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ORIGINAL RESEARCH
Evaluation of phytocannabinoids from high-potency Cannabis
sativa using in vitro bioassays to determine structure–activity
relationships for cannabinoid receptor 1 and cannabinoid
receptor 2
Afeef S. Husni •Christopher R. McCurdy •Mohamed M. Radwan •
Safwat A. Ahmed •Desmond Slade •Samir A. Ross •
Mahmoud A. ElSohly •Stephen J. Cutler
Received: 9 October 2013 / Accepted: 17 February 2014 / Published online: 26 April 2014
ÓSpringer Science+Business Media New York 2014
Abstract Cannabis has been around for thousands of
years and has been used recreationally, medicinally, and
for fiber. Over 500 compounds have been isolated from
Cannabis sativa with approximately 105 being cannabi-
noids. Of those 105 compounds, D
9
-tetrahydrocannabinol
has been determined as the primary constituent, which is
also responsible for the psychoactivity associated with
Cannabis. Cannabinoid receptors belong to the large
superfamily of G protein-coupled receptors. Targeting the
cannabinoid receptors has the potential to treat a variety of
conditions such as pain, neurodegeneration, appetite,
immune function, anxiety, cancer, and others. Developing
in vitro bioassays to determine binding and functional
activity of compounds has the ability to lead researchers to
develop a safe and effective drug that may target the
cannabinoid receptors. Using radioligand binding and
functional bioassays, a structure–activity relationship for
major and minor cannabinoids was developed.
Keywords Cannabis Tetrahydrocannabinol
Structure–activity relationship Cannabinoid
Cannabinoid receptor 1 Cannabinoid receptor 2
Introduction
Marijuana, also known as Cannabis, is defined as any
preparation of the Cannabis plant used to elicit psychoactive
effects whether it is recreational or medicinal. According to
the 2004 World Drug Report, 3.7 % of the population
15–64 years of age consumed marijuana from 2001–2003
(2004 World Drug Report; 2010 World Drug Report). The
use of marijuana is associated with numerous pharmaco-
logical effects; most, but not all may be attributed to
D
9
-tetrahydrocannabinol (D
9
-THC) (Gaoni and Mechoulam,
1964). The combination of D
9
-THC and other compounds
from Cannabis, such as cannabidiol (CBD), may exhibit
specific pharmacological effects. Since D
9
-THC is primarily
responsible for the psychoactive effects of Cannabis, sci-
entists have learned how to genetically increase the con-
centration of D
9
-THC within plants to produce a higher
percentage (ElSohly, 2000). Since 1993, the concentration of
D
9
-THC within marijuana has increased from 3.4 % to
approximately 8.8 % in 2008 (Mehmedic et al., 2010).
Cannabis use has been reported for thousands of years
and is not only associated with recreational or medicinal
use, but it is also used for fiber and seeds. Cannabis pro-
duces a durable fiber, called hemp, for the manufacturing
of rope and fabric. Along with the production of hemp, the
seeds of Cannabis are rich in unsaturated fatty acids. The
use of Cannabis dates back to around 2000 BC when the
Chinese invented hemp paper (Peters and Nahas, 1999). In
Dr. Mahmoud ElSohly’s book published in 2010, Mari-
juana and the Cannabinoids, it is noted that Cannabis
A. S. Husni C. R. McCurdy S. J. Cutler (&)
Department of Medicinal Chemistry, School of Pharmacy,
University of Mississippi, University, MS 38677, USA
e-mail: cutler@olemiss.edu
C. R. McCurdy M. M. Radwan S. A. Ahmed D. Slade
S. A. Ross M. A. ElSohly S. J. Cutler
National Center for Natural Products Research, School
of Pharmacy, University of Mississippi, University,
MS 38677, USA
S. A. Ross
Department of Pharmacognosy, School of Pharmacy, University
of Mississippi, University, MS 38677, USA
M. A. ElSohly
Department of Pharmaceutics, School of Pharmacy, University
of Mississippi, University, MS 38677, USA
123
Med Chem Res (2014) 23:4295–4300
DOI 10.1007/s00044-014-0972-6
MEDICINAL
CHEMISTR
Y
RESEARCH
serves as a recreational drug and, more importantly, as a
potential therapeutic treatment for numerous diseases such
as wasting syndrome, obesity, and multiple sclerosis
(Clarke and Watson, 2010).
