Identifying Functional Hotspot Residues for Biased Ligand Design in G-Protein-Coupled Receptors

Abstract

G-protein-coupled receptors (GPCRs) mediate multiple signaling pathways in the cell, depending on the agonist that activates the receptor and multiple cellular factors. Agonists that show higher potency to specific signaling pathways over others are known as "biased agonists" and have been shown to have better therapeutic index. Although biased agonists are desirable, their design poses several challenges to date. The number of assays to identify biased agonists seems expensive and tedious. Therefore, computational methods that can reliably calculate the possible bias of various ligands ahead of experiments and provide guidance, will be both cost and time effective. In this work, using the mechanism of allosteric communication from the extracellular region to the intracellular transducer protein coupling region in GPCRs, we have developed a computational method to calculate ligand bias ahead of experiments. We have validated the method for several -arrestin-biased agonists in-adrenergic receptor (2AR), serotonin receptors 5-HT1B and 5-HT2B and for G-protein-biased agonists in the -opioid receptor. Using this computational method, we also performed a blind prediction followed by experimental testing and showed that the agonist carmoterol is-arrestin-biased in 2AR. Additionally, we have identified amino acid residues in the biased agonist binding site in both2AR and -opioid receptors that are involved in potentiating the ligand bias. We call these residues functional hotspots, and they can be used to derive pharmacophores to design biased agonists in GPCRs.

Novel M2‐selective, Gi‐biased agonists of muscarinic acetylcholine receptors

“…Receptor conformations specific to compounds 6A and 7A were further confirmed by the smaller distance between TM5 and TM7 upon binding of these compounds in comparison to iperoxo or carbachol (Figure ). Taken together, the absence of a hydrogen bond of compounds 6A and 7A with Asn404, their differential allosteric interaction network, and finding of conformations specific for compounds 6A and 7A support the theory of functional hot spots (Nivedha et al, ) as well as variation among allosteric interaction networks in dependence on receptor conformation (Bhattacharya & Vaidehi, ; Miao, Nichols, Gasper, Metger, & McCammon, ). A common mechanism of GPCR activation is “opening” of the receptor G‐protein interface by moving TM6 away from TM3 and TM7 away from TM5 (Deupi & Kobilka, ; Weis & Kobilka, ).…”

“…A specific signalling pathway may be activated by the interaction of an agonist with a specific amino acid within the orthosteric binding site. Such residues are termed functional hot spots (Nivedha et al, ). Relatively small agonists may bind only to some, not all, functional hot spots in the binding site, and thus activate only a subset of signalling pathways that result in ligand‐mediated signalling bias.…”

“…Residues in the binding site that mediate activation of a specific signalling pathway are termed functional hot spots (Nivedha et al, ). Binding of an agonist to one or a subset of functional hot spots within the binding site results in activation of a subset of signalling pathways and thus in ligand‐mediated signalling bias.…”

Molecular determinants of β-arrestin coupling to formoterol-bound β1-adrenoceptor

“…Experimental data suggest that formoterol, carmoterol and carvedilol are arrestin biased ligands and that isoprenaline is a balanced agonist signalling equally between the G protein and arrestin pathways 5,34 (see Fig. 4d for ligand structures).…”

The future of bronchodilation: looking for new classes of bronchodilators

“…Conveniently, ligands could be biased or "functionally selective" toward either a G-protein or β-arrestin-mediated pathway (figure 4) [107,108]. Agonists that show a higher potency to specific signalling pathways over others are known as "biased agonists" and have a better therapeutic index [109].…”