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Superagonism at muscarinic acetylcholine receptors: methodical challenges and structural basis Superagonism connotes receptor activation with greater intrinsic efficacy than the endogenous agonist (Smith et al., 2010). Until now superagonism has rarely been described for GPCRs. Here, we introduce iperoxo, the most potent activator of muscarinic acetylcholine receptors (Dallanoce et al., 1999), as the first superagonist at M2 muscarinic acetylcholine receptors. In addition we present a novel molecular mechanism underlying this superagonism. The signalling of iperoxo and newly synthesized derivatives was compared with acetylcholine (ACh) and oxotremorine M in CHO hM2-cells applying whole cell dynamic mass redistribution (DMR in Hanks’ balanced salt solution supplemented with 20 mM HEPES, pH 7.0, 28 °C), and measurement of G protein activation by performing [35S]GTPγS experiments with membrane homogenates from CHO-hM2 cells (10 mM HEPES, 10 mM MgCl2, 100 mM NaCl, 10 µM GDP, pH 7.4, 30 °C, 1 h). All presented data are means ± SEM of n experiments. Iperoxo displays a 100-1000 fold higher potency than the endogenous activator acetylcholine, e.g. iperoxo-pEC50 in DMR = 10.10 ± 0.22 (n = 6) in comparison to ACh-pEC50 = 7.60 ± 0.16 (n = 4), reflecting Gi protein activation in CHO-hM2 cells (Schröder et al., 2010). These assays underlie strong amplification mechanisms during signal transduction from the active receptor protein to the functional readout. Therefore, we also determined whole cell binding affinities of the investigated compounds in order to calculate coupling efficiencies of the agonist-bound receptor proteins. While Iperoxo, acetylcholine and all other agonists induce a similar maximum response in the functional assays applied, coupling efficacies τ disclose that iperoxo clearly exceeds acetylcholine in Gi-/Gs-signalling competence in CHO-hM2 cells: Gi-activation iperoxo-log(τ) = 2.64 ± 0.08 (n = 6) vs. ACh-log(τ) = 1.80 ± 0.07 (n = 4); Gs-activation iperoxo-log(τ) = 1.15 ± 0.07 (n = 6) vs. ACh-log(τ) = 0.41 ± 0.04 (n = 4); in both cases the difference was highly significant: p < 0.0001; Student’s t-test). In a loss-of-function mutant CHO hM2-Y1043.33A (Antony et al., 2009), DMR by iperoxo and iperoxo-like derivatives is hardly compromised (iperoxo-Emax = (83 ± 3) % of the maximum iperoxo-induced response at CHO-hM2 wild type; n = 5) in contrast to acetylcholine (ACh-Emax = (8 ± 5) % of the maximum iperoxo-induced response at CHO-hM2 wild type; n = 5). Thus, this assay visualizes superagonism directly, without calculation of coupling efficiencies. Moreover, structure-activity relationships of iperoxo-derivatives with modified cationic head-group imply that superagonism by iperoxo is mechanistically based on parallel activation of the receptor protein via two activation points. Taken together, superagonism at the muscarinic acetylcholine receptors is shown for the first time. Additionally our study suggests that superagonism at other GPCRs might have been overlooked due to methodical reasons. Exploitation of superagonism opens an avenue towards novel biological probes and therapeutic tools.
Literature Antony, J. et al. (2009) Dualsteric GPCR targeting: a novel route to binding and signaling pathway selectivity. FASEB J. 23: 442–450. Dallanoce, C. et al. (1999) Synthesis and functional characterization of novel derivatives related to oxotremorine and oxotremorine-M. Bioorg. Med. Chem. 7: 1539–1547. Schröder, R. et al. (2010) Deconvolution of complex G protein-coupled receptor signaling in live cells using dynamic mass redistribution measurements. Nat. Biotech. 28: 943-949. Smith, N.J. et al. (2011) When simple agonism is not enough: emerging modalities of GPCR ligands. Mol. Cell. Endocrinol. 331: 241–247.
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