The CB1 receptor is encoded by the CNR1 gene, and is
widely expressed throughout the brain. It is also expressed
in the spinal cord, pituitary gland, thyroid gland, adrenal
gland, fat cells, muscle cells, liver cells, digestive tract,
lungs, kidneys, and male and female reproductive organs.
Gerrard et al. (1991) cloned the rat cannabinoid receptor;
shortly after, isolation of a human CB1 receptor cDNA was
reported. The amino acid sequence showed 472 total amino
acids, one less than other mammalian species (Matsuda,
1991). This receptor has been the target of much research
due to the pharmacological effects associated with its
activation (Pertwee, 1997).
Shortly after characterizing and cloning the human CB1
receptor, the CB2 receptor was cloned (Devane, 1992). The
CNR2 gene encodes the CB2 receptor, and the amino acid
sequence shows approximately 360 total amino acids. The
CB1 and CB2 receptors have approximately 44 % simi-
larity of their amino acid sequences (Munro et al., 1993).
The CB2 receptors are widely expressed throughout the
peripheral tissues of the immune system, spleen, tonsils,
thymus, and gastrointestinal system. Further investigation
of CB2 receptors led to the discovery that these receptors
are also expressed within the brain (Onaivi et al., 2006).
The CB2 receptors play a major role in inflammatory dis-
eases due to their interaction with these receptors in the
immune system (Cabral and Griffin-Thomas, 2009).
This misuse of Cannabis negatively affects the people
who need help with unwanted side effects associated with
cancer chemotherapy and AIDS. Cannabis is not only used
to help those suffering from cancer chemotherapy and
AIDS (Harrigan, 2001) (Berry and Mechoulam, 2002), but
it also lowers intraocular pressure for those with glaucoma,
acts as a pain reliever, and more recently has been found to
help with symptoms of multiple sclerosis, Alzheimer’s, and
depression (Benito, 2003). Therefore, researchers are
attempting to formulate synthetic cannabinoids that
resemble the compounds isolated from Cannabis, but do
not express psychotropic properties.
During the past century, and especially in the past
20 years, researchers have investigated D
9
-Tetrahydrocan-
nabinol (THC), the primary active constituent in marijuana,
and it derivatives, for medical uses (Ahmed et al., 2008;
Ross et al., 2005). These uses include wasting-syndrome in
AIDS patients, anti-anxiety, antiemetic (in patients
receiving cancer chemotherapy), analgetic (especially in
cancer pain), anti-inflammatory, and neuroprotective
effects, among others. The development of treatment
strategies for these disorders remains a high priority. The
broad effects associated with THC and other cannabinoids
are directly related to the endocannabinoid system, which
is a major regulatory system of the central and peripheral
nervous system. The endocannabinoid system, composed
of cannabinoid receptors (CB1 and CB2), their endogenous
ligands, and the enzymes responsible for metabolizing
these ligands, is linked to the control of various physio-
logical processes. These include depression, anxiety, and
drug addiction, among others, and it is clear that the
endocannabinoid system provides a valuable new thera-
peutic target for a variety of disorders (Lambert, 2009).
Though much effort has been exerted in discovering and
developing cannabinoid receptor ligands, there are still few
marketed drugs in this category, and hence there is great
potential and urgency for application of rational drug
design for discovery of novel cannabinoid ligands. This
study will help scientist gain a better understanding of the
structure–activity relationship (SAR) of ligands binding
affinity for these receptors.
Materials and methods
Plant material
Cannabis sativa plants were grown from high-potency
Mexican seeds. The seeds and plants were authenticated by
Dr. Suman Chandra, University of Mississippi, and a
specimen (S1310V1) was deposited at the Coy Waller
Complex, National Center for Natural Products Research,
School of Pharmacy, University of Mississippi. Whole
buds of mature female plants were harvested, air-dried, and
packed in barrels; and stored at -24 °C.
