in co-expressing cells, the opioid antagonists naloxone and diprenorphine failed to compete [3H]OFQ/N binding.     

Differential effects of cyanogen bromide on ligand binding by mu, delta and kappa opioid receptors

Guinea pig brain membranes treated with cyanogen bromide (CNBr) demonstrate a loss in the number of mu opioid receptors and a lower binding affinity of delta opioid receptors. These receptor changes are irreversible. Results from ligand protection experiments support the hypothesis that the location of the methionine groups, the sites at which CNBr cleaves peptides, differs between these two types of opioid receptors. Kappa receptors are significantly less sensitive to the action of CNBr than mu or delta receptors.     

Distribution Pattern of Metorphamide Compared with Other Opioid Peptides from Proenkephalin and Prodynorphin in the Bovine Brain

Metorphamide is a [Met]-enkephalin-containing opioid octapeptide with a C-terminal alpha-amide group. It is derived from proenkephalin and is, so far, the only endogenous opioid peptide with a particularly high affinity for mu opioid (morphine) receptors, a somewhat lesser affinity for kappa opioid receptors, and a relatively low affinity for delta opioid receptors. The concentrations of metorphamide in the bovine caudate nucleus, the hypothalamus, the spinal cord, and the neurointermediate pituitary were determined by radioimmunoassay and chromatography separation procedures. Metorphamide concentrations were compared with the concentrations of eight other opioid peptides from proenkephalin and prodynorphin in identical extracts. The other opioid peptides were [Met]-enkephalyl-Arg6-Phe7 and [Met]-enkephalyl-Arg6-Gly7-Leu8 from proenkephalin; alpha-neoendorphin, beta-neoendorphin, dynorphin A(1-8), dynorphin A(1-17), and dynorphin B from prodynorphin; and [Leu]-enkephalin, which can be derived from either precursor. All opioid peptides were present in all four bovine neural tissues investigated. Metorphamide concentrations were lower than the concentrations of the other proenkephalin-derived opioid peptides. They were, however, similar to the concentrations of the prodynorphin-derived opioid peptides in the same tissues. Marked differences in the relative ratios of the opioids derived from prodynorphin across brain regions were observed, a finding suggesting differential posttranslational processing. Differences in the ratios of the proenkephalin-derived opioids across brain regions were less pronounced. The results from this study together with previous findings on metorphamide's mu opioid receptor binding and bioactivities suggest that the amounts of metorphamide in the bovine brain are sufficient to make this peptide a candidate for a physiologically significant endogenous mu opioid receptor ligand.     

Synthesis and binding properties to DNA and to opioid receptors of enkephalin-ellipticinium conjugates

In order to specifically direct cytotoxic agents against tumor cells bearing delta opioid receptors, the DNA intercalating agents ellipticine and 9-OH-ellipticine were coupled by quaternarization of the pyridine nitrogen to an enkephalin modified pentapeptide through a short chemical linker. The ellipticine ring of these conjugates was shown to intercalate into DNA, with DNA affinity constants close to those of the non-conjugated ellipticines. Despite the addition of a polycyclic ring to the C-terminal amino acid, the D-Ala2-D-Leu5-enkephalin-ellipticine conjugates bind to the opioid receptor from rat brain and NG 108-15 cells with an affinity constant close to 10(8) M-1. Other derivatives were synthesized as a control using a tripeptide which does not bind to the opioid receptor.     

Redefining the structure-activity relationships of 2,6-methano-3-benzazocines. 5. Opioid receptor binding properties of N-((4'-phenyl)-phenethyl) analogues of 8-CAC

A series of aryl-containing N-monosubstituted analogues of the lead compound 8-[N-((4'-phenyl)-phenethyl)]-carboxamidocyclazocine were synthesized and evaluated to probe a putative hydrophobic binding pocket of opioid receptors. Very high binding affinity to the mu opioid receptor was achieved though the N-(2-(4'-methoxybiphenyl-4-yl)ethyl) analogue of 8-CAC. High binding affinity to mu and very high binding affinity to kappa opioid receptors was observed for the N-(3-bromophenethyl) analogue of 8-CAC. High binding affinity to all three opioid receptors were observed for the N-(2-naphthylethyl) analogue of 8-CAC.     

125I][D-Ala2]deltorphin-I: a high affinity, delta-selective opioid receptor ligand.

