Terry Kenakin

An important aspect of drug therapy is the presentation of the drug to the target in vivo, i.e. drugs must enter the receptor compartment to produce therapeutic effect. The drug usually is brought ...

Sodium ions are classically conceptualized as negative allosteric modulators for G protein-coupled receptors, although there have been reports of either positive allosteric modulation or no effect of sodium on GPCR function. Here, we identified opposing actions of sodium on μ and κ opioid receptors. We utilized a variety of methods including radioligand binding, real-time conformational monitoring of transitions using bioluminescence resonance energy transfer, and signaling assays using the TRUPATH resource. At the μ receptors, sodium behaved as a negative allosteric modulator of binding, conformational transitions, and signaling. Intriguingly, bitopic μ agonists displayed transducer-specific effects on conformational transitions and signaling sodium concentrations. By contrast, at the κ opioid receptor, sodium negatively modulated agonist binding and positively modulated conformational transitions and signaling. Taken together, these findings support the notion that the differential sensitivities to sodium concentrations will result in opposing effects on the cell surface and intracellular signaling.

This chapter considers biased signaling as a natural function of G protein-coupled receptors (GPCRs) in the form of probe dependence. Thus, any ligand that changes the conformation of the receptor (agonist, antagonist, or allosteric modulator) has the potential to change the natural signaling of the receptor through unequal conformational alterations in the receptor structure. This gives an added dimension to agonist selectivity beyond extracellular recognition, namely the ability of agonists to emphasize certain signaling pathways in the cell at the expense of others. Given this, selectivity is discussed in terms of varying intrinsic efficacy and selective stabilization of receptor states with methods to detect and measure these effects. Last, the translation of in vitro to complex in vivo systems will be considered.

Sodium ions are classically conceptualized as negative allosteric modulators for G protein coupled receptors (GPCRs), although there have been reports of either positive allosteric modulation or no effect of sodium on GPCR function. Here we identified opposing actions of sodium on the μ and κ opioid receptors. We utilized a variety of methods including radioligand binding, real-time conformational monitoring of transitions using bioluminescence resonance energy transfer and signaling assays using the TRUPATH resource. At the μ receptors, sodium behaved as a negative allosteric modulator of binding, conformational transitions and signaling. Intriguingly, bitopic μ agonists were unaffected by sodium concentrations. By contrast, at the κ opioid receptor sodium negatively modulated agonist binding and positively modulated conformational transitions and signaling. Taken together, these findings support the notion that the differential sensitivities to sodium concentrations will result in opposing effects on cell surface and intracellular signaling.

With the advent of functional screening, more allosteric molecules are being discovered and developed as possible therapeutic entities. Allosteric proteins are unique because of two specific properties: (1) separate binding sites for allosteric modulators and guests, and (2) mandatory alteration of receptor conformation upon binding of allosteric modulators. For GPCRs, these properties produce many beneficial effects on pharmacologic systems that are described here. Allosteric discovery campaigns also bring with them added considerations that must be addressed for the endeavor to be successful and these are described herein as well. Significance Statement Recent years have seen the increasing presence of allosteric molecules as possible therapeutic drug candidates. The scientific procedures to characterize these are unique and require special techniques so it is imperative that scientists understand the new concepts involved in allosteric function. This review reviews the reasons why allosteric molecules should be considered as new drug entities and the techniques required to optimize the discovery process for allosteric molecules.

SUMMARY: The NTSR1 neurotensin receptor (NTSR1) is a G protein coupled receptor (GPCR) found in the brain and peripheral tissues with neurotensin (NTS) being its endogenous peptide ligand. In the brain, NTS modulates dopamine neuronal activity, induces opioid-independent analgesia, and regulates food intake. Recent studies indicate that biasing NTSR1 toward β-Arrestin signaling can attenuate the actions of psychostimulants and other drugs of abuse. Here we provide the cryoEM structures of NTSR1 ternary complexes with heterotrimeric Gq and Go with and without the brain penetrant small molecule SBI-553. In functional studies, we discovered that SBI-553 displays complex allosteric actions exemplified by negative allosteric modulation for G proteins that are G α subunit selective and positive allosteric modulation and agonism for β-Arrestin translocation at NTSR1. Detailed structural analysis of the allosteric binding site illuminated the structural determinants for biased allosteric modulation of SBI-553 on NTSR1. These insights promise to both accelerate the structure-guided design of more effective NTSR1 therapeutics and provide insights into the complexities of GPCR allosteric modulation.

G protein-coupled receptors modulate a plethora of physiological processes and mediate the effects of one-third of FDA-approved drugs. Depending on which ligand activates a receptor, it can engage different intracellular transducers. This ‘biased signaling’ paradigm requires that we now characterize physiological signaling not just by receptors but by ligand-receptor pairs. Ligands eliciting biased signaling may constitute better drugs with higher efficacy and fewer adverse effects. However, ligand bias is very complex, making reproducibility and description challenging. Here, we provide guidelines and terminology for any scientists to design and report ligand bias experiments. The guidelines will aid consistency and clarity, as the basic receptor research and drug discovery communities continue to advance our understanding and exploitation of ligand bias. Scientific insight, biosensors, and analytical methods are still evolving and should benefit from and contribute to the implementation of the guidelines, together improving translation from in vitro to disease-relevant in vivo models.