Xylazine Interaction with Opioid Receptors: Binding and Signaling

Project Summary/Abstract
This resubmitted R21 proposal is in response to the Notice of Special Interest (NOSI): Xylazine:
Understanding Its Use and Consequences, NOT-DA-24-012. The goal of this study is to determine if
xylazine and its metabolites act as agonists or positive allosteric modulators (PAMs) at the mu and
kappa opioid receptors (MOR, KOR). Additional studies will determine if xylazine and its metabolites
are agonists, antagonists, or inverse agonists at the sigma 1 (s1R) and sigma 2 receptors (s2R).
Xylazine binds with nM affinity to the sigma receptors. Illegal drugs, particularly fentanyl and stimulants, are
being mixed with xylazine to enhance drugs effects or increase street value by increasing weight. Xylazine
is not approved for human use. Xylazine is historically regarded as an α2-adrenergic receptor agonist based
on physiological studies using α2-adrenergic receptor antagonists. However, a recent studies showed that
the opioid antagonist naloxone induced withdrawal from xylazine in mice and xylazine had micromolar affinity
for the α2-adrenergic receptor. Our preliminary data shows that xylazine had a Ki value of 330 ± 13 nM
for inhibiting binding to the human KOR. Xylazine also inhibited forskolin-induced cyclic AMP levels
in CHO cells expressing the human KOR and stimulated G protein activity as measured by a BRET
assay. The following Specific Aims will characterize the potency and pharmacological properties of xylazine.
Aim 1 will determine if xylazine and its metabolites act as PAMs at the MOR and KOR as shown with G protein
and β-arrestin signaling. Bias signaling through the six Gαi/o/z proteins will be determined and compared with
results obtained with β-arrestin recruitment. In addition, experiments will determine if xylazine and its metabolites
inhibit forskolin-stimulated cAMP as an agonist or as a PAM. Radioligand binding experiments will determine if
xylazine acts as a PAM by increasing the affinity of agonists such as fentanyl for the MOR or if xylazine increases
the potency of an agonist in a functional assay by enhancing coupling of the receptor with the G protein or β-
arrestin without affecting the affinity. Experiments will determine if the opioid receptor PAM BMS986122 will
increase the potency of xylazine at the KOR and MOR. Xylazine binds to s1R and s2R with Ki values of less
than 200 nM. The pharmacological properties of xylazine at the s1R and s2R are not known. The proposed
experiments will use a BRET assay and cell proliferation assays to determine if xylazine and its metabolites are
agonists, antagonists, or PAMs at the s1R and s2R. Collectively, this study will determine the
pharmacological properties of xylazine and its metabolites, which may help explain physiological effects
observed in people taking illegal drugs mixed with xylazine.

Grant Number: 1R21DA061044-01A1
NIH Institute/Center: NIH
Principal Investigator: JEAN BIDLACK