REVIEW
published: 13 September 2016
doi: 10.3389/fnins.2016.00406
Frontiers in Neuroscience | www.frontiersin.org 1 September 2016 | Volume 10 | Article 406
Edited by:
Marialessandra Contino,
University of Bari Aldo Moro, Italy
Reviewed by:
Aurel Popa-Wagner,
University of Rostock, Germany
Michael Decker,
University of Würzburg, Germany
Claudia Mugnaini,
University of Siena, Italy
*Correspondence:
Rafael Franco
rfranco@ub.edu;
rfranco123@gmail.com
Specialty section:
This article was submitted to
Neurodegeneration,
a section of the journal
Frontiers in Neuroscience
Received: 25 May 2016
Accepted: 22 August 2016
Published: 13 September 2016
Citation:
Navarro G, Morales P,
Rodríguez-Cueto C, Fernández-Ruiz J,
Jagerovic N and Franco R (2016)
Targeting Cannabinoid CB
2
Receptors
in the Central Nervous System.
Medicinal Chemistry Approaches with
Focus on Neurodegenerative
Disorders. Front. Neurosci. 10:406.
doi: 10.3389/fnins.2016.00406
Targeting Cannabinoid CB
2
Receptors in the Central Nervous
System. Medicinal Chemistry
Approaches with Focus on
Neurodegenerative Disorders
Gemma Navarro
1, 2, 3
, Paula Morales
4, 5
, Carmen Rodríguez-Cueto
2, 6, 7
,
Javier Fernández-Ruiz
2, 6, 7
, Nadine Jagerovic
4
and Rafael Franco
1, 2, 3
*
1
Department of Biochemistry and Molecular Biomedicine, University of Barcelona, Barcelona, Spain,
2
Centro de
Investigación en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Instituto de Salud Carlos III, Madrid, Spain,
3
Cell and Molecular Neuropharmacology, Institut de Biomedicina (IBUB), Universitat de Barcelona, Barcelona, Spain,
4
Instituto de Química Médica, Consejo Superior de Investigaciones Científicas, Madrid, Spain,
5
Center for Drug Discovery,
University of North Carolina at Greensboro, Greensboro, NC, USA,
6
Departamento de Bioquímica, Facultad de Medicina,
Instituto Universitario de Investigación en Neuroquímica, Universidad Complutense, Madrid, Spain,
7
Instituto Ramón y Cajal
de Investigación Sanitaria, Madrid, Spain
Endocannabinoids activate two types of specific G-protein-coupled receptors (GPCRs),
namely cannabinoid CB
1
and CB
2
. Contrary to the psychotropic actions of agonists
of CB
1
receptors, and serious side effects of the selective antagonists of this receptor,
drugs acting on CB
2
receptors appear as promising drugs to combat CNS diseases
(Parkinson’s disease, Huntington’s chorea, cerebellar ataxia, amyotrohic lateral sclerosis).
Differential localization of CB
2
receptors in neural cell types and upregulation in
neuroinflammation are keys to understand the therapeutic potential in inter alia diseases
that imply progressive neurodegeneration. Medicinal chemistry approaches are now
engaged to develop imaging tools to map receptors in the living human brain, to
develop more efficacious agonists, and to investigate the possibility to develop allosteric
modulators.
Keywords: heteromer, microglia, astroglia, M0/M1/M2 phenotype, neuroprotection, neurorestoration, GPCR,
amyotrophic lateral sclerosis
INTRODUCTION
To d ate only two cannabinoid receptors have been identified and completely accepted as key
members of the endocannabinoid signaling. The CB
1
receptor (CB
1
R) is mainly expressed in the
central nervous system (CNS) (
Hu and Mackie, 2015), whereas, t he CB
2
receptor (CB
2
R) is mainly
expressed in the periphery, especially in blood cells, and in blood-cell producing organs (Onaivi
et al., 1999; Atwood and Mackie, 2010; Atwood et al., 2012). Other re ceptors, e.g., GPR55, the cation
channel TRPV1 and the nuclear receptors of the PPAR family, are also under discussion as possible
members of the endocannabinoid receptor family. CB
1
R and CB
2
R belong to the most populated
family of the human proteome, i.e., to the family of receptors coupled to heterotrimeric G proteins
(GPCRs). More specifically they are members of class A GPCRs, which are characterized by being
structurally similar to rhodopsin, for having an extracellular N-terminal domain, a seven α-helical
transmembrane domain, and a C-terminal domain of 73 (for CB
1
R) or of 59 (for CB
2
R) amino
acids. Total length of the most common
1
protein products is 472 for CB
1
R and 360 for CB
2
R. The
1
Isoforms of endocannabinoid receptors have been identified (details available at www.uniprot.org.)
