ORL1 and opioid receptor preferences of nociceptin and dynorphin A analogues with Dmp substituted for N-terminal aromatic residues.

Nociceptin (NOC) and dynorphin A (DYN) analogues containing 2',6'-dimethylphenylalanine (Dmp) in place of Phe or Tyr in position 1 and/or 4 were synthesized and their metabolic stability and receptor-binding properties were investigated. [Dmp1]NOC(1-13)-NH2 (1) possessed high ORL1 receptor affinity comparable to that of the parent peptide with substantially improved affinities for kappa-, mu-, and delta-opioid receptors. However, Dmp4 substitution of NOC peptide (2) reduced ORL1 receptor affinity. [Dmp1]DYN(1-13)-NH2 (4) and its Dmp4 analogue (5) possessed a 3-fold greater kappa-opioid receptor affinity and improved kappa-receptor selectivity compared to the parent peptide. Analogue 4 however exhibited an unexpectedly low in vitro bioactivity (GPI assay), suggesting, the phenolic hydroxyl group at the N-terminal residue in DYN peptide is extremely important for activation of the kappa-opioid receptor. Analogue 5 possessed an improved kappa-opioid receptor selectivity with an IC50 ratio of 1(kappa)/509(mu)/211598(delta); thus, this peptide may serve as a highly selective kappa-receptor agonist for pharmacological study. Dmp1 substitution in both the NOC and DYN peptides improved metabolic stability toward these peptides, while Dmp4 substitution provided no additional metabolic stability.

Dermorphin tetrapeptide analogues with 2',6'-dimethylphenylalanine (Dmp) substituted for aromatic amino acids have high mu opioid receptor binding and biological activities.

To investigate the value of the 2',6'-dimethylphenylalanine (Dmp) residue as an aromatic amino acid substitution, we prepared analogues of the mu opioid receptor-selective dermorphin tetrapeptide Tyr-D-Arg-Phe-betaAla-NH(2) (YRFB) in which Dmp or its D-isomer replaced Tyr(1) or Phe(3). Replacing Phe(3) with Dmp essentially tripled mu receptor affinity and the receptor's in vitro biological activities as determined with the guinea pig ileum (GPI) assay but did not change delta receptor affinity. Despite an inversion of the D configuration at this position, mu receptor affinity and selectivity remained comparable with those of the L-isomer. Replacing the N-terminal Tyr residue with Dmp produced a slightly improved mu receptor affinity and a potent GPI activity, even though the substituted compound lacks the side chain phenolic hydroxyl group at the N-terminal residue. Dual substitution of Dmp for Tyr(1) and Phe(3) produced significantly improved mu receptor affinity and selectivity compared with the singly substituted analogues. Subcutaneous injection of the two analogues, [Dmp(3)]YRFB and [Dmp(1)]YRFB, in mice produced potent analgesic activities that were greater than morphine in the formalin test. These lines of evidence suggest that the Dmp residue would be an effective aromatic amino acid surrogate for both Tyr and Phe in the design and development of novel opioid mimetics.

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.