Cell culture
Parental HEK293 cells were stably transfected via elec-
troporation with full-length human recombinant cDNA for
cannabinoid receptor subtypes 1 and 2. The human
recombinant cDNA was obtained from Origene. Once
transfected, the cells were maintained at 37 °C and 5 %
CO
2
in a Dulbecco’s Modified Eagle’s medium (DMEM)
nutrient mixture F-12 HAM supplemented with
2mML-glutamine, 10 % fetal bovine serum (FBS), 0.5 %
penicillin–streptomycin, and G418 (Geneticin, 600 mg/
mL). A single cell was picked from the parental plate and
forced to replicate on its own in a fresh plate with the
appropriate media. Membranes were prepared by scraping
the cells in a 50 mM Tris–HCl buffer, homogenized via
sonication, and centrifuged for 40 min at 13,650 rpm at
4°C. The membranes were stored at -80 °C. Protein
concentrations for each membrane preparation were found
using the Bradford protein assay.
4296 Med Chem Res (2014) 23:4295–4300
123
Competitive binding assay
The binding assays were performed using slight modifi-
cations to previously published methods (Pertwee, 1999).
Using 0.5 nM
3
H-CP-55940, 10 lM test compound (unless
dose–response then first well is 100 lM followed by
appropriate dilutions), and 10 lg protein of membrane for
a total assay volume of 210 lL. Binding was initiated by
the addition of 10 lg protein of CB1 or CB2 cell mem-
branes. Assays were carried out at 37 °C for 90 min before
termination via rapid vacuum filtration through Whatman
GF/C glass-fiber filters, presoaked with 0.3 % BSA, using a
Perkin Elmer 96-well Unifilter Harvester (Perkin Elmer
Life Sciences Inc., Boston, MA, U.S.A.). Each assay plate
was washed seven times with ice-cold wash buffer (50 mM
Tris, 154 mM NaCl, 20 mM disodium ethylenediaminete-
traacetic acid (EDTA), 0.2 % BSA, and pH =7.4). Filters
were allowed to dry overnight at room temperature (25 °C)
and then radioactive counts were extracted from the filters
using a scintillation cocktail before quantification using a
Perkin Elmer TopCount (Perking Elmer Life Sciences Inc.,
Boston, Mass. U.S.A). There results were calculated using
GraphPad Prism (GraphPad Software, San Diego, CA,
U.S.A.) to obtain K
i
and IC
50
values. Total binding was
defined as binding in the presence of 0.1 % dimethylsulf-
oxide (DMSO). Non-specific binding was the binding
observed in the presence of 0.1 lM CP-55940. Specific
binding was defined as the difference between total and
non-specific binding.
GTPcS functional assay
The functional assays were performed using slight modi-
fications to previously published methods (Xiong et al.,
2011). The assay buffer for the GTPcS functional assay
consisted of 50 mM Tris–HCl, 0.2 mM ethylene glycol
tetraacetic acid (EGTA), 9 mM MgCl
2
, 150 mM NaCl, and
1.4 g BSA. Binding took place under the following con-
ditions: 50 lL compound diluted to the desired concen-
trations in the dose–response curve was mixed with 20 lg
CB1 or CB2 membrane, 50 lM GDP, 0.5 nM
35
S-labeled
GTP, and 300 lL assay buffer for a total volume of 500 lL
per well. Plates were incubated for 120 min at 37 °C. The
reaction was terminated via rapid vacuum filtration through
Whatman GF/B filters using a Perkin Elmer 96-well Uni-
filter Harvester (Perkin Elmer Life Sciences Inc., Boston,
MA, U.S.A.). Each assay plate was washed four times with
ice-cold wash buffer (10 mM Tris–HCl, pH 7.4). Filter
plates were allowed to dry overnight at room temperature
(25 °C) and then radioactive counts were extracted from
the filters using a scintillation cocktail before quantification
using a Perkin Elmer TopCount (Perking Elmer Life
Sciences Inc., Boston, Mass. U.S.A). Basal binding was
defined as binding in the presence of assay buffer. Non-
specific binding was the binding observed in the presence
of 40 lM unlabeled GTPcS salt. Emax binding was
defined as binding in the presence of 1 lM CP-55940. K
i
and EC
50
values were calculated using Graph Pad Prism
(GraphPad Software, San Diego, CA, U.S.A.).
