Vol. 282, Issue 2, 513-520, 1997

Regulation of Neuronal and Recombinant GABA_A Receptor Ion Channels by Xenovulene A, a Natural Product Isolated from Acremonium strictum

P. Thomas, H. Sundaram, B. J. Krishek, P. Chazot , X. Xie, P. Bevan, S. J. Brocchini, C. J. Latham, P. Charlton, M. Moore, S. J. Lewis, D. M. Thornton, F. A. Stephenson and T. G. Smart

The School of Pharmacy, Departments of Pharmacology (P.T., B.J.K., X.X., T.G.S.) and Pharmaceutical Chemistry (H.S., P.C., F.A.S.), London, WC1N 1AX, and Xenova Ltd., (P.B., S.J.B., C.J.L., P.C., M.M., S.J.L., D.M.T.), Slough, SL1 4EF, United Kingdom

Xenovulene A (XR368) is a natural product exhibiting little structural resemblance with classical benzodiazepines yet is able to displace high-affinity ligand binding to the benzodiazepine site of the -aminobutyric acid (GABA)_A receptor. We have characterized this compound and an associated congener (XR7009) by use of radioligand binding and electrophysiological methodologies with native neurons and the Xenopus oocyte expression system. Xenovulene A, and the more potent XR7009, inhibited [³H]flunitrazepam binding to rat forebrain with K_i values of 7 and 192 nM, and 1.7 and 42 nM, respectively, each site accounting for approximately 50% of the total specific binding. In cerebellar and spinal cord membranes, these ligands identified only single binding sites. These ligands demonstrated no intrinsic agonist activity at recombinant GABA_A receptors comprising 112S subunits expressed in Xenopus oocytes, yet at 1 µM both significantly potentiated the GABA-induced response and reduced the GABA EC₅₀ from 10.9 (control) to 5.1 (Xenovulene A) or 2.7 µM (XR7009). The rank potency order for enhancement of the 10 µM GABA response is: XR7009 (EC₅₀, 0.02 µM) > diazepam (0.03) > Xenovulene A (0.05) > flurazepam (0.17). The activity of XR368 and XR7009 was reduced by the benzodiazepine antagonist, flumazenil, and absent in receptors devoid of the 2 subunit. These agents exhibited receptor subtype selectivity because 312S receptors were less sensitive to these compounds relative to 1 subunit-containing receptors, whereas 612S receptors were completely insensitive. Potentiation of the response to GABA on native GABA_A receptors in cortical neurons substantiates the profile of the novel structures of Xenovulene A and XR7009 as specific benzodiazepine agonists.