The Structural Basis for GTS-21 Selectivity between Human and Rat Nicotinic 7 Receptors

The 7 nAChR-selective partial agonist 3-(2,4-dimethoxybenzylidene)anabaseine (GTS-21) is more efficacious and potent for rat receptors than for human 7 receptors. Four single amino acid differences exist between human and rat 7 in the agonist binding site, two in the C loop, and one each in the E and F loops. Reciprocal mutations were made in these three domains and evaluated in Xenopus laevis oocytes. Mutations in the C and F loops significantly increased the efficacy of GTS-21 for the human receptor mutants but not to the level of the wild-type, and reciprocal mutations in rat 7 did not decrease responses to GTS-21. Whereas mutations in the E loop alone were without effect, the Eand F-loop mutations together increased GTS-21 efficacy and potency for human receptors, but the EF mutations in the rat receptors decreased the GTS-21 potency without changing the efficacy. The only mutants that showed a full reversal of the efficacy differences between human and rat 7 contained complete exchange of all four sites in the C, E, and F loops or just the sites in the C and F loops. However, the reversal of the potency ratio seen with the EF mutants was not evident in the CEF mutants. Our data therefore indicate that the pharmacological differences between rat and human 7 receptors are caused by reciprocal differences in sites within and around the binding site. Specific features in the agonist molecule itself are also identified that interact with these structural features of the receptor agonist binding site. A crucial assumption for the translation of preclinical research from animal studies to human therapeutics is that receptor pharmacology will be consistent between species. That is, drugs shown to be useful based on their ability to work in animal (rodent) models would also have similar activity on human forms of the receptors. The neuronal 7type nicotinic acetylcholine receptor (nAChR) has been identified as a potential target for the treatment of Alzheimer’s disease (Lindstrom, 1997), and 3-(2,4-dimethoxybenzylidene) anabaseine (GTS-21; also called DMXBA), which selectively targets this receptor, has been shown to improve learning and memory in animal models of cholinergic hypofunction (Kem, 2000). This 7-selective partial agonist has also been shown to prevent the death of differentiated PC-12 cells that occurs after nerve growth factor removal and the death of cultured primary neurons that occurs after high levels of NMDA receptor activation (Martin et al., 1994; Shimohama et al., 1998). It is interesting that although GTS-21 was able to protect PC-12 cells from the cytotoxic effects of amyloid peptide exposure, it was not able to protect human-derived SK-N-SH cells from the same cytotoxic stress, although the GTS-21 4-hydroxy metabolite, 3-(4-hydroxy,2-methoxybenzylidene)anabaseine (4-OH-GTS-21), was cytoprotective in the same assay (Meyer et al., 1998a). A likely explanation for these observed differences in cytoprotective activity came from the observation that GTS-21 was far less efficacious for human 7 receptors than it was for rat 7 receptors (Briggs et al., 1997). Although 4-OH-GTS-21 also activates rat 7 receptors better than human 7 receptors, it is more efficacious than GTS-21 for both receptors, so that at a cytoprotective concentration, it produces activation of human 7 receptors that is comparable with the activation of rat receptors produced by GTS-21 at the same concentration (Papke