Discovery of tripeptide-derived multifunctional ligands possessing delta/mu opioid receptor agonist and neurokinin 1 receptor antagonist activities.

Several bifunctional peptides were synthesized and characterized based on the pentapeptide-derived ligand NP30 (1: Tyr-DAla-Gly-Phe-Gly-Trp-O-[3',5'-Bzl(CF3)2]). Modification and truncation of amino acid residues were performed, and the tripeptide-derived ligand NP66 (11: Dmt-DAla-Trp-NH-[3',5'-(CF3)2-Bzl]) was obtained based on the overlapping pharmacophore concept. The Trp(3) residue of ligand 11 works as a message residue for both opioid and NK1 activities. The significance lies in the observation that the approach of appropriate truncation of peptide sequence could lead to a tripeptide-derived chimeric ligand with effective binding and functional activities for both mu and delta opioid and NK1 receptors with agonist activities at mu and delta opioid and antagonist activity at NK1 receptors, respectively.

Truncation of the peptide sequence in bifunctional ligands with mu and delta opioid receptor agonist and neurokinin 1 receptor antagonist activities.

The optimization and truncation of our lead peptide-derived ligand TY005 possessing eight amino-acid residues was performed. Among the synthesized derivatives, NP30 (Tyr(1)-DAla(2)-Gly(3)-Phe(4)-Gly(5)-Trp(6)-O-[3',5'-Bzl(CF3)2]) showed balanced and potent opioid agonist as well as substance P antagonist activities in isolated tissue-based assays, together with significant antinociceptive and antiallodynic activities in vivo.

The biological activity and metabolic stability of peptidic bifunctional compounds that are opioid receptor agonists and neurokinin-1 receptor antagonists with a cystine moiety.

In order to improve metabolic stability, a ring structure with a cystine moiety was introduced into TY027 (Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-[3',5'-(CF(3))(2)Bzl]), which is a lead compound of our developing bifunctional peptide possessing opioid agonist and NK1 antagonist activities. TY038 (Tyr-cyclo[D-Cys-Gly-Phe-Met-Pro-D-Cys]-Trp-NH-[3',5'-(CF(3))(2)Bzl]) was found as a highly selective delta opioid agonist over mu receptor in conventional tissue-based assays, together with an effective NK1 antagonist activity and good metabolic stability with more than 24h half life in rat plasma.

A structure-activity relationship study and combinatorial synthetic approach of C-terminal modified bifunctional peptides that are delta/mu opioid receptor agonists and neurokinin 1 receptor antagonists.

A series of bifunctional peptides with opioid agonist and substance P antagonist bioactivities were designed with the concept of overlapping pharmacophores. In this concept, the bifunctional peptides were expected to interact with each receptor separately in the spinal dorsal horn where both the opioid receptors and the NK1 receptors were found to be expressed, to show an enhanced analgesic effect, no opioid-induced tolerance, and to provide better compliance than coadministration of two drugs. Compounds were synthesized using a two-step combinatorial method for C-terminal modification. In the method, the protected C-terminal-free carboxyl peptide, Boc-Tyr( tBu)- d-Ala-Gly Phe-Pro-Leu-Trp(Boc)-OH, was synthesized as a shared intermediate using Fmoc solid phase chemistry on a 2-chlorotrityl resin. This intermediate was esterified or amidated in solution phase. The structure-activity relationships (SAR) showed that the C-terminus acted as not only a critical pharmacophore for the substance P antagonist activities, but as an address region for the opioid agonist pharmacophore that is structurally distant from the C-terminal. Among the peptides, H-Tyr- d -Ala-Gly-Phe-Pro-Leu-Trp-NH-Bzl ( 3) demonstrated high binding affinities at both delta and mu receptors ( K i = 10 and 0.65 nM, respectively) with efficient agonist functional activity in the mouse isolated vas deferens (MVD) and guinea pig isolated ileum (GPI) assays (IC 50 = 50 and 13 nM, respectively). Compound 3 also showed a good antagonist activity in the GPI assay with substance P stimulation ( K e = 26 nM) and good affinity for the hNK1 receptor ( K i = 14 nM). Consequently, compound 3 is expected to be a promising and novel type of analgesic with bifunctional activities.

Design, synthesis, and biological evaluation of novel bifunctional C-terminal-modified peptides for delta/mu opioid receptor agonists and neurokinin-1 receptor antagonists.

A series of bifunctional peptides that act as agonists for delta and mu opioid receptors with delta selectivity and as antagonist for neurokinin-1 (NK1) receptors were designed and synthesized for potential application as analgesics in various pain states. The peptides were characterized using radioligand binding assays and functional assays using cell membrane and animal tissue. Optimization was performed on the fifth residue which serves as an address moiety for both receptor recognitions. It had critical effects on both activities at delta/mu opioid receptors and NK1 receptors. Among the synthesized peptides, H-Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-O-3,5-Bzl(CF3) 2 (5) and H-Tyr-D-Ala-Gly-Phe-Nle-Pro-Leu-Trp-O-3,5-Bzl(CF3)2 (7) had excellent agonist activity for both delta opioid and mu opioid receptors and excellent antagonist activity for NK1 receptors. These results indicate that the rational design of multifunctional ligands with opioid agonist and neurokinin-1 antagonist activities can be accomplished and may provide a new tool for treatment of chronic and several pain states.

