Ginkgolide X Is a Potent Antagonist of Anionic Cys-loop Receptors with a Unique Selectivity Profile at Glycine Receptors

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 IC50 values, except the rho 1 receptor at which it was a significantly weaker antagonist. Ginkgolide X also displayed high nanomolar/low micromolar IC50 values at the homomeric alpha 1 and alpha 2 GlyRs, whereas it was inactive at the heteromeric alpha 1 beta and alpha 2 beta subtypes at concentrations up to 300 mu M. 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 GABAAR. Conformational analysis and docking of low-energy conformations of the native ginkgolide A and ginkgolide X into a alpha 1 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.