The intrinsic efficacy of a ligand acting at a 7-transmembrane (TM) domain, G-protein-coupled receptor (GPCR) describes its ability to shift the equilibrium from inactive receptor (R) in favor of active receptor states (R*). Thus, intrinsic efficacy is the parameter that distinguishes agonists, partial agonists, antagonists, and inverse agonists and is a major determinant of pharmacological activity of a drug molecule, along with receptor affinity. This is particularly highlighted by the mu-opioid receptor (µ-OR), a GPCR responsible for both the pain relieving and unwanted effects of opioid drugs, such as life-threatening respiratory depression and the rewarding properties that underlie addiction to drugs such as morphine, oxycodone, and heroin. Even though opioid drugs bind to the same orthosteric site on µ-OR, the physiological outcomes observed are determined by their degree of intrinsic efficacy and the efficacy requirements of the system (Morgan and Christie, 2011). For example, the discriminative stimulus of opioid agonists varies depending on efficacy (Walker et al., 2004), suggesting that opioid agonist efficacy is a determinant of abuse potential. Thus, it has been shown that higher efficacy ligands have greater addictive liability compared to lower-efficacy opioid ligands (Center for Substance Abuse Treatment, 2004). In order to be able to predict the ability of µ-OR agonists to cause various physiological effects, an understanding of their intrinsic efficacy is required.

Current approaches to determine the intrinsic efficacy of agonists rely upon measurement of signaling downstream of the receptor in tissue- or cell-based systems. In spite of the use of methods to account for signal amplification which can cause system and measurement bias the calculated intrinsic efficacy of ligands can vary based on the species of the tissue or cellular background, ligand bias, and temporal effects (Kenakin, 2002; Kenakin and Christopoulos, 2011; Luttrell and Kenakin, 2011; Klein Herenbrink et al., 2016). For example, using the same cellular background, the putatively biased opioid ligand TRV130 initiates arrestin recruitment (74% of morphine) downstream of mouse µ-OR, less with human µ-OR (14% of morphine), and shows undetectable levels using rat µ-OR (DeWire et al., 2013). Complications such as these make correlations of physiological effects to values of efficacy and ‘bias factors’ (Rajagopal et al., 2010; Kenakin et al., 2012) difficult to interpret. Therefore, in this study we sought to establish a method to evaluate the intrinsic efficacy of opioid ligands utilizing a cell-free assay, thereby removing confounds of signaling outputs and signal amplification.

The recently solved crystal structure of µ-OR in complex with the highly efficacious agonist BU72 utilized nanobody 39 (Nb39), a camelid antibody fragment, to stabilize active µ-OR (Huang et al., 2015). Nb39 enhances agonist affinity at µ-OR and stabilizes conformational changes in the receptor associated with a signaling-competent, active-like state, including an outward movement of TM6 (Farrens et al., 1996; Palczewski et al., 2000; Rasmussen et al., 2011b). Nanobodies are small, monomeric proteins that can be utilized as conformational biosensors and have therefore been used as tools to monitor formation of active-state β2-adrenergic receptors in live cells (Irannejad et al., 2013). Consequently, we sought to use Nb39 as a G protein mimic and a probe to detect active-state conformation of purified monomeric µ-OR in reconstituted high density lipoprotein (rHDL) particles, comprised of the lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol and Apolipoprotein-A1 (Apo-A1) (Kuszak et al., 2009), using a variety of orthosteric ligands. We predicted that the intrinsic efficacy of an agonist will determine the rate of Nb39 binding to µ-OR.

We also wished to utilize Nb39 to probe the mechanism by which small molecule positive allosteric modulators (PAMs) alter the activity of orthosteric µ-OR ligands. µ-PAMS, exemplified by BMS-986122 (Burford et al., 2013), enhance the affinity and/or efficacy of orthosteric ligands for µ-OR, but show a distinct dependence on which orthosteric ligand is used to probe the allosteric interaction, with the degree of cooperativity being sensitive to the intrinsic efficacy of the orthosteric ligand (Burford et al., 2013; Livingston and Traynor, 2014). We have suggested that the mechanism of allosteric enhancement of orthosteric agonist activity involves a disruption of binding of the endogenous negative allosteric modulator Na⁺, thereby stabilizing an agonist-bound active (R*) state of the µ-OR (Livingston and Traynor, 2014).