Results
The importance of developing a structure–activity rela-
tionship for the cannabinoid receptors is due to the lack of
understanding of the receptor binding sites. Developing
in vitro bioassays to evaluate binding affinity and func-
tional activity for each of the cannabinoid receptors is
critical to understanding the pharmacology behind these
receptors. Currently, there is no crystal structure that exists
for the cannabinoid receptors active binding site. This
structure–activity relationship of phytocannabinoids may
help further understand the pharmacology of these recep-
tors along with requirements for their binding (Fig. 1).
In most cases, if a ligand binds to a receptor then it
would also functionally activate the receptor; however, this
is not always true. Although compounds may bind tightly
to a specific receptor, they do not always produce a bio-
logical response. It is not uncommon for GPCRs to dis-
sociate upon binding of a ligand (Carlsson, 2010).
Conversely, some ligands may not bind to the specific
receptor yet cause a functional response. This is thought to
be because of an allosteric binding site, a binding site other
than the two known cannabinoid receptors, in which the
ligand still produces a functional effect via the endocan-
nabinoid system pathway.
Binding
The use of radiolabeled competitive binding assays is a
common technique for evaluating compounds and their
binding affinities to specific receptors (Table 1). D
9
-THC
displayed binding affinity within the low nanomolar range
for both CB1 and CB2 receptors, 18 and 42 nM, respec-
tively. The isolated compounds within the D
9
-THC family
all displayed weaker affinity for CB1 and CB2. D
8
-THC
displays slightly lesser binding affinity than D
9
-THC for
the CB1 receptor, suggesting that the location of the double
bond has a role in binding affinity. Interestingly, 11 dis-
played strong binding affinity for the CB2 receptor with a
Ki value of 11 nM. Therefore, substitutions at the C-8
position of cannabinol may have an influential effect on
selectivity when binding to CB2. Compounds 12–23 did
not warrant significant binding affinities.
Med Chem Res (2014) 23:4295–4300 4297
123
Functional
Using radiolabeled functional bioassays allows for easily
determining if a compound is acting as an agonist, partial
agonist, antagonist, or inverse agonist. All phytocannabi-
noids mentioned in this study were determined to act as
agonists using the GTPcS functional bioassay (Table 1).
Three compounds displayed single nanomolar activity for
either CB1 or CB2 receptors. Cannabichromanone D (21)
warranted an EC
50
of 8 nM for the CB1 receptor. This
compound differs from the other compounds in the cann-
abichromanone class because the aliphatic chain cyclizes
with the phenolic hydroxy to form a third ring. Though the
cannabichromanone derivatives have not been evaluated
for their ability to induce psychoactivity, it is safe to say
that these compounds will induce psychoactivity, depend-
ing on the dose, because of their ability to functionally
stimulate the CB1 receptor. However, cannabidivarin (23)
displayed preferential activity for CB2 in the low nano-
molar range, 3 nM. The importance of showing preference
for the CB2 receptor is ideal in order to negate the side
effects associated with activation of CB1, such as psy-
choactivity. Contrary to the side chain length associated
with D
9
-THC, a decrease in the side chain length to three
AB
C
E
F
D
Fig. 1 Chemical structures of compounds isolated from Cannabis sativa organized via family substructure. aD
9
-THC, bD
8
-THC, cCannabinol,
dCannabigerol, eCannabichromanone, fCannabidiol
4298 Med Chem Res (2014) 23:4295–4300
123
carbons of cannabidivarin causes a dramatic increase in
selectivity and potency for the CB2 receptor.
Discussion
Cannabinoid receptors portray different pharmacological
properties when activated by an agonist, antagonist, or
inverse agonist. Agonists that stimulate CB1 cause some of
the unwanted side effects associated with Cannabis, such
as psychoactivity (Hensen, 2005). Furthermore, inverse
agonists that stimulate CB1 cause a loss of appetite, which
in turn helps with the treatment of obesity. Rimonabant is
an example of CB1-selective inverse agonist, and was
marketed in Europe for the treatment of obesity until three
years later when it was removed from the market. Removal
of Rimonabant was due to those patients taking the drug
having suicidal thoughts (Katoch-Rouse, 2003). Since it
was selective for CB1, it is hypothesized that stimulation of
the CB1 receptor may be the link to causing any type of
negative side effects associated with the endocannabinoid
system, the system responsible for activating the cannabi-
noid receptors.