Navarro et al. CB
2
Receptors as Targets in Neurodegenerative Diseases
difference in receptor length comes from the bigger N-terminal
domain of the CB
1
R (116 vs. 33 amino acids).
Soon after its discovery and the realization of the relevant
role of endogenous cannabinoids, the CB
1
R was considered a
potential target to combat CNS diseases. In fact, the CB
1
R is
considered the class A GPCR member with the highest expression
in the CNS. In sharp contrast, controversy surrounds expression
of CB
2
R in the CNS, and until recently this receptor was not
considered as target for neurological or neuropsychiatric diseases
(
Atwood and Mackie, 2010; Atwood et al., 2012). This paper
scans the literature that supports th e view that CB
2
R may
have now more potential than CB
1
R to combat some CNS
disorders, in particular those related to neuroinflammatory, and
neurodegenerative events. The paper also informs on current
developments in medicinal chemistry aspects of CB
2
R-b ased
CNS drug discovery.
BETTER PROSPECTS FOR CB
2
R THAN
FOR CB
1
R IN CNS DISEASES
GPCRs constitute the target of approximately 40% of approved
drugs. Drug development programs are still heavily relying
on the potential of GPCRs for a huge variety of diseases.
Agonists, which are able to activate the receptor and compete
with the endogenous agonist, and antagonists, which block the
receptor and impede activation by th e endogenous agonist, have
therapeutic potential. However, the number of medic at ions that
consist of GPCR antagonists outnumbers that of GPCR agonists.
In general terms, t he higher success of antagonists means that
they have fewer side effec ts than agonists, although other causes
overlay. The endocannabinoid system is a very special case as
endogenous compounds produced by neurons and acting on
central CB
1
Rs are absolutely required for higher brain functions,
but any synthetic or natural (e.g., 1
9
-tetrahydrocannabinol)
agonist reaching the brain and hitting CB
1
R has proved to have
psychotropic actions in animal models of disease and in humans.
Therefore, the potential of CB
1
Rs as ta rgets for diseases of the
CNS, and also peripheral disorders, has been limited by the
psychoactive side effects derived from their agonists, and for
the need to consider the risk-benefit balance. In this context,
some researchers wanted to develop CB
1
R ant agonists (including
inverse agonists) as a safer alternative in those pathologies having
an overactivity of the endocannabinoid system (e.g., obesity,
addiction, schizophrenia), although side effects were a lso evident
with such strategy (see below).
The first two molecules targeting CB
1
R that reached the
therapeutic market (in the 80s) were 1
9
-tetrahydrocannabinol,
also known as dronabinol (marketed as Marinol
R
), and nabilone
(marketed as Cesamet
R
) (Figure 1), both prescribed to combat
nausea and vomiting, as well anorexia, derived from cancer,
and AIDS treatments, respe ct ively (
Green et al., 1989), but
their use was limited. By contrast, a CB
1
R antagonist/inverse
agonist, rimonabant (Acomplia
R
), was approved in 2006 to
treat obesity, and metabolic syndrome (
Carai et al., 2006) and
generated extremely high expectations. Unfortunately, the drug
had to be retired due to side effects, especially due to reports
of suicide (
Sam et al., 2011). Consequently, chances, that other
CB
1
R select ive drug may advance though regulatory bodies, and
reach the market have dramatically diminished. In this context,
the CB
2
R has taken the lead in the race to find novel cannabinoid-
related drugs for CNS diseases. On the one hand, CB
1
R is
expressed in almost any brain region, and in many neuronal
cell types, whereas CB
2
R expression in neurons is restricted to
few areas. Accordingly, fewer side effects are expected when
drugs are targeting receptors with restricted expression than
when drugs are targeting receptors widely expressed in the CNS.