In this report, we demonstrate that the rate of Nb39 binding to purified µ-OR simulates the action of heterotrimeric G protein and provides a measure of the intrinsic efficacy of opioid ligands, and that the efficacy rankings determined using this second messenger- and cell/tissue-independent assay are significantly correlated with other measures of efficacy. Moreover, we observed differences in Nb39 dissociation suggestive of ligand-specific R* states. Finally, we show that µ-PAMs can alone promote Nb39 binding, indicating that they stabilize formation of R* to increase the apparent efficacy of partial agonist drugs such as morphine.

We have described a method for examining the efficacy of orthosteric and allosteric ligands of a prototypic class A GPCR, µ-OR, which relies upon the ability of the conformationally-selective sensor Nb39 to recognize active (R*) state(s) of the receptor as confirmed using heterotrimeric G protein. Understanding the effects of ligands on the distribution of receptor conformations is crucial in predicting their activity at downstream signaling outputs and in vivo. Direct measurement of Nb39 binding to µ-OR using interferometry is independent of signaling and does not require calculations of efficacy from parameters determined in signaling assays. Further, the output of the interferometry assay is not subject to amplification and is dependent only upon receptor-ligand interaction, enabling detection of fine distinctions in agonist action that may be masked when measuring a downstream signaling output (Black and Leff, 1983; Ehlert, 1985). Importantly, the technique has the potential to detect differences in agonist-induced receptor conformations that may predict biased signaling without relying upon traditional calculations of ‘bias factors’ (Kenakin et al., 2012; Stott et al., 2016). Finally, the method can be applied to positive allosteric modulation of orthosteric ligands and provides insight into the mechanism of action of PAMs at µ-OR by showing these molecules, although acting at a distinct site, stabilize active (R*) receptor states even in the absence of orthosteric agonist. Overall, the method provides a way of quantitatively examining the efficacy of µ-OR orthosteric and allosteric ligands to stabilize R*, and can readily be applied to other GPCRs with conformationally-selective nanobodies available (Staus et al., 2014; 2016).

As predicted based on data from previous studies with nanobodies (Rasmussen et al., 2011a; Irannejad et al., 2013; Huang et al., 2015), orthosteric agonists resulted in robust Nb39 binding, while the orthosteric antagonists naloxone or diprenorphine failed to promote detectable Nb39 binding. We show the ability of an agonist to promote Nb39 binding is correlated with its ability to promote signal transduction through G_i/o protein as measured by GTPγ³⁵S binding. In fact, measurement of Nb39 binding is more sensitive than GTPγ³⁵S binding, as some low-efficacy ligands, including nalbuphine and the µ-OR PAMs, were able to promote Nb39 binding but failed to show measurable G protein activity using the GTPγ³⁵S-binding assay (Livingston and Traynor, 2014). While theoretically measuring the same process, that is µ-OR active-state promotion by agonists, leading to a protein-protein interaction at the receptor’s cytoplasmic face, the output of the GTPγ³⁵S assay can vary dramatically based upon nucleotide concentration, receptor:Gi_/o protein ratios, MgCl₂ concentration, NaCl concentrations, time, and temperature (Traynor and Nahorski, 1995; Szekeres and Traynor, 1997; Remmers et al., 2000; Heusler et al., 2016). In comparison, Nb39 binding is not an enzymatic process like nucleotide exchange, but instead represents a bimolecular binding event. However, it is recognized that measured efficacy in a cellular context will depend on the signal measured and the environment of the receptor.