The results presented in this manuscript indicate that
phytocannabinoids have the potential to become lead
therapeutic compounds to help patients suffering from
cancer chemotherapy, AIDS, multiple sclerosis, glaucoma,
and several other major diseases, while negating unwanted
side effects associated with CB1 stimulation. Some side
effects associated with Cannabis use include, but are not
limited to psychoactivity, dependence, and increased heart
rate. This manuscript identifies and evaluates 23 isolates
from Cannabis sativa. The effort to develop compounds
useful for medication requires structural diversity in order
to achieve therapeutic success. Using in vitro bioassays has
led to two potential compounds that need further testing to
determine pharmacological effects in vivo.
Acknowledgments This study was supported by Grant Number
P20GM104931 from the National Institute of General Medical Sci-
ences (NIGMS), a component of the National Institutes of Health
(NIH) and its contents are solely the responsibility of the authors and
do not necessarily represent the official view of NIGMS or NIH. This
Table 1 Binding affinities and functional activities of all compounds isolated from Cannabis sativa
Compound Binding affinity (nM) Functional activity (nM)
CB1 CB2 CB1 CB2
D
9
-THC, 118 ±442±9 269 ±36 327 ±43
D
9
-tetrahydrocannabinolic acid, 21,292 ±89 1,650 ±163 [10,000 [10,000
D
9
-tetrahydrocannabivarin, 322 ±5 105 ±21 [10,000 [10,000
10-a-OH-THC, 43,293 ±445 2,771 ±488 4,425 ±1,229 7,264 ±1,565
Cannabiripsol, 55,668 ±1,324 2,143 ±353 [10,000 [10,000
10-a-OH-D
9,11
-hexahydrocannabinol, 6117 ±16 129 ±13 [10,000 [10,000
10-b-OH-D
9,11
-hexahydrocannabinol, 7[10,000 [10,000 [10,000 [10,000
D
8
-THC, 878 ±512±2 5,820 ±782 524 ±70
10-a-OH-D
8
-THC, 931 ±630±4[10,000 2,622 ±352
Cannabinol, 10 75 ±473±4 307 ±29 289 ±38
8-OH-cannabinol, 11 8,063 ±1,986 11 ±1 1,438 ±399 5,099 ±725
Cannabivarin, 12 565 ±138 4,780 ±331 [10,000 [10,000
Cannabigerol, 13 3,090 ±583 2,919 ±752 [10,000 1,158 ±221
Cannabigerolic acid, 14 4,526 ±953 [10,000 182 ±32 118 ±27
6,7-Epoxy-cannabigerol, 15 [10,000 4,718 ±87 1,192 ±330 [10,000
5-Methoxy cannabigerol, 16 [10,000 3,989 ±772 235 ±51 1,572 ±376
4-OH-5-acetoxy-cannabigerol, 17 1,409 ±162 388 ±67 618 ±106 1,743 ±443
2-Geranyl-5-n-pentyl-1,4-benzoquinone, 18 [10,000 [10,000 [10,000 2,592 ±519
Cannabichromanone B, 19 3,470 ±601 4,371 ±1,119 965 ±268 [10,000
Cannabichromanone C, 20 8,681 ±1,404 5,789 ±685 483 ±121 138 ±36
Cannabichromanone D, 21 7,117 ±1,090 2,828 ±569 8 ±0.9 3,945 ±1,106
Cannabidiol, 22 151 ±28 4,582 ±613 1,469 ±197 104 ±14
Cannabidivarin, 23 503 ±58 3,970 ±976 [10,000 3 ±0.8
All the compounds evaluated displayed agonistic activity in the GTPcS functional assay for both CB1 and CB2 receptors
Med Chem Res (2014) 23:4295–4300 4299
123
investigation was conducted in a facility constructed with support
from research facilities improvement program C06RR14503 from the
NIH National Center for Research Resources.