Furthermore, CB
2
R are upregulated in a variety of CNS diseases
that course with activated microglia or astroglia. Then the CB
2
R
but not the CB
1
R is a promising candidate to consider in
diseases with a neuroinflammatory component. It is even possible
that the activation of CB
2
Rs may explain recent controversies
in relation with the consumption of cannabis as a factor
either increasing risk or preventing against spontaneous brain
insults (e.g., intracerebral hemorrhage). Recent epidemiological
studies suggest a potential protective effect of cannabis to
the modulation of C-reactive protein response in intracerebral
hemorrhage (
Di Napoli et al., 2012, 2016; Alshaarawy and
Anthony, 2015), an effect that could be possibly related to CB
2
R
activation, although this has not been investigated. Advantages of
developing CB
2
R selective drugs to prevent neurodegeneration in
cases of neuroinflammation are presented later in this article.
As macrophages express CB
2
R and mic roglia is somehow
a similar cell type, these receptors were soon identified in
microglial cells, but further research demonstrated that they can
be also found in other types of glial cells (see below). There is
however, some controversy on the degree of CB
2
R expression
in resting vs. activated microglial cells. Also the activated
microglial phenotype is different in macrophages filtered from
the blood into the CNS and in resident microglia that becomes
activated due to, inter alia, accumulation of protein aggregates
such as alpha-synuclein, or ß-amyloid. Remarkably, (see Franco
and Fernández-Suárez, 2015 and references therein) a better
understanding of the expression and role of CB
2
R in t h e different
microglial phenotypes (M0, M1, M2) will help in designing
CB
2
R selective ligands able to induce the neuroprotective/anti-
inflammatory-skewed phenotype(s).
CB
2
R may be also expressed by CNS neurons. The role of
CB
2
Rs in schizophrenia, depression, food consumption, and
drug addiction has been demonstrated in different laboratories
and the results are consistent with neuronal expression of the
receptor (
Onaivi et al., 2008a,b,c; Hu et al., 2009; García-
Gutiérrez et al., 2010; Ishiguro et al., 2010a,b; García-Gutiérrez
and Manzanares, 2011; Ortega-Alvaro et al., 2011; Aracil-
Fernández et al., 2012; Navarrete et al., 2012, 2013; Bahi
et al., 2014; Blanco-Calvo et al., 2014; Ortega-Álvaro et al.,
2015; Rodríguez-Arias et al., 2015; García-Cabrerizo and García-
Fuster, 2016). The receptor is significantly expressed in neurons
in the brain stem (Van Sickle et al., 2005), in the cerebellum
(Skaper et al., 1996; Ashton et al., 2006; Gong et al., 2006;
Rodríguez-Cueto et al., 2014
) in the internal and the external
segments of the globus pallidus of the non-human primate
(
Lanciego et al., 2011), and in the substantia nigra (in humans,
not in rodents) (
García et al., 2016; Gómez-Gálvez et al., 2016).
Frontiers in Neuroscience | www.frontiersin.org 2 September 2016 | Volume 10 | Article 406
Navarro et al. CB
2
Receptors as Targets in Neurodegenerative Diseases
FIGURE 1 | Chemical structure of 1
9
-THC, nabilone, and the CB
2
R ligands: JWH133, 0-1966, AEA , BCP, SMM-189, PM226,
1-butyl-3-[(cyclohexylamino)methylidene]-8-methylquinoline-2,4(1H,3H)-dione, [
11
C]NE40, [
11
C]KD2, [
11
C]RS-016,
2-{2-chloro-[5-(4-[
18
F]-d
2
-methoxy)-6-(4-fluorophenethylamino)-1,3,5-triazin-2-yl]phenyl}propan-2-ol.
Frontiers in Neuroscience | www.frontiersin.org 3 September 2016 | Volume 10 | Article 406
Navarro et al. CB
2
Receptors as Targets in Neurodegenerative Diseases
Different laboratories working with rodents or primates have
also identified receptor expression in neurons of the prefrontal
cortex and hippocampus (Callén et al., 2012; den Boon et al.,
2012; Sierra et al., 2015; García-Cabrerizo and García-Fuster,
2016
). Expression of CB
2
R in the basal ganglia show promise
in Parkinson’s disease and Huntington’s chorea; t h e presence
of the receptor in hippocampus and prefrontal cortex makes it
attractive for Alzheimer’s disease and the expression in brain stem
and cerebellum opens novel therapeutic avenues for a variety of
diseases such as hereditary spinocerebellar ataxias. Last but not
least, t h e data on CB
2
R-mediated endocannabinoid regulation of
microglial activation makes the receptor attractive for diseases
with a neuroinflammatory component.
