| pA2 online © Copyright 2004 The British Pharmacological Society |
074P
University of Bath Summer Meeting July 2004 |
|
Pharmacological characterisation of caloporoside and analogues using [35S] TBPS binding to adult rat forebrain GABAA recepters S. Abuhamdah1,2, A. Fürstner3, G. Lees2 and P.L. Chazot1. 1School of Biological and Biomedical Sciences, Durham University, UK; 2Sunderland Pharmacy School, University of Sunderland, UK; 3Max-Planck-Institute, Mulheim, Germany |
|
Print abstractCaloporoside is a natural active fungal metabolite, which was isolated several years ago from fermentation of Caloporous dichrous and was described to exhibit antibacterial, antifungal and phospholipase C inhibitory activity (Weber et al, 1994). Chemical synthesis of caloporoside and a number of analogues has been described (Fürstner et al, 1996, 1998). We have previously reported evidence that related compounds, lactose and octyl-ß-D-mannoside, bind and functionally modulate rodent GABAA receptors, respectively (Rezai et al., 2003; Lees, Chazot et al., 2000).
In this present study, we have characterized the pharmacology of caloporoside and two further congeners, 2-Hydroxy-6-{[(16R)-beta-D-mannopyranosyloxy) heptadecyl]} benzoic acid and octyl-beta-D-glucoside on GABAA receptors using [35S]-t-butylbicyclophosphoorothionate radioligand binding assay. The assay was performed in 50 mM Tris-buffer supplemented with 0.2 M NaCl (pH 7.4). Well-washed adult rat Sprague Dawley strain forebrain membranes (100 µg) were incubated in the presence of 25 nM [ 35S] TBPS radioligand, at 25 Cº for 90 min. Non-specific binding was defined in the presence of 100 µM picrotoxinin. [3H] flunitrazepam binding assays were performed as described in Rezai et al., 2002. The reactions were terminated by rapid filtration using a Brandell cell harvester.
Caloporoside and 2-Hydroxy-6-{[(16R)-ß-D-mannopyranosyloxy) heptadecyl]} benzoic acid produced concentration-dependent complete inhibition of specific [S35] TBPS binding with overall apparent IC50 values of (147 ± 1 µM) and (142 ± 1 µM) respectively, and steep pseudo Hill coefficients (nH = -2.78 ± 1.4 and -2.96 ± 0.84, respectively) (mean ± SD for three determinations). In contrast, octyl-ß-D-glucoside elicited a concentration-dependent stimulation of specific [S35] TBPS binding (Emax = 144 ± 4%; EC50 = 390 ± 230 nM, mean ± SD for three independent experiments). The level of stimulation was greater than that elicited by flunitrazepam (Emax = 120 ± 3%; EC50 = 4.3 ± 3.1 nM, mean ± SD for three independent experiments), determined in parallel experiments. However, the three test compounds elicited no significant effect (positive or negative) upon [3H] flunitrazepam binding, indicating that these compounds did not bind directly, or allosterically couple, to the benzodiazepine site of the GABAA receptor. This study suggests that octyl-ß-D-glucoside is a high affinity positive GABAA receptor channel modulator, and may act in a similar fashion to octyl-ß-D-mannoside (Lees, Chazot et al., 2000).
Lees G., et al., (2000) Bioorg. Med. Chem. Letts. 10, 1759-1761
Fürstner, A. et al., (1996) Tetrahedron, 52, 15071-15078
Fürstner A. et al., (1998) J. Org. Chem. 63, 3072-3080
Rezai, N. et al., (2003) Biochem. Pharmacol. 65, 619-623
Weber, W. et al., (1994) J. Antibiotic, 47, 1188-1194