References
Ahmed S, Ross S, Slade D, Radwan M, Zulfiqar F, ElSohly M (2008)
Cannabinoid ester constituents from high-potency Cannabis
sativa. J Nat Prod 71:536–542
Benito C (2003) Cannabinoid CB2 receptors and fatty acid amide
hydrolase are selectively overexpressed in neuritic plaque-
associated glia in Alzheimer’s disease brains. J Neuro 23:136
Berry E, Mechoulam R (2002) Tetrahydrocannabinol and endocan-
nabinoids in feeding and appetite. Pharm Ther 95:185–190
Cabral G, Griffin-Thomas L (2009) Emerging role of the cannabinoid
receptor CB2 in immune regulation: therapeutic prospects for
neuroinflammation. Expert Rev Mol Med 11:e3
Carlsson J (2010) Structure-based discovery of A adenosine receptor
ligands. J Med Chem 53:3748–3755
Clarke R, Watson D (2010) Cannabis and Natural Cannabis
Medicines. In: ElSohly MA (ed) Marijuana and the cannabi-
noids. Humana Press Inc, Totowa, pp 1–16
Devane W (1992) Isolation and structure of a brain constituent that
binds to the cannabinoid receptor. Science 258:1946–1949
ElSohly MA (2000) Potency trends of delta-9-THC and other
cannabinoids in confiscated marijuana from 1980–1997.
J Foren Sci 45:24–30
Gaoni Y, Mechoulam R (1964) Isolation, structure, and partial
synthesis of an active constituent of hashish. J Am Chem
86:1646–1647
Gerrard CM (1991) Molecular cloning of a human cannabinoid
receptor, which is also expressed in testis. Biochem J 279:129
Harrigan M (2001) Cerebral salt wasting syndrome. Crit Care Clin
17:125–138
Hensen B (2005) Cannabinoid therapeutics: high hopes for the future.
Drug Discov 10:459
Katoch-Rouse R (2003) Synthesis, structure activity relationship, and
evaluation of SR141716 analogues: development of central
cannabinoid receptor ligands with lower lipophilicity. J Med
Chem 46:642–645
Lambert D (2009) Cannabis and endocannabinoids: the old man and
the teenagers. In: Lambert DM (ed) Preface of cannabinoids in
nature and medicine. Wiley-VCH, Weinheim
Matsuda LA (1991) Structure of a cannabinoid receptor and
functional expression of the cloned cDNA. Nature 346:561–564
Mehmedic Z, Chandra S, Slade D, Denham H, Foster S, Patel A, Ross
S, Kahn I, ElSohly M (2010) Potency trends of D
9
-THC and
other cannabinoids in confiscated cannabis preparations from
1993 to 2008. J Forensic Sci 55:1209–1217
Munro S, Thomas K, Abu-Shaar M (1993) Molecular characterization
of a peripheral receptor for cannabinoids. Nature 365:61–65
Onaivi E, Ishiguro H, Patel S, Perchuk A, Meozzi P, Myers L, Mora
Z, Tagliaferro P, Gardner E, Brusco A, Akinshola B, Liu Q,
Hope B, Iwasaki S, Arinami T, Teasenfitz L, Uhl G (2006)
Discovery of the presence and functional expression of CB2
receptors in brain. Annals 1074:514–536
Pertwee R (1997) Pharmacology of cannabinoid CB1 and CB2
receptors. Pharmacol Ther 74:129–180
Pertwee R (1999) Pharmacology of cannabinoid receptor ligands.
Curr Med Chem 6:635–664
Peters H, Nahas G (1999) A brief history of four millenia (B.C.
2000—A.D. 1974). In: Nahas GG (ed) Marihuana and Medicine.
Humana Press Inc, Totowa, pp 3–7
Ross S, ElSohly M, Sultana G, Mehmedic Z, Hossain C, Chandra S
(2005) Flavonoid glycosides and cannabinoids from the pollen of
Cannabis sativa L. Phyto Anal 16:45–48
Xiong W, Cheng K, Cui T, Godlewski G, Rice K, Xu Y, Zhang L
(2011) Cannabinoid potentiation of glycine receptors contributes
to cannabis-induced analgesia. Nat Chem Biol 7:295–303
4300 Med Chem Res (2014) 23:4295–4300
123
