Carole Lemieux

Cyclic enkephalin analogs containing a cystine bridge

Abstract Two conformationally constrained enkephalin analogs were synthesized by substitution of cysteines in positions 2 and 5 and oxidative disulfide bond formation. In the guinea pig ileum assay the obtained cyclic analogs, [D-Cys 2 -L-Cys 5 ]enkephalinamide and [D-Cys 2 -D-Cys 5 ]enkephalinamide, showed potency ratios of 37.9 ± 0.8 and 73.3 ± 0.9, respectively, relative to [Met 5 ]enkephalin. The extremely high potency of the analogs was shown to be a consequence of the conformational restrictions introduced by cyclization. Rat brain membrane binding studies with [ 3 H]naloxone and [ 3 H](D-Ala 2 , D-Leu 5 )enkephalin as radiolabels revealed a moderate preference of both analogs for μ-receptors over δ-receptors. Furthermore, the cystine-containing analogs were shown to be highly resistant to enzymatic degradation.

A novel cyclic opioid peptide analog showing high preference for μ-receptors

The side-chain to side-chain cyclized opioid peptide analogs H-Tyr-D-Orn-Phe-Asp-NH2 (I) and H-Tyr-D-Lys-Phe-Glu-NH2 (II) were synthesized and tested in the guinea pig ileum and mouse vas deferens assays and in binding assays based on displacement of mu- and delta-opioid receptor-selective radioligands from rat brain membranes. The more rigid cyclic analog I containing a 13-membered ring structure showed very high preference for mu-receptors over delta-receptors, whereas the more flexible cyclic peptide II (15-membered ring) was non-selective. These results indicate that variation in the degree of conformational restriction of opioid peptides can produce drastic shifts in their receptor selectivity profile. Because of its high mu-receptor selectivity and rigidity cyclic analog I will be useful for determining the conformational requirements of mu-opioid receptors.

Evidence of a peptide backbone contribution toward selective receptor recognition for leucine enkephalin thioamide analogs.

The in vitro opioid activities of a series of leucine enkephalin analogs containing a thioamide linkage in place of the peptide bond at various positions of the backbone were determined in mu- and delta-receptor-selective bio- and binding-assays. Thioamide substitution in the 1-2 position resulted in an inactive compound, whereas the same modification in the 2-3 and 4-5 position produced potency enhancement. Most interestingly, the 2-3 modified analog showed a 3 to 5 times higher preference for delta- over mu-receptors than natural leucine enkephalin. These results suggest that subtle backbone modifications can have a profound effect on receptor affinity and selectivity of biologically active peptides.

Comparison of μ-, δ- and κ-receptor binding sites through pharmacologic evaluation of p-nitrophenylalanine analogs of opioid peptides

Abstract For the purpose of comparing the structural requirements of opioid receptor subsites interacting with phenylalanine residues of opioid peptides, analogs containing p-nitrophenylalanine (Phe (pNO 2 )) were synthesized and tested in the guinea pig ileum (GPI) and mouse vas deferens (MVD) assay as well as in μ- and δ-receptor selective binding assays. Whereas substitution of Phe(pNO 2 ) in position 4 of the μ-selective analog H-Tyr-c[-N ϵ -D-Lys-Gly-Phe-Leu-] and of the δ-selective analog H-Tyr-D-Ser-Gly-Phe-Leu-Thr-OH produced a significant potency increase in all assays, the corresponding substitution in the κ-selective dynorphin-(1–13) fragment resulted in a decreased potency in the GPI-assay. These findings suggest that the electronic requirements of the Phe 4 binding site at the μ- and δ-receptor are identical, but different from those of the corresponding site at the κ-receptor. The observation that Phe(pNO 2 ) substitution in position 3 of morphiceptin and dermorphin also produces a drastic potency drop indicates that the Phe 3 side-chain of the latter peptides at the μ- and δ-receptor may bind to a site different from that interacting with the Phe 4 side-chain of enkephalins.

Blood-Brain Barrier Penetration by Two Dermorphin Tetrapeptide Analogues : Role of Lipophilicity vs Structural Flexibility

Two dermorphin analogues having an almost identical structure but different structural flexibility were compared for opioid activity. In 1 the aromatic side chains were incorporated into a lactam structure, while in 2 N-amide alkylation was retained but the side chains were flexible. Both compounds produced comparable antinociceptive effects in the mouse tail flick test after peripheral administration. This indicates that lipophilicity, rather than side chain flexibility, is the key determinant for blood-CNS barrier penetration.

Structural Modifications of the N-terminal Tetrapeptide Segment of [D-Ala2]Deltorphin I: Effects on Opioid Receptor Affinities and Activities In Vitro and on Antinociceptive Potency

