Biochemical pharmacology of the positive allosteric modulation of the GABAb receptor "in Vitro" and "in Vivo"

Allosteric modulators of G-protein coupled receptors (GPCRs) interact with binding sites on
the receptor molecule that are topographically distinct from the classic orthosteric site. Having
only a marginal effect by themselves, they induce conformational changes of receptors that
result in the modulation of agonist-induced function in either a stimulating or an inhibiting
way, depending on whether they are positive or negative allosteric modulators, respectively.
Their mechanism of action is, thus, in synchrony with the frequency and the magnitude of
physiological signaling. This is the main reason why allosteric modulators are considered to
have a better side-effect profile and to be less prone to induction of tolerance than classic
orthosteric agonists. Allosteric modulators have gained significance in the scientific
community in the past decade.
This thesis comprises four parts and focuses on the positive allosteric modulation of the
GABAB receptors. Two prototypal positive allosteric modulators CGP7930 and GS39783
have recently been discovered and characterized in Novartis Pharma (Urwyler et al. 2001 and
2003). A number of questions regarding their further characterization, namely their effects on
orthosteric ligands with distinct intrinsic properties, the role allosteric modulation plays in
GABAB receptor desensitization and biochemical effects of GS39783 in vivo are addressed in
this thesis.
Mechanisms of allosteric modulation at GABAB receptors by CGP7930 and GS39783:
effects on affinities and efficacies of orthosteric ligands with distinct intrinsic properties
The first part of this thesis shows that, as it is predicted by theoretical models of receptor
activation, all GABAB ligand species are amenable to allosteric modulation. A number of
selective GABAB receptor ligands were tested in the presence and the absence of positive
allosteric modulators CGP7930 and GS39783 in in vitro assays, such as radioligand binding,
GTP(γ)S and cellular cyclic AMP (cAMP) measurements. A decrease in affinity of
antagonists was observed in radioligand binding experiments, without a change of the
receptor number, oppositely to increases in affinity of partial agonists. In the GTP(γ)S
experiment the presence of CGP7930 and GS39783 revealed intrinsic efficacies for
CGP35348 and 2-OH-saclofen, two “silent” GABAB receptor antagonists. In the cAMP
measurements, an even more sensitive experimental system, the two abovementioned
compounds acted as partial agonists, with increased efficacies in the presence of positive
allosteric modulators. Inverse agonistic tendencies were observed with the “silent” antagonist
CGP52432. In this part of the thesis, the positive allosteric modulators GS39783 and
CGP7930 have been shown to be useful experimental tools for elucidating intrinsic properties
of orthosteric ligands. (Chapter 5, Section 5.1.)
Receptor activation involving positive allosteric modulation, unlike full agonism, does
not result in GABAB receptor desensitization: an in vitro study
To inspect the role of the positive allosteric modulator GS39783 in GABAB receptor
desensitization, receptor function and cell surface receptor density were examined in a
recombinant GABAB cell line and in primary neuronal cultures upon persistent treatments
with GABAB agonists, and combinations of agonists and GS39783. While the GABAB
receptor desensitized after lasting pretreatments with saturating concentrations of GABAB
agonists GABA or R(-)-baclofen, the combined treatment with low concentration of agonists
and GS39738 did not lead to desensitization, despite activating the receptor to the same extent
as desensitization-inducing agonists. These results indicate that it is the degree of occupancy
of the orthosteric binding site that determines desensitization, rather than the degree of
receptor activation. Desensitization experiments with the GABAB receptor and GS39783 in
this study demonstrate that, according to predictions, positive allosteric modulation as a
therapeutic principle may indeed be more promising than orthosteric agonism, having less
propensity for developing tolerance due to receptor desensitization. (Chapter 5, Section 5.2.)
Changes in behavior of allosteric and orthosteric GABAB receptor ligands after a
continuous agonist pretreatment
Investigating the effects of GS39783 on GABAB receptor desensitization, interesting findings
revealed changes in ligand behavior upon receptor desensitization in the GABAB recombinant
cell line. “Silent” antagonists such as CGP62349, CGP52432, CGP56999 and SCH50911
were found to have inverse agonistic properties, the partial agonist 2-OH-saclofen was devoid
of positive intrinsic efficacy and the positive allosteric modulator GS39738 was acting in a
manner of an allosteric agonist. The possibility of residual GABA present from the
pretreatment and responsible for these effects was ruled out. All observed phenomena point
toward an increase in constitutive activity of the receptor. Increase of constitutive receptor
activity after lasting agonist pretreatments have previously been reported for the β2-adrenergic
and the opioid receptors. This is, however, the first such finding for the GABAB receptor,
which might be important in elucidating the valence of orthosteric ligands as well as their
effects upon a chronic drug treatment. It would be interesting to see whether the same
phenomena would be observed also for other members of GPCR family 3. (Chapter 5,
Section 5.3.)
The positive allosteric modulator GS39783 enhances GABAB receptor-mediated
inhibition of cyclic AMP formation in rat striatum in vivo
In the last part of this thesis, I provide the first biochemical evidence of in vivo activity of a
positive allosteric modulator of GPCRs. By using in vivo microdialysis in striata of freely
moving rats, changes in extracellular levels of cAMP following GABAB receptor activation
were monitored. Locally applied GABAB receptor agonist R(-)-baclofen inhibited cAMP
formation stimulated by 7β-forskolin in a concentration-dependent manner, which was
reversed by the co-application of the selective GABAB antagonist CGP56999. Orally applied
positive allosteric modulator GS39783 lacked effects on its own but, together with a threshold
concentration of R(-)-baclofen, it significantly decreased cAMP formation in a dosedependent
fashion. Effects of GS39783 were revoked with CGP56999, showing dependence
on concomitant GABAB receptor activation by an agonist and suggesting allosteric
modulation as its mechanism of action in vivo. (Chapter 5, Section 5.4.)