74 research outputs found
Comparative potencies of CGP 47654A and CGP 46165A as GABA (B) receptor antagonists in rat brain
No full textIn rat neocortical slices maintained in Mg2+-free Krebs medium, the gamma-aminobutyric acid (GABAB) receptor agonist baclofen concentration-dependently depressed the frequency of spontaneous discharges (EC50 = 6.1 microM). This was reversibly antagonised by 3-aminopropyl-(1,1-difluoro-n-butyl)-phosphinic acid (25, 100, 500 microM) (CGP 47654A) and 3-aminopropyl-P-(alpha-hydroxybenzyl)-phosphinic acid (CGP 46165A) (50, 100, 400 microM) which produced rightwards shifts of the baclofen concentration-response curves, with respective pA2 values of 4.9+/-0.2 and 4.6+/-0.15. Although relatively potent on GABAB heteroreceptors studied here, CGP 47654A and CGP 46165A were 5 and 50 times weaker, respectively, as GABAB autoreceptor antagonists [Froestl, W., Mickel, S.J., Von Sprecher, G., Diel, P.J., Hall, R.G., Maier, L., Strub, D., Melillo, V., Baumann, P.A., Bernasconi, R., Gentsch, C., Hauser, K., Jaekel, J., Karlsson, G., Klebs, K., Maitre, L., Marescaux, C., Pozza, M.F., Schmutz, M., Steinmann, M.W., Van Riezen, H., Vassout, A., Mondadori, C., Olpe, H.R., Waldmeier, P.C., Bittiger, H., 1995. Phosphinic acid analogues of GABA. 2. Selective, orally active GABAB antagonists. J. Med. Chem. 38: 3313-3331.], representing potentially useful ligands for differentiating GABA hetero- and autoreceptors
Ligands for the isolation of GABAB receptors W. Froestl would like to dedicate this work to the first GABAB chemist, Cr Heinrich Keberle, on the occasion of his 77th birthday.
No full textThe development of medication for alcohol-use disorders targeting the GABAB receptor system
No full textThe present paper summarizes experimental and clinical data suggesting the therapeutic potential of the prototypic GABAB receptor agonist, baclofen, for the treatment of alcohol-use disorders (AUDs). Numerous studies have reported baclofen-induced suppression of alcohol drinking, relapse-like drinking, and alcohol reinforcing, rewarding, stimulating, and motivational properties in rats and mice. The majority of clinical surveys conducted to date have demonstrated the capacity of baclofen to suppress alcohol consumption, craving for alcohol, and alcohol withdrawal symptomatology in alcohol-dependent patients. More recently, the discovery of a positive allosteric modulatory binding site, together with the synthesis of in vivo effective ligands, provided a new tool for pharmacological manipulations of the GABAB receptor. Accumulating lines of preclinical evidence suggest that positive allosteric modulators of the GABAB receptor (GABAB PAMs), such as GS39783, display a high therapeutic index and retain baclofen’s capacity to suppress alcohol consumption and alcohol reinforcing and motivational properties. The present paper also summarizes the most relevant patents on GABAB receptor agonists and GABAB PAMs as possible pharmacotherapies for AUDs
Reducing effect of the positive allosteric modulator of the GABA(B) receptor, GS39783, on alcohol self-administration in alcohol-preferring rats
No full textRationals & Objectives The positive allosteric modulator of the GABA(B) receptor, GS39783, has recently been found to suppress acquisition and maintenance of alcohol drinking behavior in selectively bred Sardinian alcohol-preferring (sP) rats exposed to the standard, homecage two-bottle "alcohol vs water" choice regimen. The present study was designed to extend the characterization of the "anti-alcohol" effects of GS39783 to oral self-administration of alcohol under an operant procedure.
Materials & Methods Two separate groups of male sP rats were trained to lever-press (on an FR4 schedule) to orally self-administer alcohol (15%, v/v) or sucrose (0.3%, w/v) in daily 30-min sessions. Once lever-pressing behavior reached stable levels, the effect of GS39783 (0, 25, 50, and 100 mg/kg, i.g.) on responding for alcohol and sucrose was determined.
