7 research outputs found
Identification of Crucial Amino Acid Residues Involved in Agonist Signaling at the GPR55 Receptor
GPR55
is a newly deorphanized class A G-protein-coupled receptor
that has been implicated in inflammatory pain, neuropathic pain, metabolic
disorder, bone development, and cancer. Few potent GPR55 ligands have
been identified to date. This is largely due to an absence of information
about salient features of GPR55, such as residues important for signaling
and residues implicated in the GPR55 signaling cascade. The goal of
this work was to identify residues that are key for the signaling
of the GPR55 endogenous ligand, l-α-lysophosphatidylinositol
(LPI), as well as the signaling of the GPR55 agonist, ML184 {CID 2440433,
3-[4-(2,3-dimethylphenyl)piperazine-1-carbonyl]-<i>N</i>,<i>N</i>-dimethyl-4-pyrrolidin-1-ylbenzenesulfonamide}.
Serum response element (SRE) and serum response factor (SRF) luciferase
assays were used as readouts for studying LPI and ML184 signaling
at the GPR55 mutants. A GPR55 R* model based on the recent δ-opioid
receptor (DOR) crystal structure was used to interpret the resultant
mutation data. Two residues were found to be crucial for agonist signaling
at GPR55, K2.60 and E3.29, suggesting that these residues form the
primary interaction site for ML184 and LPI at GPR55. Y3.32F, H(170)F,
and F6.55A/L mutation results suggested that these residues are part
of the orthosteric binding site for ML184, while Y3.32F and H(170)F
mutation results suggest that these two residues are part of the LPI
binding pocket. Y3.32L, M3.36A, and F6.48A mutation results suggest
the importance of a Y3.32/M3.36/F6.48 cluster in the GPR55 signaling
cascade. C(10)A and C(260)A mutations suggest that these residues
form a second disulfide bridge in the extracellular domain of GPR55,
occluding ligand extracellular entry in the TMH1–TMH7 region
of GPR55. Taken together, these results provide the first set of discrete
information about GPR55 residues important for LPI and ML184 signaling
and for GPR55 activation. This information should aid in the rational
design of next-generation GPR55 ligands and the creation of the first
high-affinity GPR55 radioligand, a tool that is sorely needed in the
field
Thienopyrimidine Derivatives as GPR55 Receptor Antagonists: Insight into Structure–Activity Relationship
GPR55 is an orphan G-protein coupled receptor involved
in various
pathophysiological conditions. However, there are only a few noncannabinoid
GPR55 ligands reported so far. The lack of potent and selective GPR55
ligands precludes a deep exploration of this receptor. The studies
presented here focused on a thienopyrimidine scaffold based on the
GPR55 antagonist ML192, previously discovered by high-throughput screening.
The GPR55 activities of the new synthesized compounds were assessed
using β-arrestin recruitment assays in Chinese hamster ovary
cells overexpressing human GPR55. Some derivatives were identified
as GPR55 antagonists with functional efficacy and selectivity versus
CB1 and CB2 cannabinoid receptors
The Importance of Hydrogen Bonding and Aromatic Stacking to the Affinity and Efficacy of Cannabinoid Receptor CB<sub>2</sub> Antagonist, 5‑(4-chloro-3-methylphenyl)-1-[(4-methylphenyl)methyl]‑<i>N</i>‑[(1<i>S</i>,2<i>S</i>,4<i>R</i>)‑1,3,3-trimethylbicyclo[2.2.1]hept-2-yl]-1H-pyrazole-3-carboxamide (SR144528)
Despite
the therapeutic promise of the subnanomolar affinity cannabinoid
CB<sub>2</sub> antagonist, 5-(4-chloro-3-methylphenyl)-1-[(4-methylphenyl)methyl]-<i>N</i>-[(1<i>S</i>,2<i>S</i>,4<i>R</i>)-1,3,3-trimethylbicyclo[2.2.1]hept-2-yl]-1<i>H</i>-pyrazole-3-carboxamide (SR144528, <b>1</b>), little is known about its binding site interactions and
no primary interaction site for <b>1</b> at CB2 has been identified.
We report here the results of Glide docking studies in our cannabinoid
CB<sub>2</sub> inactive state model that were then tested via compound
synthesis, binding, and functional assays. Our results show that the
amide functional group of <b>1</b> is critical to its CB2 affinity
and efficacy and that aromatic stacking interactions in the TMH5/6
aromatic cluster of CB2 are also important. Molecular modifications
that increased the positive electrostatic potential in the region
between the fenchyl and aromatic rings led to more efficacious compounds.
This result is consistent with the EC-3 loop negatively charged amino
acid, D275 (identified via Glide docking studies) acting as the primary
interaction site for <b>1</b> and its analogues
Chromenopyrazole, a Versatile Cannabinoid Scaffold with in Vivo Activity in a Model of Multiple Sclerosis
A combination
of molecular modeling and structure–activity
relationship studies has been used to fine-tune CB<sub>2</sub> selectivity
in the chromenopyrazole ring, a versatile CB<sub>1</sub>/CB<sub>2</sub> cannabinoid scaffold. Thus, a series of 36 new derivatives covering
a wide range of structural diversity has been synthesized, and docking
studies have been performed for some of them. Biological evaluation
of the new compounds includes, among others, cannabinoid binding assays,
functional studies, and surface plasmon resonance measurements. The
most promising compound [<b>43</b> (PM226)], a selective and
potent CB<sub>2</sub> agonist isoxazole derivative, was tested in
the acute phase of Theiler’s murine encephalomyelitis virus-induced
demyelinating disease (TMEV-IDD), a well-established animal model
of primary progressive multiple sclerosis. Compound <b>43</b> dampened neuroinflammation by reducing microglial activation in
the TMEV
Mapping Cannabinoid 1 Receptor Allosteric Site(s): Critical Molecular Determinant and Signaling Profile of GAT100, a Novel, Potent, and Irreversibly Binding Probe
One of the most abundant
G-protein coupled receptors (GPCRs) in
brain, the cannabinoid 1 receptor (CB1R), is a tractable therapeutic
target for treating diverse psychobehavioral and somatic disorders.