Discovery of a potent and efficacious peptide derivative for δ/µ opioid agonist/neurokinin 1 antagonist activity with a 2',6'-dimethyl-L-tyrosine: in vitro, in vivo, and NMR-based structural studies.

Multivalent ligands with δ/µ opioid agonist and NK1 antagonist activities have shown promising analgesic potency without detectable sign of toxicities, including motor skill impairment and opioid-induced tolerance. To improve their biological activities and metabolic stability, structural optimization was performed on our peptide-derived lead compounds by introducing 2',6'-dimethyl-L-tyrosine (Dmt) instead of Tyr at the first position. The compound 7 (Dmt-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-[3',5'-(CF(3))(2)-Bzl]) showed improved multivalent bioactivities compared to those of the lead compounds, had more than 6 h half-life in rat plasma, and had significant antinociceptive efficacy in vivo. The NMR structural analysis suggested that Dmt(1) incorporation in compound 7 induces the structured conformation in the opioid pharmacophore (N-terminus) and simultaneously shifts the orientation of the NK1 pharmacophore (C-terminus), consistent with its affinities and activities at both opioid and NK1 receptors. These results indicate that compound 7 is a valuable research tool to seek a novel analgesic drug.

Biological and conformational evaluation of bifunctional compounds for opioid receptor agonists and neurokinin 1 receptor antagonists possessing two penicillamines.

Neuropathic pain states and tolerance to opioids can result from system changes in the CNS, such as up-regulation of the NK1 receptor and substance P, lead to antiopioid effects in ascending or descending pain-signaling pathways. Bifunctional compounds, possessing both the NK1 antagonist pharmacophore and the opioid agonist pharmacophore with delta-selectivity, could counteract these system changes to have significant analgesic efficacy without undesirable side effects. As a result of the introduction of cyclic and topological constraints with penicillamines, 2 (Tyr-cyclo[d-Pen-Gly-Phe-Pen]-Pro-Leu-Trp-NH-[3',5'-(CF(3))(2)-Bzl]) was found as the best bifunctional compound with effective NK1 antagonist and potent opioid agonist activities, and 1400-fold delta-selectivity over the mu-receptor. The NMR structural analysis of 2 revealed that the relative positioning of the two connected pharmacophores as well as its cyclic and topological constraints might be responsible for its excellent bifunctional activities as well as its significant delta-opioid selectivity. Together with the observed high metabolic stability, 2 could be considered as a valuable research tool and possibly a promising candidate for a novel analgesic drug.

Improving metabolic stability by glycosylation: bifunctional peptide derivatives that are opioid receptor agonists and neurokinin 1 receptor antagonists.

In order to obtain a metabolically more stable analgesic peptide derivative, O-beta-glycosylated serine (Ser(Glc)) was introduced into TY027 (Tyr-d-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-3',5'-Bzl(CF(3))(2)) which was a previously reported bifunctional compound with delta/micro opioid agonist and neurokinin-1 receptor antagonist activities and with a half-life of 4.8 h in rat plasma. Incorporation of Ser(Glc) into various positions of TY027 gave analogues with variable bioactivities. Analogue 6 (Tyr-d-Ala-Gly-Phe-Nle-Pro-Leu-Ser(Glc)-Trp-NH-3',5'-Bzl(CF(3))(2)) was found to have effective bifunctional activities with a well-defined conformation with two beta-turns based on the NMR conformational analysis in the presence of DPC micelles. In addition, 6 showed significant improvement in its metabolic stability (70 + or - 9% of 6 was intact after 24 h incubation in rat plasma). This improved metabolic stability, along with its effective and delta selective bifunctional activities, suggests that 6 could be an interesting research tool and possibly a promising candidate as a novel analgesic drug.

The importance of micelle-bound states for the bioactivities of bifunctional peptide derivatives for delta/mu opioid receptor agonists and neurokinin 1 receptor antagonists.

To provide new insight into the determining factors of membrane-bound peptide conformation that might play an important role in peptide-receptor docking and further biological behaviors, the dodecylphosphocholine (DPC) micelle-bound conformations of bifunctional peptide derivatives of delta-preferring opioid agonists and NK1 antagonists (1: Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-O-3,5-Bzl(CF 3) 2; 2: Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-3,5-Bzl(CF 3) 2; 3: Tyr-D-Ala-Gly-Phe-Met-Pro-Leu-Trp-NH-Bzl) were determined based on 2D NMR studies. Although the differences in the primary sequence were limited to the C-terminus, the obtained NMR conformations were unexpectedly different for each compound. Moreover, their biological activities showed different trends in direct relation to the compound-specific conformations in DPC micelles. The important result is that not only were the NK1 antagonist activities different (the pharmacophore located at the C-terminus)but the opioid agonist activities (this pharmacophore was at the structurally preserved N-terminus) also were shifted, suggesting that a general conformational change in the bioactive state was induced due to relatively small and limited structural modifications.