Bridging the gap: bitopic ligands of G-protein-coupled receptors J. Robert Lane, Patrick M. Sexton, Arthur Christopoulos  Trends in Pharmacological Sciences  Volume 34, Issue 1, Pages 59-66 (January 2013) DOI: 10.1016/j.tips.2012.10.003 Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 1 Features of allosteric modulation and stimulus bias at G-protein-coupled receptors (GPCRs). ‘Classical’ allosteric modulation is traditionally associated with the interaction of structurally distinct ligands across different binding sites (located extracellularly or intracellularly); non-conserved allosteric sites can allow for selective targeting of modulator ligands. The magnitude of the observed allosteric effect is determined by the cooperativity between orthosteric and allosteric sites, and approaches a limit (ceiling level) when both sites are occupied. However, differences in the magnitude and direction of the allosteric effect can occur due to the promotion of different conformations by structurally distinct ligands (probe dependence). Distinct GPCR conformations stabilized by orthosteric, allosteric, or co-bound ligands can bias the resulting stimulus, which can then cause selection of specific signaling pathways to the exclusion of others for pleiotropically coupled receptors. Trends in Pharmacological Sciences 2013 34, 59-66DOI: (10.1016/j.tips.2012.10.003) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 2 Theoretical advantages associated with the design of bitopic ligands. (a) Bitopic ligand design as a means to ‘increase binding affinity’. Effects of the bitopic ligand THRX-160209 and its constituent orthosteric (3-BHP) or allosteric (4-ABP) moieties on the binding of the orthosteric probe NMS at the M2 muscarinic receptor. Adapted from [13]. (b) ‘Gain in subtype selectivity’, as exemplified by the bitopic ligand, ‘hybrid 2’ (2-{3-[1-(6-{1,1-dimethyl-1-[4-(isoxazol-3-yloxy)but-2-ynyl]-ammonium}hexyl)-1,1-dimethylammonio]-2,2-dimethylpropyl}-benzo[de] isoquinoline-1,3-dione dibromide), relative to the non-selective, orthosteric, agonist iperoxo, at native muscarinic acetylcholine receptors expressed in guinea pig left atrium (M2), rabbit vas deferens (M1), and guinea pig ileum (M3). Adapted from [8]. (c) Promotion of ‘stimulus bias’. Attachment of the orthosteric adenosine-derived agonist, LUF5519, to the positive allosteric modulator (2-amino-4,5-dimethyl-3-thienyl) [3(trifluoromethyl)phenyl]methanone yields the bitopic ligand, LUF6258, at the adenosine A1 receptor. Profiling of this compound in both an ERK1/2 phosphorylation assay and an assay to measure [35S]GTPγS binding revealed a distinct bias profile for LUF6258 compared with LUF5519 or a derivative of LUF6258 with a linker length too short to bridge both sites simultaneously (LUF6234). Bias is illustrated by a bias plot [53] in which the response in either assay to equimolar concentrations of agonist is plotted. Data reanalyzed from [14]. Trends in Pharmacological Sciences 2013 34, 59-66DOI: (10.1016/j.tips.2012.10.003) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 3 Potential modes of putative bitopic ligand interactions. A true bitopic mode involves the ligand making simultaneous interaction with an orthosteric and an allosteric site. Alternatively, ligands may distribute between orthosteric or allosteric orientations via a ‘flip-flop’ mechanism, or concomitantly by binding cooperatively to each site on a single receptor. Trends in Pharmacological Sciences 2013 34, 59-66DOI: (10.1016/j.tips.2012.10.003) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 4 Structurally similar G-protein-coupled receptors (GPCRs) possess multiple possible binding cavities. The program, CAVER [54], was used to identify cavities connecting the ligand binding site (situated within the transmembrane domains) to the extracellular surface in the high-resolution crystal structures of the following biogenic amine receptors: dopamine D3 receptor (PDB 3PBL, orange); muscarinic M2 acetylcholine receptor (PDB 3UON, green); histamine H1 receptor (PDB 3RZE, pink); and the β2 adrenergic receptor (PDB 2RH1, yellow) [17,36,37,55]. These cavities are shown superimposed on the structure of the β2 adrenergic receptor, which is rendered in gray and has loops removed for clarity. Views of the cavities are shown from the top (a, extracellular) and side (b). Trends in Pharmacological Sciences 2013 34, 59-66DOI: (10.1016/j.tips.2012.10.003) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 5 Logistic flowchart of considerations in the design of bitopic ligands. Trends in Pharmacological Sciences 2013 34, 59-66DOI: (10.1016/j.tips.2012.10.003) Copyright © 2012 Elsevier Ltd Terms and Conditions