Adverse on-target effects associated with small-molecule CB1R orthosteric
agonists and inverse agonists/antagonists have plagued their translational
potential. Allosteric CB1R modulators offer a potentially safer modality
through which CB1R signaling may be directed for therapeutic benefit.
Rational design of candidate, druglike CB1R allosteric modulators
requires greater understanding of the architecture of the CB1R allosteric
endodomain(s) and the capacity of CB1R allosteric ligands to tune
the receptor’s information output. We have recently reported
the synthesis of a focused library of rationally designed, covalent
analogues of Org27569 and PSNCBAM-1, two prototypic CB1R negative
allosteric modulators (NAMs). Among the novel, pharmacologically active
CB1R NAMs reported, the isothiocyanate GAT100 emerged as the lead
by virtue of its exceptional potency in the [<sup>35</sup>S]GTPγS
and β-arrestin signaling assays and its ability to label CB1R
as a covalent allosteric probe with significantly reduced inverse
agonism in the [<sup>35</sup>S]GTPγS assay as compared to Org27569.
We report here a comprehensive functional profiling of GAT100 across
an array of important downstream cell-signaling pathways and analysis
of its potential orthosteric probe-dependence and signaling bias.
The results demonstrate that GAT100 is a NAM of the orthosteric CB1R
agonist CP55,940 and the endocannabinoids 2-arachidonoylglycerol and
anandamide for β-arrestin1 recruitment, PLCβ3 and ERK1/2
phosphorylation, cAMP accumulation, and CB1R internalization in HEK293A
cells overexpressing CB1R and in Neuro2a and ST<i>Hdh</i><sup>Q7/Q7</sup> cells endogenously expressing CB1R. Distinctively,
GAT100 was a more potent and efficacious CB1R NAM than Org27569 and
PSNCBAM-1 in all signaling assays and did not exhibit the inverse
agonism associated with Org27569 and PSNCBAM-1. Computational docking
studies implicate C7.38(382) as a key feature of GAT100 ligand-binding
motif. These data help inform the engineering of newer-generation,
druggable CB1R allosteric modulators and demonstrate the utility of
GAT100 as a covalent probe for mapping structure–function correlates
characteristic of the druggable CB1R allosteric space
CB2-Selective Cannabinoid Receptor Ligands: Synthesis, Pharmacological Evaluation, and Molecular Modeling Investigation of 1,8-Naphthyridin-2(1<i>H</i>)‑one-3-carboxamides
We
have recently identified 1,8-naphthyridin-2(1<i>H</i>)-one-3-carboxamide
as a new scaffold very suitable for the development
of new CB2 receptor potent and selective ligands. In this paper we
describe a number of additional derivatives in which the same central
scaffold has been variously functionalized in position 1 or 6. All
new compounds showed high selectivity and affinity in the nanomolar
range for the CB2 receptor. Furthermore, we found that their functional
activity is controlled by the presence of the substituents at position
C-6 of the naphthyridine scaffold. In fact, the introduction of substituents
in this position determined a functionality switch from agonist to
antagonists/inverse agonists. Finally, docking studies showed that
the difference between the pharmacology of these ligands may be in
the ability/inability to block the Toggle Switch W6.48(258) (χ1 <i>g+</i> → <i>trans</i>) transition
Identification of the GPR55 Antagonist Binding Site Using a Novel Set of High-Potency GPR55 Selective Ligands
GPR55 is a class A G protein-coupled
receptor (GPCR) that has been
implicated in inflammatory pain, neuropathic pain, metabolic disorder,
bone development, and cancer. Initially deorphanized as a cannabinoid
receptor, GPR55 has been shown to be activated by non-cannabinoid
ligands such as l-α-lysophosphatidylinositol (LPI).
While there is a growing body of evidence of physiological and pathophysiological
roles for GPR55, the paucity of specific antagonists has limited its
study. In collaboration with the Molecular Libraries Probe Production
Centers Network initiative, we identified a series of GPR55 antagonists
using a β-arrestin, high-throughput, high-content screen of
∼300000 compounds. This screen yielded novel, GPR55 antagonist
chemotypes with IC<sub>50</sub> values in the range of 0.16–2.72
μM [Heynen-Genel, S., et al. (2010) Screening for Selective
Ligands for GPR55: Antagonists (ML191, ML192, ML193) (Bookshelf ID
NBK66153; PMID entry 22091481)]. Importantly, many of the GPR55 antagonists
were completely selective, with no agonism or antagonism against GPR35,
CB1, or CB2 up to 20 μM. Using a model of the GPR55 inactive
state, we studied the binding of an antagonist series that emerged
from this screen. These studies suggest that GPR55 antagonists possess
a head region that occupies a horizontal binding pocket extending
into the extracellular loop region, a central ligand portion that
fits vertically in the receptor binding pocket and terminates with
a pendant aromatic or heterocyclic ring that juts out. Both the region
that extends extracellularly and the pendant ring are features associated
with antagonism. Taken together, our results provide a set of design
rules for the development of second-generation GPR55 selective antagonists