2 research outputs found
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]ÂGTPγS
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]ÂGTPγS 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
Enantiospecific Allosteric Modulation of Cannabinoid 1 Receptor
The
cannabinoid 1 receptor (CB1R) is one of the most widely expressed
metabotropic G protein-coupled receptors in brain, and its participation
in various (patho)Âphysiological processes has made CB1R activation
a viable therapeutic modality. Adverse psychotropic effects limit
the clinical utility of CB1R orthosteric agonists and have promoted
the search for CB1R positive allosteric modulators (PAMs) with the
promise of improved drug-like pharmacology and enhanced safety over
typical CB1R agonists. In this study, we describe the synthesis and <i>in vitro</i> and <i>ex vivo</i> pharmacology of the
novel allosteric CB1R modulator GAT211 (racemic) and its resolved
enantiomers, GAT228 (<i>R</i>) and GAT229 (<i>S</i>). GAT211 engages CB1R allosteric site(s), enhances the binding of
the orthosteric full agonist [<sup>3</sup>H]ÂCP55,490, and reduces
the binding of the orthosteric antagonist/inverse agonist [<sup>3</sup>H]ÂSR141716A. GAT211 displayed both PAM and agonist activity in HEK293A
and Neuro2a cells expressing human recombinant CB1R (hCB1R) and in
mouse-brain membranes rich in native CB1R. GAT211 also exhibited a
strong PAM effect in isolated vas deferens endogenously expressing
CB1R. Each resolved and crystallized GAT211 enantiomer showed a markedly
distinctive pharmacology as a CB1R allosteric modulator. In all biological
systems examined, GAT211’s allosteric agonist activity resided
with the <i>R-</i>(+)-enantiomer (GAT228), whereas its PAM
activity resided with the <i>S</i>-(−)-enantiomer
(GAT229), which lacked intrinsic activity. These results constitute
the first demonstration of enantiomer-selective CB1R positive allosteric
modulation and set a precedent whereby enantiomeric resolution can
decisively define the molecular pharmacology of a CB1R allosteric
ligand