Cannabinoids (CBs), analgesic drugs used for thousands of years, were first found
in Cannabis sativa, and the multiple CBs used medicinally, such as tetrahydrocannabinol
(THC), cannabidiol (CBD) and dozens more, have complex structures. In addition to
their production by plants, CBs are naturally present in the nerves and immune systems
of humans and animals. Both exogenous and endogenous CBs carry out a variety of physiological
functions by engaging with two CB receptors, the CB1 and CB2 receptors, in the human
endocannabinoid system (ECS). Both CB1 and CB2 are G protein-coupled receptors that
share a 7-transmembrane (7TM) topology. CB1, known as the central CB receptor, is
mainly distributed in the brain, spinal cord, and peripheral nervous system. CB1 activation
in the human body typically promotes the release of neurotransmitters, controls pain
and memory learning, and regulates metabolism and the cardiovascular system. Clinically,
CB1 is a direct drug target for drug addiction, neurodegenerative diseases, pain,
epilepsy, and obesity. Unlike the exclusive expression of CB1 in the nervous system,
CB2 is mainly distributed in peripheral immune cells. Selective CB2 agonists would
have therapeutic potential in the treatment of inflammation and pain and avoid side
effects caused by currently used clinical drugs. Although significant progress has
been made in developing agonists toward CB receptors, efficient clinical drugs targeting
CB receptors remain lacking due to their complex signaling mechanisms. The recent
structural elucidation of CB receptors has greatly aided our understanding of the
activation and signal transduction mechanisms of CB receptors.
Structural characterization of CB receptors at the atomic level began in 2016, when
Professor Zhi-jie Liu’s laboratory and Dr. Zhenhua Shao in the Rosenbuam laboratory
solved the crystal structure of CB1.
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This structural information greatly facilitated the understanding of CB1 ligand recognition
and signal transduction mechanisms. Continuing this progress, Professor Zhi-jie Liu’s
laboratory determined two agonist-bound CB1 crystal structures, which not only uncover
the agonist-CB1 interactions within the orthosteric ligand-binding pocket but also
disclose the different structural features of agonist-bound and antagonist-bound CB1.
In 2019, Brian Kobilka’s group and Skiniotis’s group reported the cryo-electron microscopy
structure of CB1 bound to an agonist, FUB, and downstream heterotrimeric Gi protein.
The agonist, FUB, exhibited a high affinity for the orthostatic ligand-binding pocket
of the CB1 receptor, maintaining the CB1 receptor in an active configuration to form
a stable complex with nucleotide-free heterotrimeric Gi protein. The highly conserved
orthosteric binding pocket of CB1 poses a great challenge for the rational drug design
of potent CB1 agonists with high selectivity. Therefore, avoiding the orthosteric
site and developing allosteric regulators of CB1 have become CB1 research hotspots.
To address this issue, a collaborative effort by the teams of Dr. Shao Zhenhua and
Dr. Rosenbuam resulted in solution of the crystal structure of CB1 in complex with
an allosteric ligand.
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In this structure, the allosteric modulator ORG27569 was identified at the outside
of the 7TM bundle of the receptor, buried in the cell membrane. This new discovery
undoubtedly provides a new route for drug development toward the CB1 receptor.
Along with progress made in CB1 research, the study of another CB receptor, CB2, has
also achieved great breakthroughs. In 2019, Professor Zhi-jie Liu’s laboratory solved
the crystal structure of CB2 in complex with a rationally designed antagonist AM10257.
This structure reveals the distinct antagonist-binding mode in CB2 and provides the
molecular basis for the high-degree subtype selectivity of antagonists between CB1
and CB2. In January 2020, due to their combined efforts, the groups of Huaqiang Xu,
Xiangquan Xie, and Cheng Zhang published the three-dimensional structure of CB2 bound
to the agonist WIN 55212-2 and heterotrimeric Gi protein,
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revealing the mechanisms by which the specific agonist WIN 55212-2 activates CB2 and
CB2 interacts with the Gi protein. In the same issue of Cell, Zhi-jie Liu’s group
reported a systematic study on the structures of both CB1 and CB2 engaged with G proteins,
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and the crystal structure of agonist-bound CB2. By simultaneously solving the three-dimensional
structures of the AM12033-CB2-Gi and AM841-CB1-Gi complexes, they revealed the structural
basis for the activation of CB1 and CB2, as well as their coupling to downstream G
proteins.
CB1 and CB2 share 44% sequence homology and are simultaneously activated by many natural
CB molecules. Consistently, recent structures of CB receptors have provided the structural
basis for this phenomenon and shown that these two receptors share very similar ligand-binding
pockets at orthosteric sites, generating great challenges in the design of selective
agonists. However, structural identification of the allosteric ligand-binding sites
in CB receptors provides new hope for the development of small compounds to selectively
modulate CB receptor functions. These recent structural studies suggest that CB receptors
adopt at least three states, an antagonistic state (inactive), intermediate state
(active-like) and active state, which serves as the structural basis for complex signaling
downstream of CB receptors (Fig. 1). Recent structural characterizations of CB receptors
will greatly facilitate the design of new ligands to modulate the selective functions
of CB receptors. Notably, the CBD was approved by the Food and Drug Administration
(FDA) in 2018 to treat epilepsy. We now look forward to more drugs targeting these
two CB receptors for clinical usage in the near future.
Fig. 1
Structural understanding of Cannabinoid receptors.
Recent structural studies have revealed that two cannabinoid receptors (CB1 and CB2)
shared a conserved orthostatic binding pocket for their agonists. Notably, an extra
allosteric binding pocket was found for CB1 receptor. Both endogenous molecule cholesterol
or synthetic ligand ORG27569 was able to bind to allosteric pockets, thus regulate
activation state of CB1. Collectively, crystallographic and Cryo-EM studies have identified
at least three structural states for CB receptors, which are inactive, active-like
(intermediate) and active, indicating complex mechanisms underlying CB receptors’
activation and signaling transduction.