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GPCR heterodimer, via orthosteric or allosteric ligands. This assay measures
time-dependent ligand occupancy of radiolabeled orthosteric or (with adaptations)
allosteric ligands as modulated by either small molecules or receptor dimer partners
bound or unbound with their own ligands.
INTRODUCTION AND RATIONALE
Heterotrimeric G proteins remain the best-characterized allosteric modulator of
G protein-coupled receptors (GPCRs), through their capacity to modulate ligand-
binding affinity. Early models described the effects of G protein recruitment to a
ligand-bound receptor as the “ternary complex model” (
De Lean, Stadel, &
Lefkowitz, 1980; Limbird, 2004
). This analysis is also relevant when the heterotri-
meric G protein is substituted by an allosteric ligand, which differentially modulates
the activated receptor bound by orthosteric agonist (
May, Leach, Sexton, &
Christopoulos, 2007
). By binding to topographically distinct binding sites with re-
spect to the orthosteric binding site, allosteric ligands have rapidly become a viable
alternative to modulate the selectivity and functionality of receptors and more re-
cently ligand-directed signaling (
Christopoulos, 2002; Kenakin, 2010; Kenakin &
Miller, 2010; Urwyler, 2011
). Allosteric modulators, as such, have been around a
long time but were initially described as noncompetitive antagonists (
Litschig
et al., 1999; Varney et al., 1999
). Such noncompetitive antagonism was often re-
ferred as insurmountable, because the receptor-antagonist complex was a new entity
in its own right, distinct from the agonist-receptor complex (
Vauquelin, Van Liefde,
Birzbier, & Vanderheyden, 2002
). Allosteric modulators can affect GPCRs at mul-
tiple levels: (1) the binding of orthosteric ligand to the receptor, (2) the transmission
of ligand-binding information to other parts of the receptor, or (3) the signaling
downstream of receptor activation (
Fig. 9.1
A and B). First, allosteric ligands were
shown to modulate the affinity of orthosteric drugs for their binding sites. Coopera-
tivity (also denoted as the “
” factor) between the two binding sites can be neutral (no
a
effect or
a
¼
1), positive (leftward shift of the binding curve or
1), or negative
a>
(rightward shift of the binding curve or
1). Interestingly, allosteric effects are sat-
urable, that is, when all the allosteric sites are occupied, no further allosteric mod-
ulation is observed. Moreover, the regulation of the different signaling modes
downstream of a given receptor can be achieved by modulating either the efficacy
or potency of the response in question, with or without modulation of the binding
affinity of the orthosteric ligand, or the coupling efficacy between the G protein
and the receptor. For all the modes of modulation described in the preceding text,
an allosteric ligand can therefore be a positive allosteric modulator (PAM) or a neg-
ative allosteric modulator (NAM). One of the major characteristics of PAMs and
NAMs is their relative inability to trigger GPCR-induced responses in the absence
of the orthosteric ligand. There are, however, ago-allosteric ligands or allosteric ag-
onists, which behave like agonists but through binding sites distinct from orthosteric
ligands. Finally, there are also synthetic ligands that can simultaneously bind to both
a<