The selective delta opioid agonist [D-Ala2]deltorphin-I was radioiodinated and the product purified using reverse phase HPLC. The binding characteristics and distribution profile of [125I][D-Ala2]deltorphin-I were assessed in mouse brain using homogenate binding techniques and quantitative autoradiography. [125I][D-Ala2]deltorphin-I bound with high affinity to a single class of sites (KD = 0.5 nM) in brain membrane preparations and striatal sections. Competition studies indicated that [125I][D-Ala2]deltorphin-I was selectively labeling delta opioid receptors as shown by the ratio of apparent affinities for mu and delta receptors (KI mu/KI delta = 1388). The autoradiographical distribution profile of [125I][D-Ala2]deltorphin-I binding sites was also consistent with that of other delta-selective radioligands. The data indicate that [125I][D-Ala2]deltorphin-I binds to delta opioid receptors with high affinity and selectivity. Because of its very high specific activity, it can be detected rapidly with high sensitivity by autoradiographic emulsion.     

Mixed kappa agonists and mu agonists/antagonists as potential pharmacotherapeutics for cocaine abuse: synthesis and opioid receptor binding affinity of N-substituted derivatives of morphinan

A series of new N-substituted derivatives of morphinan was synthesized and their binding affinity for the three opioid receptors (mu, delta, and kappa) was determined. A paradoxical effect of N-propargyl (MCL-117) and N-(3-iodoprop-(2E)-enyl) (MCL-118) substituents on the binding affinities for the mu and kappa opioid receptors was observed. All of these novel derivatives showed a preference for the mu and kappa versus delta binding.     

Desipramine modulation of sigma and opioid peptide receptor expression in glial cells.

Exposure of C6 glial cell cultures to desipramine induced the appearance of opioid receptors and up-regulated sigma receptors. Opioid binding was demonstrated with 3H-etorphine and 3H-dihydromorphine (DHM), but was not observed with the mu, delta and kappa ligands 3H-DAMGE, 3H-DADLE or 3H-(-)ethylketocyclazocine in the presence of specific blockers, respectively. Competition experiments with 3H-DHM and either (-)naloxone or (+)naloxone indicated the presence of authentic opioid receptors. In similar studies with beta-endorphin, its truncated form (1-27) or their N-acetyl derivatives, beta-endorphin proved to have the highest affinity. Opioid receptors in glial cell aggregates were primarily kappa, with few mu and delta sites. Desipramine increased Bmax values for kappa but not mu and delta.     

Characterization of [3H]naltrindole binding to delta opioid receptors in rat brain.

[3H]Naltrindole binding characteristics were determined using homogenized rat brain tissue. Saturation binding studies at 25 degrees C measured an equilibrium dissociation constant (Kd) value of 37.0 +/- 3.0 pM and a receptor density (Bmax) value of 63.4 +/- 2.0 fmol/mg protein. Association binding studies showed that equilibrium was reached within 90 min at a radioligand concentration of 30 pM. Naltrindole, as well as the ligands selective for delta (delta) opioid receptors, such as pCI-DPDPE and Deltorphin II inhibited [3H]naltrindole binding with nanomolar IC50 values. Ligands selective for mu (mu) and kappa (kappa) opioid receptors were only effective in inhibiting [3H]naltrindole binding at micromolar concentrations. From these data, we conclude that [3H]naltrindole is a high affinity, selective radioligand for delta opioid receptors.     

Specific cross-linking of Lys233 and Cys235 in the mu opioid receptor by a reporter affinity label

The first example of the use of a reporter affinity label (NNA) that contains a fluorogenic naphthalene dialdehyde moiety to identify neighboring lysine and cysteine residues at a recognition site is described. The opioid receptors have served as the proof-of-concept because they contain multiple lysine and cysteine residues. The kinetics of isoindole formation resulting from covalent binding of NNA to wild-type and mutant opioid receptors were followed in cultured cells using flow cytometry. The finding that NNA bound to mutant mu opioid receptors (K233R and C235S) without producing specific fluorescence enhancement suggested that covalent bonding occurred at these positions to produce an isoindole fluorophore in the wild-type mu receptor. The similar kinetics of fluorophore formation for wild-type mu, delta, and kappa opioid receptors suggest that these conserved residues are the cross-linking sites in all three types of opioid receptors. The combined utilization of a reporter affinity label and site-directed mutagenesis offers a more expeditious method of identifying cross-linking at a recognition site when compared to classical procedures.