Rational design of dualsteric GPCR ligands: quests and promise

Abstract

Dualsteric ligands represent a novel mode of targeting G protein-coupled receptors (GPCRs). These compounds attach simultaneously to both, the orthosteric transmitter binding site and an additional allosteric binding area of a receptor protein. This approach allows the exploitation of favourable characteristics of the orthosteric and the allosteric site by a single ligand molecule. The orthosteric interaction provides high affinity binding and activation of receptors. The allosteric interaction yields receptor subtype-selectivity and, in addition, may modulate both, efficacy and intracellular signalling pathway activation. Insight into the spatial arrangement of the orthosteric and the allosteric site is far advanced in the muscarinic acetylcholine receptor, and the design of dualsteric muscarinic agonists has now been accomplished. Using the muscarinic receptor as a paradigm, this review summarizes the way from suggestive evidence for an orthosteric/allosteric overlap binding to the rational design and experimental validation of dualsteric ligands. As allosteric interactions are increasingly described for GPCRs and as insight into the spatial geometry of ligand/GPCR-complexes is growing impressively, the rational design of dualsteric drugs is a promising new approach to achieve fine-tuned GPCR-modulation.

Figure 1

‘Blue-print’ for the design of dualsteric receptor activators with subtype-selectivity. (A) Side view on the upper part of the muscarinic M₂ acetylcholine receptor. The free volume of the ligand binding pocket is lined by the grid structure. The model is based on the crystal structure of bovine rhodopsin in the inactive state. An inverse agonist (N-methylscopolamine) is docked into the orthosteric site to stabilize an inactive receptor conformation in the molecular dynamics simulation (modified from Voigtländer et al., 2003). The circle marks the allosteric core region which harbours non-conserved subtype-selectivity providing amino acids. (B) Design concept for subtype-selective allosteric/orthosteric hybrid agonists. The allosteric building block is derived from the alkane-bis-ammonio (ABA)-type compound W84, iperoxo (agonist d) is the orthosteric building block, the resulting hybrid compound is 1d.

Figure 2

Structures of ligands with a suggested allosteric/orthosteric binding mode at muscarinic receptors.

Figure 4

Binding modes of muscarinic dualsteric agonists in the absence and presence of an orthosteric radioligand probe – related experimental evidence and functional consequences. Centre: a symbolized muscarinic receptor contains in its binding cavity the inner orthosteric and the outer allosteric binding region (grey shaded). The dualsteric ligand such as compound 1d (Figure 3) consists of an orthosteric agonist building block (green triangle) which is connected by a linker with an allosteric building block (blue rectangle); orthosteric probe: red triangle. Boxes, right and bottom: experimental evidence for dualsteric binding arising from radioligand binding assays in wild-type and point-mutated human M₂ muscarinic receptors (right box) and from G protein-activation experiments ([³⁵S]GTPγS-assay, bottom box). Boxes, left: functional consequences related to dualsteric binding arising from radioligand binding assays, G protein-activation assays, label-free signalling pathway analysis by dynamic mass redistribution and organ-bath experiments. For details see Antony et al. (2009) and Kebig et al. (2009).

Figure 3

Potency, efficacy and muscarinic subtype-selectivity of building blocks and resulting allosteric/orthosteric hybrid compounds. Potency is indicated as minus log EC₅₀ and minus log K_B in the case of agonism and antagonism respectively, for the action of orthosteric agonists a–d (upper panel), allosteric inverse agonist W84 (middle panel) and hybrids 1a–1d (lower panel) in isolated organ models for M₁ (rabbit vas deferens), M₂ (guinea pig left atrium), M₃ (guinea pig ileum). Hybrids consist of the fragment 1 from W84 and the respective agonist molecules a–d. Efficacy is indicated by dot colour – green: agonist activity, red: antagonist activity. Values for a–d are taken from Dallanoce et al. (1999), for W84 from Tränkle et al., 1998, for 1a–1c from Disingrini et al. (2006) and for 1d from Antony et al. (2009).

Figure 5

‘Functional misfit’ of dualsteric ligands which consist of an orthosteric agonist building block and an allosteric inverse agonist building block. Middle panel: the ligand-free receptor may switch spontaneously from the inactive (top) into the active (conformation) which stimulates a G protein (GP); the inactive-to-active conformational transition includes the orthosteric and the allosteric binding site (symbolized as white areas in the ligand binding pocket of the receptor protein). The inactive conformation (left panel) is stabilized by inverse agonist ligands that may bind either to the allosteric (left panel, top) or to the orthosteric site (left panel, bottom). Likewise, the active conformation (right panel) can be stabilized by appropriate allosteric and orthosteric ligands. The building blocks of the hybrid compounds shown in Figure 3 prefer functionally opposing receptor conformations (stippled line).