Results Pretreatment with GS39783 resulted in a significant, dose-dependent reduction in responding for alcohol; at the dose of 100 mg/kg GS39783, the number of lever responses for alcohol was reduced by approximately 50% in comparison to vehicle-treated rats. The effect of GS39783 on alcohol self-administration was specific, as responding for sucrose was completely unaffected by pretreatment with GS39783.
Conclusions These data demonstrate the capability of GS39783 to attenuate the reinforcing properties of alcohol in alcohol-preferring rats. These data constitute a further piece of experimental evidence in support of the hypothesized role for the GABA(B) receptor in the control of alcohol drinking and reinforcement
Ligands for expression cloning and isolation of GABA(B) receptors
No full textOutlined is the rationale behind the syntheses of radioligands [125I]CGP64213 and [125I]CGP71872, which led to the identification of cloned GABA(B) receptors 1a and 1b 17 years after the first pharmacological characterisation of native GABA(B) receptors by Bowery et al. [Nature 283 (1980) 92-94]. More recently it was shown that the N-terminal extracellular domains of GABA(B) receptors 1a and 1b contain the binding sites for agonists and antagonists [B. Malitschek et al., Mol. Pharmacol. 56 (1999) 448-454]. In order to isolate the extracellular domain(s) of GABA(B) receptors 1a (or 1b) and to purify and crystallise these proteins a third ligand [125I]CGP84963 was designed, which combines, in one molecule, a GABA(B) receptor binding part, an azidosalicylic acid as photoaffinity moiety and 2-iminobiotin, which binds to avidin in a reversible, pH-dependent fashion [W. Froestl et al., Neuropharmacology 38 (1999) 1641-1646]
GABAB receptors in Schwann cells influence proliferation and myelin protein expression
No full textThe location and the role of gamma-aminobutyric acid type B (GABA(B)) receptors in the central nervous system have recently received considerable attention, whilst relatively little is known regarding the peripheral nervous system. In this regard, here we demonstrate for the first time that GABA(B) receptor isoforms [i.e. GABA(B(1)) and GABA(B(2))] are specifically localized in the rat Schwann cell population of the sciatic nerve. Using the selective GABA(B) agonist [i.e. (-)-baclofen] and the antagonists (i.e. CGP 62349, CGP 56999 A, CGP 55845 A), such receptors are shown to be functionally active and negatively coupled to the adenylate cyclase system. Furthermore, exposure of cultured Schwann cells to (-)-baclofen inhibits their proliferation and reduces the synthesis of specific myelin proteins (i.e. glycoprotein Po, peripheral myelin protein 22, myelin-associated glycoprotein, connexin 32), providing evidence for a physiological role of GABA(B) receptors in the glial cells of the peripheral nervous system
Reducing effect of the positive allosteric modulators of the GABAB receptor, CGP7930 and GS39783, on alcohol intake in alcohol-preferring rats
No full textThe gamma-aminobutyric acidB (GABA(B)) receptor full agonists, baclofen and CGP44532, have been found to suppress different aspects of alcohol drinking behavior, including acquisition and maintenance, in selectively bred Sardinian alcohol-preferring (sP) rats. The present study was designed to assess whether this capability extends to the recently synthesized, positive allosteric modulators of the GABA(B) receptor, 2,6-Di-tert-butyl-4-(3-hydroxy-2,2-dimethyl-propyl)-phenol (CGP7930) and N,N'-dicyclopentyl-2-methylsulfanyl-5-nitro-pyrimidine-4,6-diamine (GS39783). In the "acquisition" experiments, CGP7930 (0, 25, 50 and 100 mg/kg; i.g.) and GS39783 (0, 6.25, 12.5 and 25 mg/kg; i.g.) were administered for 5 consecutive days to alcohol-naive sP rats. In the "maintenance" experiments, (0, 50 and 100 mg/kg; i.g.) and GS39783 (0, 50 and 100 mg/kg; i.g.) were administered for 5 consecutive days to alcohol-experienced