It is important to note that the fact we see differences in K_obs for the various agonists indicates that each agonist stabilizes a different distribution of active states of μ-OR, but that these states are all recognized by Nb39. The range of active µ-OR conformations formed by the orthosteric agonists and/or allosteric modulators recognized by Nb39 is, as of yet, unknown, and within a cellular context will depend on the environment and state of the receptor, but the crystal structure of active µ-OR bound to Nb39 shows that the nanobody does stabilize an active state of the receptor displaying the prototypic outward movement of TM6 associated with activation of GPCRs (Farrens et al., 1996; Scheerer et al., 2008; Rasmussen et al., 2011b; Kruse et al., 2013; Huang et al., 2015). Also, the affinities of the agonist BU72 for Nb39-bound µ-OR and Gαi-bound µ-OR are similar (Huang et al., 2015), suggesting the conformations of µ-OR stabilized by Nb39 and Gαi are also comparable, a finding confirmed by our current data with heterotrimeric G protein. The correlation with other efficacy measures leaves room for interpretation on the nature of receptor states that Nb39 can bind. If Nb39 was able to recognize and bind all active-like conformations capable of initiating downstream signal transduction, one might expect perfect correlations with other measures of efficacy. As evidenced, the correlation with Ehlert’s efficacy values shows this is not the case. Although a well-accepted method for determining intrinsic efficacy, Ehlert’s measure relies upon data collected from a signaling assay, therefore if a ligand has a signaling bias, the results may differ depending on which signaling output is chosen. The Ehlert equation relies upon a potency value (EC₅₀), affinity value, and an E_max that is relative to a chosen standard. The EC₅₀ of an assay will be heavily reliant on the system: receptor reserve, time of incubation, and inherent system maximum and is therefore not an absolute number (Strange, 2008). Additionally, the affinity of the ligand is not a factor in the interferometry assay as all ligands are present at saturating concentrations. This is likely why the correlation with τ as calculated from the operational model is stronger (Figure 4). By calculating τ alone, ligand affinity is removed as a descriptor of efficacy.

Data from the interferometry experiments correlated strongly with the shift in affinity of orthosteric agonists in the presence of Na⁺ ions and Guanine nucleotides. Affinity is ideally system-independent and so the Na⁺/GTPγS shift appears to be more reflective of true intrinsic efficacy, or ability of a ligand to discriminate between R and R*, with no dependence on the selection and measurement of a signaling output. It should be noted that two agonists, etorphine and BU72, are unique in that they exhibit little sensitivity to Na⁺/GTPγS despite their high efficacy. It is possible that their insensitivity to Na⁺ ions is not absolute. Rather, etorphine and BU72 may be extremely negatively cooperative with Na⁺ binding, thereby reducing the potency of Na⁺ to alter the affinity of these ligands to such a degree that no shift was observed at the Na⁺ concentration tested (100 mM). Nonetheless, the lack of the ability of Na⁺/GTPγS to alter affinity of etorphine and BU72 is in agreement with the lack of allosteric effects of the PAMs on these ligands (Livingston and Traynor, 2014), suggesting they drive formation of R* too efficiently to be further enhanced by allosteric ligands. Indeed, NMR data have suggested that BU72 is a ‘superagonist’ capable of stabilizing conformational changes in µ-OR, in particular increased dynamics in intracellular loop 1 and helix 8, that are not seen with other full agonists (Sounier et al., 2015). Etorphine may act in a similar fashion.

The rationale of the present study was to directly compare agonist ability to generate an active conformation of the μ-OR as a determinant of intrinsic efficacy without the need to measure a downstream signal. The comparative measures above are all indicative of Gα function. However, if measures are taken downstream that involve the Gβγ subunit then the actual value of the efficacy measure might vary but the rank order should stay the same since Nb39 mimics the action of heterotrimeric G proteins. It is possible that non-G-protein-mediated actions, that could include β-arrestin recruitment, would show a different rank order. Consequently, we also compared the kinetics of Nb39 binding induced by several agonists with published agonist efficacy data for β-arrestin recruitment (McPherson et al., 2010) and saw a significant correlation suggesting that Nb39 may recognize μ-OR states that recruit β-arrestin as well as those that recruit G protein, or that recruitment of β-arrestin to μ-OR must be preceded by transition to a Nb39-recognizable state. We did not necessarily expect this finding given that nanobodies which promote high affinity agonist binding of the β2AR recognize a conformation of the receptor similar to the conformation which binds G protein (Rasmussen et al., 2011a). On the other hand the result does support the finding that some ligands, including the biased agonist PZM21, show different kinetics. It will be important in the future to develop the system to follow β-arrestin recruitment to purified μ-OR directly. However, this will require the purification of homogenous, phosphorylated receptor.