Cannabinoid neuroregulation is mainly based on retrograde
signaling (
Alger, 20 02), i.e., endocannabinoids come from post-
synaptic elements to activate presynaptic receptors. However,
postsynaptic CB
2
Rs have been also reported (
Brusco et al., 2008).
The combination of restricted neuronal expression with the
possibility of targeting pre- or postsynaptic receptors, makes the
CB
2
R a really attractive target.
CB
2
R IN NEURODEGENERATIVE
DISORDERS. RELEVANCE OF
DIFFERENTIAL EXPRESSION OF CB
2
R IN
NEURAL CELLS
The preser vation of neuronal integrity and survival is
one of the most promising therapeutic possibilities of
CB
2
R-targeting cannabinoids (
Atwood et al., 2012). There
is potential in pain and in numerous acute or chronic
neurodegenerative/neuroinflammatory conditions (Jhaveri
et al., 2007; Micale et al., 2007; Campillo and Páez, 2009). The
neuroprotective potential of compounds targeting the CB
2
R is,
first of all, the logical consequence of their location in key cell
types (e.g., in specific neuronal subsets, activated astrocytes,
reactive microglia, perivascular microglia, oligodendrocytes, and
neural progenitor cells), and also in some structures (e.g., the
blood-brain barrier (BBB)) that are critical for the maintenance
of the CNS integrity (Amenta et al., 2012 ; Chung et al., 2016)
(Figure 2A). Such variety of locations enable compounds capable
to selectively activate the CB
2
R to exert a selective control over
the specific functions fulfilled by these cells in degeneration,
protection and/or repair (
Fernández-Ruiz et al., 2014). For
example, BBB function is under the control of CB
2
R-mediated
signals (Fujii et al., 2014), which maintain the integrity of tig h t
junctions, inhibit leukocyte infiltration, and facilitate β-amyloid
clearance (Vendel and de Lange, 2014).
CB
2
Rs in glial cells recruited to the site of the
neurodegeneration, appear to be critical for preserving the
neuronal integrity and function (Savonenko et al., 2015). In fact,
CB
2
R may be absent of these cells in resting conditions, with
a weak expression in the healthy brain. As the receptors are
strongly up-regulated when glial cells are activated in conditions
of neurodegeneration (
Fernández-Ruiz et a l., 2007, 2015), they
have potential from a th erapeutic point of view (Figure 2B).
Up-regulation may occur in both astrocytes and microglial
cells, but the CB
2
R-mediated signaling may vary depending
inter alia on the type of pathology and the experimental model.
CB
2
R-mediated neuroprote ct ion/neurorestoration mechanisms
are of special interest in disorders that affect movement-related
areas, such as (i) Parkinson’s and Huntington’s diseases (affecting
the basal ganglia, and producing rigidity, postural instability,
bradykinesia, tremor, and chorea), (ii) autosomal dominant
spinocerebellar ataxias (affecting the cerebellum and its afferent
and efferent connections, and producing loss of b ala nce, and
motor incoordination), and (iii) amyotrophic lateral sclerosis
(ALS) (affecting upper and lower spinal motor neurons, and
producing mus c le denervation and atrophy, which results in a
progressive weakness and paralysis affecting voluntary muscles).
For example, in this last disorder, CB
2
Rs become up-regulated in
microglial cells recruited at the spinal cord of patients (
Yiangou
et al., 2 006), a fact corroborated by studies in the TDP-43 mouse
model of the disease (
Espejo-Porras et al., 2015). However, apart
from microglial cells, other CB
2
R-positive cells were found
in this murine model (
Espejo-Porras et al., 2015). In another
murine model of ALS, (the SOD-1 mouse), CB
2
R also become
up-regulated, but the study did not characterize the type of cell
that was expressing the receptors (Shoemaker et al., 2007).