sP rats. Alcohol intake was evaluated under the standard, homecage 2-bottle "alcohol (10%, v/v) vs water" regimen with unlimited access for 24 h/day. Both CGP7930 and GS39783 dose-dependently suppressed the acquisition of alcohol drinking behavior. In the "maintenance" experiments, CGP7930 and GS39783 reduced daily alcohol intake by 30-40% only at the highest dose when compared to vehicle-treated rats; this effect tended to vanish on continuing treatment. The results of the present study suggest that positive allosteric modulation of the GABA(B) receptor produced an effect on alcohol drinking behavior similar to that produced by GABA(B) receptor full agonists. These data also suggest that positive allosteric modulation of the GABA(B) receptor may constitute a potential strategy for developing new drugs for treating alcohol dependence
Pharmacological re-evaluation of a GABAB receptor antagonist CGP 47332A in rat brain
No full textIn rat neocortical slices maintained in Mg(2+)-free Krebs medium, the gamma-aminobutyric acid (GABA(B)) receptor agonist baclofen concentration-dependently depressed the frequency of spontaneous discharges (EC(50)=12 microM). This was reversibly antagonised by (R, S)-3-amino-2-hydroxy-propyl-P-n-butyl-phosphinic acid (CGP 47332A) (25, 100, 300 microM) which produced rightwards shifts of the baclofen concentration-response curves (pA(2) value=4.8+/-0.1). In electrically stimulated slices preloaded with [3H]GABA, CGP 47332A increased its release (EC(150)=100 microM) through antagonism of GABA(B) autoreceptors. Although CGP 47332A was some six times weaker on GABA(B) auto- than on heteroreceptors, yet its congener lacking the beta-hydroxy substituent displays equal potency in both binding (IC(50)=38 microM) and GABA(B) autoreceptor functional studies (EC(150)=38 microM) as previously reported [Froestl, W., Mickel, S.J. , Von Sprecher, G., Diel, P.J., Hall, R.G., Maier, L., Strub, D., Melillo, V., Baumann, P.A., Bernasconi, R., Gentsch, C., Hauser, K., Jaekel, J., Karlsson, G., Klebs, K., Maitre, L., Marescaux, C., Pozza, M.F., Schmutz, M., Steinmann, M.W., Van Riezen, H., Vassout, A., Mondadori, C., Olpe, H.R., Waldmeier, P.C., Bittiger, H., Phosphinic acid analogues of GABA: 2. Selective, orally active GABA(B) antagonists. J. Med. Chem. 38 (1995) 3313-3331.]
Activation of the GABAB receptor prevents nicotine-induced locomotor stimulation in mice
No full textRecent studies demonstrated that activation of the GABAB receptor, either by means of orthosteric agonists or positive allosteric modulators (PAMs), inhibited different nicotine-related behaviors, including intravenous self-administration and conditioned place preference, in rodents. The present study investigated whether the anti-nicotine effects of the GABAB receptor agonist, baclofen, and GABAB PAMs, CGP7930 and GS39783, extend to nicotine stimulant effects. To this end, CD1 mice were initially treated with baclofen (0, 1.25, and 2.5 mg/kg, i.p.), CGP7930 (0, 25, and 50 mg/kg, i.g.), or GS39783 (0, 25, and 50 mg/kg, i.g.), then treated with nicotine (0 and 0.05 mg/kg, s.c.), and finally exposed to an automated apparatus for recording of locomotor activity. Pretreatment with doses of baclofen, CGP7930, or GS39783 that did not alter locomotor activity when given with nicotine vehicle fully prevented hyperlocomotion induced by 0.05 mg/kg nicotine. These data extend to nicotine stimulant effects the capacity of baclofen and GABAB PAMs to block the reinforcing, motivational, and rewarding properties of nicotine. These data strengthen the hypothesis that activation of the GABAB receptor may represent a potentially useful, anti-smoking therapeutic strategy
Biochemical pharmacology of the positive allosteric modulation of the GABAb receptor "in Vitro" and "in Vivo"
No full textAllosteric 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.
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