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.

Receptor activation involving positive allosteric modulation, unlike full agonism, does not result in GABAB receptor desensitization.

Allosteric modulators act more physiologically than orthosteric ligands, targeting only endogenously activated receptors and not their whole population, which is why they are expected to produce less side effects and tolerance. To inspect the role of the positive allosteric modulator GS39783 in GABAB receptor desensitization, we examined receptor function and cell surface expression in a recombinant GABAB cell line and in primary neuronal cultures upon persistent treatments with GABAB agonists, and combinations of agonists and GS39783. The potency of GABA to inhibit 7beta-forskolin-induced cAMP formation in recombinant cells decreased after the exposure to a saturating GABA concentration, but not after a combination of a low GABA concentration and GS39783, that activated the receptor to the same extent. Concordantly, a significant decrease of cell surface receptors was found after GABA-induced desensitization, unlike after the combined treatment with GABA and GS39783. Similar observations regarding receptor function were found in primary neurons for baclofen-induced inhibition of spontaneous Ca2+ oscillations. However, the cell surface receptor density remained unaffected upon baclofen-induced desensitization in the primary neurons, possibly due to different mechanisms of desensitization in the neurons and the recombinant cell line. These findings indicate that the degree of occupancy of the orthosteric site determines desensitization rather than the degree of receptor activation. In summary, our results conform to predictions that positive allosteric modulators have less propensity for the development of tolerance due to receptor desensitization than classical agonists

Mechanisms of allosteric modulation at GABAB receptors by CGP7930 and GS39783: effects on affinities and efficacies of orthosteric ligands with distinct intrinsic properties.

We determined the effects of the allosteric gamma-aminobutyric acid B receptor modulators CGP7930 and GS39783 on binding and function of orthosteric ligands with distinct intrinsic properties. In radioligand binding (saturation or displacement) experiments, the affinities of a number of competitive antagonists were decreased by the modulators, with no change in receptor number. The binding curves of the partial agonist CGP47656 comprised a high and a low affinity component; the affinity of the former was increased by the allosteric agents. The maximal stimulation of GTP[gamma](35)S binding via recombinant GABA(B) receptors by CGP47656 was increased 4-fold in the presence of 30 microM CGP7930 or GS39783. Two compounds known so far as "silent" competitive GABA(B) receptor antagonists, CGP35348 and 2-OH-saclofen, did not stimulate GTP[gamma](35)S binding on their own, but became low efficacy partial agonists in the presence of the two modulators. The potency of GABA to inhibit the formation of cAMP induced by a forskolin analog in a recombinant CHO cell line expressing GABA(B) receptors was increased by the modulators. CGP35348 and 2-OH-saclofen, like CGP47656, were partial agonists on their own in this assay, and the allosteric modulators increased the potency as well as the efficacy of all three compounds. With CGP52432, there was a trend towards inverse agonism in the cAMP assay. These results show that the intrinsic properties of orthosteric ligands are highly dependent on the characteristics of the assay system used and that allosteric modulators are useful tools for elucidating these properties

The positive allosteric modulator GS39783 enhances GABA(B) receptor-mediated inhibition of cyclic AMP formation in rat striatum in vivo.

We studied the effects of the positive allosteric modulator GS39783 on GABA(B) receptors at a biochemical level in vivo. Changes in extracellular levels of cyclic AMP following GABA(B) receptor activation were monitored in the striatum of freely moving rats using microdialysis. Locally applied GABA(B) agonist R(-)-baclofen inhibited cyclic AMP formation stimulated by a water-soluble forskolin analogue in a concentration-dependent manner (EC50 7.3 microM, maximal inhibition 40%). The selective GABA(B) antagonist CGP56999 reversed R(-)-baclofen-induced cyclic AMP inhibition to control levels, but not higher. Orally applied GS39783 lacked effects on its own but, together with a threshold concentration of R(-)-baclofen (1 microM), significantly decreased cyclic AMP formation in a dose-dependent fashion. Effects of GS39783 were revoked with CGP56999, showing dependence on GABA(B) receptor activation and suggesting allosteric modulation as a mechanism of action in vivo. Administered with a maximally active dose of R(-)-baclofen, GS39783 failed to further inhibit cyclic AMP formation. The data obtained with CGP56999 and the lack of effect of GS39783 alone suggest that there is no detectable endogenous activation of GABA(B) receptors controlling cyclic AMP formation in rat striatum. To our knowledge, these results provide the first biochemical demonstration of in vivo activity of a G protein-coupled receptor-positive allosteric modulator

Selected amino acids, dipeptides and arylalkylamine derivatives do not act as allosteric modulators at GABAB receptors.

Based on recent reports describing enhancing actions of arylalkylamines (fendiline [N-(3,3-diphenylpropyl)-alpha-methylbenzylamine] and prenylamine [N-(3,3-diphenylpropyl)-alpha-methylphenethylamine]), amino acids (L-phenylalanine, L-leucine and L-isoleucine), and dipeptides (L-Phe-Phe and L-Phe-Leu) on baclofen-induced responses in cortical slices, we have examined whether these compounds might act as positive allosteric modulators at GABA(B) receptors. Unlike the previously described allosteric GABA(B) receptor modulator CGP7930 (2,6-Di-tert-butyl-4-(3-hydroxy-2,2-dimethyl-propyl)-phenol), these compounds did not enhance GABA(B) receptor-mediated guanosine 5'-O-(3-thiotriphosphate) [GTP(gamma)35S] binding in native or recombinant cell membrane preparations. Similarly, in a competition binding assay using the antagonist radioligand [3H]CGP62349, CGP7930, but not the other compounds, enhanced the affinities of gamma-aminobutyric acid (GABA) for native GABA(B) receptors from rat brain cortex. Finally, in a cellular assay (Ca(2+) signaling in a recombinant cell line), CGP7930 was again the only compound found to enhance the GABA response. It is concluded that the arylalkylamines, amino acids and dipeptides tested do not act as allosteric modulators at native and recombinant GABA(B) receptors