A series of deltorphin I analogs containing D- or L-N-methylalanine (MeAla), D- or L-proline (Pro), alpha-aminoisobutyric acid (Aib), sarcosine (Sar) or D-tert-leucine (Tle) in place of D-Ala2, or phenylalanine in place of Tyr1, was synthesized. The opioid activity profiles of these peptides were determined in mu and delta opioid receptor-representative binding assays and bioassays in vitro as well as in the rat tail flick test in vivo. In comparison with the deltorphin I parent, both the L- and the D-MeAla2-analog were slightly more potent delta agonists in the mouse vas deferens (MDV) assay, and the D-MeAla2-analog showed two-fold higher antinociceptive potency in the analgesic test. In view of the fact that deltorphin analogs with an unsubstituted L-amino acid residue in the 2-position generally lack opioid activity, the observed high delta opioid potency of [L-MeAla2]deltorphin I is postulated to be due to the demonstrated presence of a conformer with a cis Tyr1-MeAla2 peptide bond, since the cis conformer allows for a spatial arrangement of the pharmacophoric moieties in the N-terminal tripeptide segment similar to that in active deltorphin analogs containing a D-amino acid residue in the 2-position. Substitution of Aib in the 2-position led to a compound, H-Tyr-Aib-Phe-Asp-Val-Val-Gly-NH2, which displayed lower delta receptor affinity than the parent peptide but higher delta selectivity and, surprisingly, three times higher antinociceptive potency. The D- and L-Pro2-, Sar2- and D-Tle2-analogs showed much reduced delta receptor affinities and were inactive in the tail flick test. Replacement of Tyr1 in deltorphin I with Phe produced a 32-fold decrease in delta receptor affinity but only a 7-fold drop in antinociceptive potency.

Variation of the net charge, lipophilicity, and side chain flexibility in Dmt(1)-DALDA: Effect on Opioid Activity and Biodistribution.

The influence of the side chain charges of the second and fourth amino acid residues in the peptidic μ opioid lead agonist Dmt-d-Arg-Phe-Lys-NH(2) ([Dmt(1)]-DALDA) was examined. Additionally, to increase the overall lipophilicity of [Dmt(1)]-DALDA and to investigate the Phe(3) side chain flexibility, the final amide bond was N-methylated and Phe(3) was replaced by a constrained aminobenzazepine analogue. The in vitro receptor binding and activity of the peptides, as well as their in vivo transport (brain in- and efflux and tissue biodistribution) and antinociceptive properties after peripheral administration (ip and sc) in mice were determined. The structural modifications result in significant shifts of receptor binding, activity, and transport properties. Strikingly, while [Dmt(1)]-DALDA and its N-methyl analogue, Dmt-d-Arg-Phe-NMeLys-NH(2), showed a long-lasting antinociceptive effect (>7 h), the peptides with d-Cit(2) generate potent antinociception more rapidly (maximal effect at 1h postinjection) but also lose their analgesic activity faster when compared to [Dmt(1)]-DALDA and [Dmt(1),NMeLys(4)]-DALDA.

Cyclic opioid peptide agonists and antagonists obtained via ring-closing metathesis.

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

[Aladan3]TIPP: A fluorescent δ-opioid antagonist with high δ-receptor binding affinity and δ selectivity†

Fluorescent analogues of the potent and highly selective δ‐opioid antagonist TIPP (HTyrTicPhePheOH) and TIP (HTyrTicPheOH) containing the exceptionally environmentally sensitive fluorescent amino acid β‐(6′‐dimethylamino‐2′‐naphthoyl)alanine (Aladan [Ald]) in place of Phe3 were synthesized. The Ald3‐ and D‐Ald3 analogues of TIPP and TIP all retained δ‐opioid antagonist properties. The most potent analogue, [Ald3]TIPP, showed a Ke value of 2.03 nM in the mouse vas deferens assay and five times higher δ vs. μ selectivity (K  μi /K  δi = 7930) than the TIPP parent peptide in the opioid receptor binding assays. Theoretical conformational analyses of [Ald3]TIPP and [Ald3]TIP using molecular mechanics calculations resulted in a number of low‐energy conformers, including some showing various patterns of aromatic ring stacking and others with the Ald side chain and a carbonyl group (fluorescence quencher) in close proximity. These ensembles of low‐energy conformers are in agreement with the results of steady‐state fluorescence experiments (fluorescence emission maxima and quantum yields) and fluorescence decay measurements (fluorescence lifetime components), which indicated that the fluorophore was either engaged in intramolecular hydrophobic interactions or in proximity of a fluorescence quencher (e.g., a carbonyl group). These fluorescent TIP(P) δ‐opioid antagonists represent valuable pharmacological tools for various applications, including studies on membrane interactions, binding to receptors, cellular uptake and intracellular distribution, and tissue distribution.

Synthesis and opioid activity profiles of cyclic dynorphin analogs

Abstract In an effort to reduce the conformational flexibility of the x-receptor selective opioid peptide dynorphin A, we synthesized the cyclic analogs dynorphin5A-(1–8) ( 1 ), -dynorphin A-(1–13) ( 2 ), dynorphin A-(1–13)( 3 ) and dynorphin A-(1–13). These side chain-to-side chain cyclized lactam analogs were prepared by the solid-phase method using a protection scheme which permitted cyclization of the peptide still attached to the solid support. In all four cases the desired cyclic analogs were obtained in reasonable yield. Aside from the cyclic monomers, the crude products also contained the side-chain-linked antiparallel cyclic dimer and higher oligomers which had been formed through intersite reaction on the resin. The obtained results demonstrate that cyclic lactam analogs containing relatively large ring structures (up to 31-membered) can be prepared by this method. In the guinea pig ileum assay analog 1 was 45 times more potent than linear dynorphin A-(l–8), whereas analogs 2 – 4 were 460–1,350 times less potent than linear dynorphin A-(l–l3). All four analogs showed Ke-values for naloxone as antagonist below 5 nM in the latter assay, indicating that they no longer interact significantly with x-receptors. In the opioid receptor binding assays cyclic analog 1 displayed extraordinarily high affinity for the μ-receptor and the determined ratios of the binding inhibition constants ( K i δ K i μ ) indicated that all four analogs are μ-receptor selective. It is concluded that the performed cyclizations resulted in overall folded conformations which are incompatible with the conformational requirements of the x-receptor.