Abstract
The novel ginkgolide analog ginkgolide X was characterized functionally at human glycine and gamma-aminobutyric acid type A receptors (GlyRs and GABA(A)Rs, respectively) in the fluorescence-based FLIPR(TM) Membrane Potential assay. The compound inhibited the signaling of all GABA(A)R subtypes included in the study with high nanomolar/low micromolar IC(50) values, except the rho 1 receptor at which it was a significantly weaker antagonist. Ginkgolide X also displayed high nanomolar/low micromolar IC(50) values at the homomeric alpha1 and alpha2 GlyRs, whereas it was inactive at the heteromeric alpha 1 beta and alpha 2 beta subtypes at concentrations up to 300 microm. Thus, the functional properties of the compound were significantly different from those of the naturally occurring ginkgolides A, B, C, J, and M but similar to those of picrotoxin. In a mutagenesis study the 6' M2 residues in the GlyR ion channel were identified as the primary molecular determinant of the selectivity profile of ginkgolide X, and a 6' M2 ring consisting of five Thr residues was found to be of key importance for its activity at the GABA(A)R. Conformational analysis and docking of low-energy conformations of the native ginkgolide A and ginkgolide X into a alpha1 GlyR homology model revealed two distinct putative binding sites formed by the 6' M2 residues together with the 2' residues and the 10' and 13' residues, respectively. Thus, we propose that the distinct functionalities of ginkgolide X compared with the other ginkgolides could arise from different flexibility and thus different binding modes to the ion channel of the anionic Cys-loop receptor.
Highlights
Introduction of the reverseS10ЈT, S13ЈT, or S10ЈT/S13ЈT mutations into the  subunit did not result in ␣1 receptors more sensitive to either ginkgolide X or picrotoxin than WT ␣1, at least not at the concentrations tested in this study
GABAARs Several human GABAAR subtypes made up from the 1 subunit, from ␣ and  subunits or from ␣, , and ␥2s subunits were expressed in tsA-201 cells by transient transfection of the respective subunit cDNAs, and the functional properties of GABA and ginkgolide X were determined at these receptors in the FMP Blue assay (Table 1 and Fig. 2)
Because transfection of cells with ␣ and  subunits gives rise to functional GABAARs, the incorporation of the ␥2s subunit in pentamers formed at the cell surface of cells transfected with ␣, , and ␥2s subunits was verified in two different ways
Summary
Introduction of the reverseS10ЈT, S13ЈT, or S10ЈT/S13ЈT mutations into the  subunit did not result in ␣1 receptors more sensitive to either ginkgolide X or picrotoxin than WT ␣1, at least not at the concentrations tested in this study (see Table 2 and Fig. 9). The binding mode of ginkgolide A to the 2Ј– 6Ј region of the ␣1 GlyR ion channel observed for the top scoring pose of the compound in the docking experiments in this study is in good agreement with the binding mode previously proposed for the native ginkgolides and supported by extensive mutagenesis work (Fig. 7D) [19, 20]. It seems to be possible for the alternative ring conformation of ginkgolide A to dock in the 6Ј–13Ј region as well (see Fig. 7E), it is slightly less favorable from an energy perspective. This preference could possibly shift in the ␣1 GlyR as the tip of ginkgolide A, due to conformational flexibility in the 3-methyldihydrofuran-2-one end of the molecule, turns hydrophobic in the alternative (ϩ0.8 kcal/mol) ring conformer and could be stabilized by hydrophobic interactions with the F6Ј residues in the heteromeric receptor
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