For most of the compounds being introduced in the preceding chapter, evidence for the proposed allosteric/orthosteric receptor binding topography came by serendipity, none of these compounds was designed from pre-existing orthosteric and allosteric building blocks. In addition, none of these compounds has efficacy for appreciable M2 receptor activation. The dualsteric compounds described in this review are the first to emerge from a rational design concept aiming at ligands that encompass orthosteric and allosteric functionalities, i.e. high affinity orthosteric receptor activation and allosteric M2 subtype-selectivity (Figure 1B). In these ligands, oxotremorine M-related receptor activators serve as orthosteric building blocks. On their own these agonists are devoid of receptor subtype selectivity (Figure 3, compounds a to d upper panel). These orthosteric building blocks are connected, by fusing the common TMA head, with fragments of ABA-type compounds such as the truncated W84 derivative (1 in Figure 3). W84 and its shortened fragments are subtype-selective for M2 (Figure 3; Antony et al., 2009). In isolated tissue preparations that serve as M1, M2 and M3 models (rabbit vas deferens, guinea pig atrium, guinea pig ileum), all hybrid compounds lose over-all potency, but gain subtype-selectivity. However, compounds 1a, 1b and 1c have almost no intrinsic efficacy. These allosteric/orthosteric hybrids behave as antagonists in the M1 and M2 model and, at best, as very weak partial agonists in the M3 model (Figure 3, lower panel; Disingrini et al., 2006). In membranes from CHO-cells overexpressing human M2 receptors, however, partial agonist-like G protein activation is observed with some of the hybrid compounds (Disingrini et al., 2006). Therefore, the latter already combine receptor subtype selectivity with weak (but physiologically irrelevant) partial agonism. Compound 1d, which includes the extremely potent agonist building block iperoxo d, acts as a full agonist in the M1, the M2 and the M3 model. With this and a closely related compound, which contains an allosteric building block from the ABA-type compound naphmethonium (cf. Antony et al., 2009), the intended M2-preferring agonism is achieved. A true dualsteric binding topography is to be validated by various experimental approaches. In particular it is necessary to exclude that the compounds bind in a purely allosteric topography, as activation of muscarinic receptors from the allosteric site has been shown for various subtypes (for review De Amici et al., in press). The M2 subtype, for instance, is activated by autoantibodies that bind to the second outer loop (e.g. Sterin-Borda et al., 1991; Goin et al., 1997; Hernandez et al., 2008). Therefore, the proposed allosteric/orthosteric binding topography has been verified (Figure 4; for details see Antony et al., 2009). In brief, binding experiments using an orthosteric antagonist radioligand prove that binding of hybrid 1d is sensitive to both, allosteric and orthosteric M2 receptor mutations. In addition, the mutation-induced changes of cooperativity between the hybrids and an orthosteric radioligand are in line with the predictions of a model for dualsteric ligand binding introduced by May et al. (2007a). Furthermore, the hybrid agonist is sensitive to the orthosteric antagonist atropine which eliminates the hybrid's intrinsic efficacy, while still allowing for hybrid binding in the purely allosteric mode. The allosteric antagonist W84, however, interacts with the hybrid in a competitive-type antagonism (Antony et al., 2009). A docking simulation shows that the receptor may well accommodate the hybrid in the orthosteric/allosteric binding topography, with the orthosteric agonist moiety being positioned in the orthosteric site and the allosteric building block in the allosteric core region (Antony et al., 2009). Taken together, the allosteric/orthosteric ligand design translates into a dualsteric receptor binding. In addition, the dualsteric binding mode goes along with biased signalling. While common M2 receptor activating agonists may trigger the Gs pathway in addition to the Gi pathway, the hybrid compound is devoid of Gs activating properties (Antony et al., 2009; Kebig et al., 2009).