‫‪Elucidating the activation mechanisms of the pain receptors, TRPA1 and TRPV1 by‬‬

‫‪cannabinoids‬‬

‫פענוח מנגנוני ההפעלה של הרצפטורים לכאב על ידי קנבינואידים‬

‫מרצה‪ :‬גלעד נוי‬

‫מנחה‪ :‬פרופ' אבי פריאל‬
‫ההרצאה תתקיים ביום חמישי‪28/04/2022 ,‬‬
‫בשעה ‪12:00‬‬

‫**הקישור לזום יישלח בהמשך‬

 Elucidating the activation mechanisms of the pain receptors, TRPA1 and TRPV1 by cannabinoids
‫פענוח מנגנוני ההפעלה של הרצפטורים לכאב על ידי קנבינואידים‬
Gilad Noy
Supervisor- prof. Avi Priel
Cannabinoids, both endogenous and exogenous, are well known to affect the pain pathway. Evidence for cannabis
as treatment for pain and inflammation can be traced back to the beginning of recorded history. To date, the most
common medically approved use of these substances is for analgesic purposes, treating the most debilitating
chronic pain disorders. However, little is known about their cellular and molecular mechanisms inducing pain
relief.
Since 1990 two cannabinoid-specific receptors, the metabotropic CB1 and CB2, have been recognized and
extensively studied. However, other receptors have been shown to play a role in the endocannabinoid system, and
an ionotropic component is hypothesized to participate, as in other neurotransmitter systems. Moreover,
pharmacological or genetic blockade of CB1 and CB2 does not abolish all effects of cannabinoids, including
cannabinoid-induced analgesia. Nevertheless, the agreed-upon criteria a "CB3" receptor should fulfill are currently
un-met by any non-CB1, non-CB2, receptor, or ion channel.
The transient receptor potential ("TRP") ion channels are involved in the transduction of a great range of stimuli.
Five types of TRP channels belonging to three subfamilies have been suggested to interact with cannabinoids of
endo or phyto origin, possibly serving as the "ionotropic cannabinoids receptors."
This work aims to provide an understanding of the molecular mechanisms of cannabinoids-evoked activation of
somatosensory TRP channels, our main goal is elucidating the molecular mechanisms by which the active
components of cannabis bind and activate TRPA1 and TRPV1. This mechanism is highly intriguing considering
that all other TRPA1 and TRPV1 activators, including various general anesthetics evoke pain sensation, while
exogenous cannabinoids induce pain relief.

TRPA1 is the only member of the mammalian TRPA sub-family. It is best known as a promiscuous electrophile
receptor, activated by irritants through reversible covalent interactions of key cysteine residues at the ankyrin repeat
domains that cause allosteric channel opening. However, several non-electrophilic compounds have been shown
to activate TRPA1 independently of the reactive cysteines, suggesting an alternative much sought-after activation
mechanism. Phytocannabinoids were described as TRPA1 agonists during the channel cloning. THC, in particular,
has been shown to activate it via a yet unknown cannabinoid binding site.
We characterized the TRPA1 THC interactions by calcium imaging and electrophysiology. We found that THC is
a potent and efficacious TRPA1 agonist, which is relatively insensitive to known antagonists. Contrary to most
known agonists, THC is non-electrophilic, does not affect the channel through the cysteine residues, and is
independent of intracellular components, making it an outstanding research tool. In order to define the
phytocannabinoids binding site, we performed site-directed mutagenesis based on the previously suggested binding
sites of other non-electrophilic agonists and antagonists. While THC activity was affected, no single or multiple
mutagenic combinations could abolish its activity. During this study, a novel binding site was described for a
synthetic non-electrophilic agonist (GNE551). We analyzed this new binding site and surprisingly abolished THC-
evoked TRPA1 activation with a single-point mutation. Using molecular docking, we found several residues in
this binding site affecting THC activity dramatically. Thus, our results unveil additional features of this novel
GNE511 binding site and point to it as the phytocannabinoid binding site of TRPA1.

TRPV1 is pivotal in detecting and responding to specific noxious stimuli such as heat, low pH, toxins, and
vanilloids. Vanilloids are a large and diverse family of pain evoking molecules that activate TRPV1
through the vanilloid binding site (VBS), a highly conserved area located in the intracellular domain. These ligands
include exogenous vanilloids such as capsaicin, the "hot" chemical in chili peppers, and endogenous cannabinoids
like anandamide. While the binding of vanilloid ligands like capsaicin and resiniferatoxin has been well studied,
the binding mode of phyto-cannabinoids at TRPV1 is less understood. CBD, the main non-psychoactive
component of the cannabis plant, is of great interest because of its anti-inflammatory, analgesic, anti-anxiety, and
anti-tumor properties. It has little to no direct effect on the cannabinoid GPCR receptors, but activates TRPV1.
We conducted a series of live-cell calcium imaging and electrophysiology testings, directly determining the CBD
dose-response, indicating that CBD is a TRPV1 partial agonist, which binds to the receptor through the VBS and
competes with other vanilloid agonists. Possibly explaining some of the phytocannabinoids pain-reducing effects,
replacing the efficacious vanilloid activation of the receptor with its low, likely sub-threshold in vivo activity.

In summary, cannabinoids have been used to treat pain and inflammation for centuries, with a surge in research
and medical use over the last decades. The results of this study identify novel molecular targets and
pharmacological mechanisms for future development of pain pathway-specific analgesic, a yet unresolved medical
need.