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Teaching an Old Dog New Tricks for Achieving CB2‑Selective
Inverse Agonism
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Morgane Mando, Christopher W. Cunningham, and Alexander J. Grenning
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Three laboratories team up to “flip the switch”
and turn a CB2R agonist into a fluorescent CB2R
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inverse agonist.
T
he endocannabinoid signaling system (ECSS) is a
“master regulator” of diverse physiologic processes
throughout the body. As such, modulating the
ECSS has broad therapeutic potential, from treating pain,
stress, and anxiety to osteoporosis, neurodegenerative
disease, and perhaps even cancer. Despite its potential as
a therapeutic target, the expression and function of the CB2
cannabinoid receptor (CB2R) in the ECSS function remain
elusive. Where is it (?), what does it do (?), and is it
druggable (?) are still “big questions” on the table!1 And
versions of these questions are old. Endocannabinoid
research dates back to the 1940s, well before there was
any knowledge of the endocannabinoid system. Phytocan-
nabinoids, natural products that we now know target the
ECSS, were being isolated, elucidated, synthesized, and
studied for therapeutic potential at this time. Furthermore,
some of this early research was carried out by none other
than organic chemistry giant Dr. Roger Adams (1889−
1971) and his research team, who can be credited with the
first total synthesis of a phytocannabinoid. 2 Large
contributions to cannabinoid research must also be linked
to the late great Dr. Raphael Mechoulam (1930−2023),
who had a storied career in the field. Among the many
important molecules that his team at Hebrew University
developed is HU-308, a cannabinoid receptor 2 (CB2R)-
selective agonist.3 In this issue of ACS Central Science, we
witness firsthand how research synergy among computa-
tional chemists, synthetic chemists, chemical biologists,
and pharmacologists can yield profound new findings for
endocannabinoid system research possible only through
Published 2024 by American Chemical
Society
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collaboration. In this case, it is teaching the old dog
(HU-308) a valuable new trick: to function as a CB2R
inverse agonist and potentially address some of these
aforementioned big questions!
We witness firsthand how
research synergy among
computational chemists,
synthetic chemists, chemical
biologists, and pharmacologists
can yield profound new findings
for endocannabinoid system
research only possible through
collaboration.
To best appreciate this scientific work, it is important to
first discuss some basic pharmacology. When an agonist
binds a receptor, it stabilizes that receptor in its active state,
which can be thought as “switching it on.” Conversely, an
inverse agonist stabilizes a constitutively active receptor
in its inactive state, thereby “switching off” its ability to
promote a signal. When a receptor is activated by an agonist,
intracellular processes can be triggered that may interfere
with receptor expression and function: pathway uncoupling,
receptor internalization, and receptor degradation can all
occur when an agonist probe is used. This is why an inverse
agonist profile is most desirable in a fluorescent probe:
binding to the receptor is unlikely to disrupt its expression
Published: May 8, 2024
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ACS Cent. Sci. 2024, 10, 946−948
ACS Central Science
Figure 1. Illustration of active (left) and inactive (right) conformations of CB2R.
at the cell surface. With the goal in mind of turning a high-
selectivity CB2R agonist into an inverse agonist, the team
focused on a region of the receptor responsible for con-
trolling the active/inactive equilibrium (Figure 1). More
specifically, one residue, TRP2586.48, serves as the toggle
switch for CB2R (and 78% of other G protein-coupled
receptors, GPCRs) activation/inactivation depending on its
conformation. Indeed, useful molecular probes that retain
their inverse agonist activity and affinity are still in need of
discovery to broaden endocannabinoid system understanding
and therapeutic development. Considering CB2R’s role in
modulating pain, precision control of function here is of timely
relevance as we continue to respond to the opioid crisis.
