The mu and delta opioid receptors (MOR, DOR) modulate many of the same brain processes in vivo,
including tolerance and anti-nociception in response to opioid drugs. Many groups have found that inhibiting
the DOR through various means decreases the side effects of MOR agonists like morphine. While the basis for
this interaction is unknown, one strong possibility is the formation of a MOR-DOR heterodimer (MDOR). Many
groups have shown that the MDOR can form in heterologous expression systems in vitro, with a unique
pharmacology and signal transduction profile when compared to the monomeric forms. Devi and colleagues
recently developed an MDOR selective antibody, and used this antibody to demonstrate MDOR upregulation in
the brains of mice chronically treated with morphine. Other experiments suggested that the MDOR promotes
tolerance, dependence, and drug seeking in vivo. While MDOR selective agonists have been developed, no
known drug-like antagonist has ever been created to our knowledge, limiting our ability to determine the role of
MDOR in vivo. To address this lack, we created a novel series of potential selective peptide MDOR
antagonists by connecting low affinity MOR (H-Tyr-Pro-Phe-D1Nal-NH2) and moderate affinity DOR (Tyr-Tic-
OH) pharmacophores with a variable length (15-42 atom) flexible polyamide spacer. We tested this preliminary
series in vitro using radioligand binding and 35S-GTP¿S coupling in antagonist mode using MOR, DOR, and
MDOR expressing cell lines. We found compelling evidence that our preliminary series selectively targets the
MDOR, with a selectivity ratio of ~91 fold for our best compound, the 24 atom spacer length. Building from this
initial success, we aim in the current proposal to explore the MDOR structure-activity relationship (SAR) of our
compound series, and improve compound potency and selectivity at the MDOR by modulating pharmacophore
affinity (increased MOR, decreased DOR) as well as linker rigidity (minimally rigid [gly-gly-pro], moderately
rigid [gly-pro]). These studies will be performed in an iterative development process, using the best compound
from each series to inform the next series, minimizing compound number. After this SAR development, we will
use our most potent and selective compound to begin to test MDOR activity in vivo. This will be accomplished
by intracerebroventricular (icv) and intrathecal (it) injection of MDOR antagonist into mice prior to tail-flick anti-
nociception evoked by MOR (DAMGO), DOR (DSLET), and MDOR (CYM51010) selective agonists. These
studies will demonstrate the selectivity of our compound in vivo. We will also pre-treat mice with MDOR
antagonist prior to the induction of tolerance and dependence with morphine to begin to explore the in vivo role
of the MDOR in these opioid side effects, which has been suggested by the literature. In the short term, this
initial optimized series will provide a useful tool to interrogate the role of the MDOR in vivo. Long term, through
modifications to improve drugability (glycosylation, etc.), these compounds may provide the basis for selective
MDOR targeted therapeutics to improve the side effect profile of opioid therapy.