Interestingly, microglial CB
2
Rs appear up-regulated in t h e
cerebellum of patients with different autosomal dominant
cerebellar ataxias, but such trend was also found in activated
astrocytes located in the cerebellar parenchyma and in
the periphery of blood vessels, and in certain neuronal
subpopulations (Rodríguez-Cueto et al., 2014). Similarly,
increased levels of CB
2
R are found in both striatal activated
astrocytes and re active microglial cells after an insult with
malonate in rats, an experimental model of Huntington’s disease
(
Sagredo et al., 2009). Although data collected from Huntington’s
disease patients or obtained in genetic models of the disease (e.g.,
R6/1, R6/2) indicated that CB
2
R were located and up-regulated
only in microglial cells (Palazuelos et al., 2009), a more recent
study situated the up-regulation of these receptors in vascular
cells, not in activated glial cells, in HD patients (Dowie et al.,
2014).
In yet another neurodegenerative condition affecting the
basal ganglia circuits, (Price et al., 2009) were the first to
demonstrate up-regulation of CB
2
R in microglial cells recruited
at the substantia nigra in MPTP-lesioned mice. In the study it
was not addressed whether there were other CB
2
R-positive cells
that do not correspond to reactive microglia. We investigated
the issue in parkinsonian patients using postmortem samples and
identified such up-regulation in microglial cells (labeled with Iba-
1) and in another unidentified cell type (
Gómez-Gálvez et al.,
2016).
CB
2
R has potential in demyelinating disorders (e.g., multiple
sclerosis; Molina-Holgado et al., 2002; Gomez et al., 2010,
2011). In fact, CB
2
R are present in oligodendrocytes, a nd more
importantly, in th eir natural precursor cells, so that they may play
a role in their survival, proliferation, a nd differentiation. CB
2
Rs
have been also identified in neural progenitor cells, and it appears
that they can play a role in the proliferation and differentiation
of these precursors (
Palazuelos et al., 2006, 2012; Goncalves
et al., 2008; Avraham et al., 2014
), opening the possibility to
Frontiers in Neuroscience | www.frontiersin.org 4 September 2016 | Volume 10 | Article 406
Navarro et al. CB
2
Receptors as Targets in Neurodegenerative Diseases
FIGURE 2 | (A). Expression of CB
2
Rs in different neural cell types and how receptor activation may impact on cell-specific functions. (B) Cellular events that explain
the therapeutic possibilities f or ligands that target CB
2
Rs, which are upregulated in activated glial cells.
facilitate neurorestoration by pharmacologically manipulating
this receptor. Lastly, the identification of CB
2
Rs in perivascular
microglial cells in the cerebellum (Núñez et al., 2004) may be
possibly related to the role attributed to these receptors at the
level of the BBB (see above).
CHALLENGES IN CB
2
R-BASED DRUG
DESIGN
Pharmacology of cannabinoid receptors is complex due to
the lipophilic nature of many natural and synthetic agonists.
Endogenous agonists of many class A GPCRs are hydrophilic,
which contrast with the lipophilic nature of endocannabinoids.
Pharmacological characterization by radioligand binding to
CB
2
R is especially complex. On the one hand, the binding site
extends deeply within the seven transmembrane domain of the
receptor, and the two available radiolabeled ligands (tritiated CP-
55940 and tritiated WIN-55212-2) do not interact with exactly
the same amino acid residues in the orthosteric center; in
particular CP-55940 does not interact with a conserved lysine
residue in the binding site (
Tao et al., 1999). Furthermore, it is
hypothesized that cannabinoids may not reach the binding site
from the outside of the cells but by lateral diffusion via the lipid
bilayer of the plasma membrane (Guo et al., 2003; Makriyannis
et al., 2005; Hurst et al., 2010). These features suggest that newly
synthesized drugs or newly discovered natural cannabinoids have
qualitatively different modes of binding to CB
2
Rs. On the other
hand, the nonspecific binding to membranes from natural CNS
sources is high and leads to low-confidence values of the amount
of receptor in neural cells. This problem is partially solved
by performing the assays in heterologous cells expressing the
human receptor; such approach provides reliable parameters for
drug discovery. The complex pharmacology is also slowing the
discovery of allosteric centers, and accordingly, of allosteric CB
2
R
modulators.
GPCR pharmacology must somehow be revisited due to
the occurrence of receptor heteromers (Cordomí et al., 2015;
Franco et al., 2016). Each heteromer is unique and functionally
different from the two constituting receptors. In fact, affinity
Frontiers in Neuroscience | www.frontiersin.org 5 September 2016 | Volume 10 | Article 406