The strategy of turning HU-308 from an agonist to an
inverse agonist is elegant in its simplicity. The team took
inspiration from two recent crystal structures of CB2R in
active4 and inactive5 conformations when HU-308 and
AM10257 are bound, respectively. Noting that a phenyl ring
of AM10257 prevents the toggle switch from reaching
an active state conformation, a “hybrid” was designed that
incorporated a stereogenic 2′-phenyl group into the
dimethylheptyl tail of HU-308. The stereogenic 2′-phenyl
group proved to be a double-edged sword: molecular
docking suggested that the resulting compound 1 would be
an inverse agonist, but including this group complicates de
novo chemical synthesis. The team devised a racemic, chiral
separation-based synthesis for these novel probes including
some synthetic flexibility for attaching additional functional
groups at molecular termini to improve affinity and selectivity
as well as for the attachment of fluorescent probes. Excitingly,
the parent structures (S)-1 and (R)-1 display chirality-specific
(in favor of the R enantiomer) and low nanomolar affinity and
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selectivity at CB2R. And with a stroke of luck, the addition of
the fluorescent probe to the parent ligand does not disrupt the
ligand affinity, efficacy, and selectivity!
The stereogenic 2′-phenyl group
proved to be a double-edged
sword: molecular docking
suggested that the resulting
compound 1 would be an inverse
agonist, but including this group
complicates de novo chemical
synthesis.
That the new scaffold (R)-1 retains its desirable phar-
macodynamic properties when complexed to different fluores-
cent probes is particularly interesting and useful. Fluorescent
probes themselves substantially impact the molecular
weight, hydrogen bonding, and ionic properties of the
probe, which can interfere with probe function.6,7 Indeed,
compounds (R)-7 and (R)-9 have attached dyes that have
multiple weakly acidic and basic functional groups that are
ionized at physiological pH. This could have disrupted
ligand binding due to potential incompatibilities with the
hydrophobic nature of the CB2R binding site. The unpredictable
nature of attaching a dye to a probe was previously reported by
another group.8 In their series, attaching different dyes and
linkers to the chromenepyrazole pharmacophore resulted in
agonists as well as inverse agonists. That scaffold 1 resulted
exclusively in CB2R inverse agonists suggests that this
pharmacophore operates in a different way and could be
amenable to many more modifications.
It remains to be seen whether the ultimate comment in
the manuscript will be realized, whether “the strategy and
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experimental framework disclosed herein may aid in the Crystal Structure of the Human Cannabinoid Receptor CB2. Cell
2019, 176, 459−467.
structure-based design of agonists, antagonists, and inverse (6) Cooper, A. G.; MacDonald, C.; Glass, M.; Hook, S.; Tyndall, J.
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Tools: Exploration of Linker and Fluorophore Attachment. Eur. J.
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CWxP here, while others include NPxxY, D/ERY, etc.�and (7) Cunningham, C. W.; Mukhopadhyay, A.; Lushington, G. H.;
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switching off the CB2R function will switch on creative (9) Filipek, S. Molecular switches in GPCRs. Curr. Opin. Struct. Biol.
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science muscles around the world, encouraging more (10) Kearney, S. E.; Gangano, A. J.; Barrus, D. G.; Rehrauer, K. J.;
collaborative teams to assemble to achieve endocannabinoid Reid, T.-E. R.; Navaratne, P. V.; Tracy, E. K.; Roitberg, A.; Ghiviriga,
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Author Information
Corresponding Authors
Christopher W. Cunningham − Department of
Pharmaceutical Sciences, Concordia University Wisconsin,
Mequon, Wisconsin 53097, United States; orcid.org/
0000-0002-4468-2213; Email: chris.cunningham@
cuw.edu
Alexander J. Grenning − Department of Chemistry, University
of Minnesota, Minneapolis, Minnesota 55455, United States;
orcid.org/0000-0002-8182-9464; Email: grenning@
umn.edu
Author
Morgane Mando − Department of Chemistry, University of
Minnesota, Minneapolis, Minnesota 55455, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acscentsci.4c00673

Funding
We gratefully acknowledge the National Institute of General
Medical Science (R35 GM137893-01) for providing support
for this work.
Notes
The authors declare no competing financial interest.

■
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