Opioid drugs such as morphine are the primary treatment for post-operative and chronic pain conditions through their actions at the mu opioid receptor (MOR). However, development of tolerance to and dependence on these drugs limits their usefulness. Thus, a novel ligand with analgesic properties but lacking tolerance and dependence liability would be of great value in clinical settings. Several published reports have documented that blockade of the delta opioid receptor (DOR), through either antagonism or knockdown of the receptor, leads to decreased development of tolerance or dependence in rodents chronically treated with the MOR agonist morphine (1
). Evidence for interactions between MOR and DOR leading to altered signaling profiles has also been discussed in reference to locomotor sensitization (5
) while other research has shown MOR and DOR cell-surface receptor expression levels to be linked (7
). The studies described above have highlighted roles for both MOR and DOR in the development of morphine-provoked analgesic tolerance and/or dependence.
For pain relief, an optimal therapeutic would be a single drug containing two opioid receptor actions: MOR agonism to promote analgesia along with DOR antagonism to prevent MOR tolerance and dependence development during chronic administration. Co-administration of two drugs acting separately at each individual receptor could be hampered by increased ‘off-target’ effects, differences in pharmacokinetic profiles, and user compliance (9
). We have therefore focused on the development and characterization of peptide ligands that simultaneously display MOR agonism and DOR antagonism while binding with equivalent affinity to each receptor.
The development of such bifunctional or mixed-efficacy ligands has become a topic of increasing interest in several therapeutic areas (10
). For example, bifunctional ligands have been proposed with DOR-KOR efficacy (12
) or MOR-cholecystokinin receptor activities (13
), targeting tolerance liability (14
) or toward novel ligands for treatment of cocaine abuse (15
). Similarly, early studies indicating that blockade of DOR reduces the development of tolerance to MOR agonists have stimulated several investigations into mixed MOR agonist/DOR antagonist compounds (18
Small peptides and, in particular, receptor-specific ligands provide a means to determine structural or conformational requirements of binding to a particular membrane-bound receptor. We have previously used molecular modeling and conformationally restricted cyclic peptide ligands as tools to analyze determinants of ligand binding to MOR, DOR, and kappa opioid receptors (KOR). Through this methodology, we developed peptides that bind selectively to different opioid receptors (21
) and have expanded these concepts to develop peptides that bind non-selectively but with differing efficacy profiles. Recent work (19
) described the development of a non-selective opioid cyclic pentapeptide that displayed MOR agonism, DOR partial agonism, and KOR agonism. The pentapeptides characterized in that study were designed to have decreased DOR efficacy compared with the parent ligand due to steric interactions inferred from ligand docking to a model of the active state of DOR. Indeed, replacement of Phe residues in position 3 or 4 of the pentapeptide with bulkier 1-naphthylalanine (1-Nal) or 2-naphthylalanine (2-Nal) residues produced analogues with decreased DOR efficacy, in agreement with our modeling studies (19
We have extended this approach by re-examining previously synthesized, non-selective opioid peptides. As our earlier studies were aimed at development of selective opioid ligands for MOR, DOR, or KOR, resulting non-selective cyclic peptides were not evaluated beyond binding affinity. Re-evaluation of these previously synthesized ligands has led to the identification of potential leads with MOR agonist/DOR antagonist properties. Among these ligands were several analogs of the MOR-selective tetrapeptide JOM-6 (Tyr-c(SCH2
)) and the DOR-selective tetrapeptide JOM-13 (Tyr-c(SS)[DCys-Phe-DPen]OH (25
) (where DPen is D-penicillamine (β, β, dimethyl-D-cysteine) and c(SCH2
S) and c(SS) denote cyclization through the side chain sulfurs of DCys and DPen via
an ethylene dithioether or a disulfide, respectively) in which Phe3
was replaced with a bulkier or more constrained aromatic residue that might be expected to bind differently to the active and inactive states of opioid receptors. Evaluation and further modification of the most promising candidates led to the two new analogs reported here, KSK-102 (Dmt-c(SCH2
) and KSK-103 (Dmt-c(SCH2
S)[DCys-Aci-DPen]OH), where Dmt is 2′, 6′-dimethyltyrosine and Aci is 2-aminoindane-2-carboxylic acid (). Of these peptides, KSK-103 displayed the desired bifunctional profile and behaved in vitro
as a MOR agonist with greater potency than the clinical standard morphine. KSK-103 was also found to be a DOR antagonist at the level of receptor-G protein stimulation and at inhibition of the downstream effector enzyme adenylyl cyclase. By comparison, DIPP(Ψ)NH2
(where Tic is tetrahydroisoquinoline-3-carboxylic acid) (20
)) and UFP-505 (Dmt-Tic-GlyNH-benzyl) (18
)), two previously described MOR agonist/DOR antagonist bifunctional peptides with reported decreased propensity to produce tolerance relative to morphine, displayed partial DOR agonism in the adenylyl cyclase assay and had less desirable receptor binding properties.
Structures of parent peptides (A) JOM-6 and (B) JOM-13 and new analogs (C) KSK-102 and (D) KSK-103.