Like the orthosteric agonists, the allosteric ligands BMS-986122 and BMS-986187 also drove Nb39 binding to µ-OR suggesting they can directly activate the receptor to form R*, even in the absence of an agonist occupying the orthosteric site. However, the rate and extent of Nb39 binding produced by the PAMs was low, comparable to low-efficacy orthosteric agonists such as nalbuphine. Compared to BMS-986122, BMS-986187 promoted Nb39 binding to a greater extent and at a faster rate, in line with its higher activity to enhance orthosteric ligand binding affinity to µ-OR in rHDL particles. The fact that BMS986187 is able to recruit Nb39, albeit to a much smaller extent than orthosteric agonists, identifies this compound as an ago-PAM, a compound that can activate receptor in the absence of orthosteric agonist and demonstrates the high sensitivity of the assay. This is supported by our recent observation that BMS986187 acts as an agonist in the adenylate cyclase assay but not in an assay for G-protein activation of β-arrestin recruitment (Livingston et al., 2018). Our proposed mechanism for PAMs at the μ-OR is to allosterically modulate the Na ⁺ ion binding site and therefore drive or help to drive a active conformational (R*) ensembles. Thus, there is a fine line between a PAM and an ago-PAM that predicts a continuum between compounds that are silent (SAMs) and bind to the allosteric site without any obvious activity, PAMs that promote agonist action and ago-PAMs. Moreover, these definitions will depend on the efficacy requirements of the system.

When added together with orthosteric ligands the PAM, BMS-986187, increased the association rate of Nb39 to µ-OR induced by morphine, but did not alter the rate of Nb39 binding in the presence of DAMGO and decreased the effect of association rate in the presence of methadone. In previous studies, we showed the efficacy of the partial agonist morphine, determined using Ehlert’s equation, was increased two-fold by BMS-986122 (from e = 0.5 to e = 1.0), whereas the efficacy of DAMGO (e = 2.0 versus 2.1) was essentially unchanged in the presence of BMS-986122 and L-methadone was slightly decreased (from e = 1.1 to 0.9). Instead with L-methadone and DAMGO there is an increase in potency. Crystallographic work is underway to determine the mode of PAM binding and to determine the structural features that govern allosteric modulation of µ-OR.

In addition to association rate constants, valuable information can be obtained from the rates of Nb39 dissociation since we can assume this rate is a product of the affinity of Nb39 for agonist-bound and active µ-OR. The dissociation rate of Nb39 was equal in the presence of most orthosteric and the allosteric ligands examined in this study, arguing that Nb39 recognizes all active (R*) conformations equally and/or that both full and partial orthosteric agonists, as well as the allosteric modulators, stabilize a common ensemble of µ-OR active conformations that is recognized by Nb39. These arguments are consistent with the exception of L-methadone, loperamide and PZM21 which show faster Nb39 dissociation. This indicates that the receptor has decreased affinity for Nb39 in the presence of these ligands, possibly reflecting a unique set of receptor conformations. In support of a differential binding mode for L-methadone, this ligand is the most sensitive orthosteric agonist toward allosteric modulation with three different chemical scaffolds (BMS-986122, BMS-986187, and MS1 [Bisignano et al., 2015]), and in turn greatly enhances the affinity of BMS-986187 for the allosteric site. This suggests that L-methadone, and possibly PZM21 and loperamide, could engage with µ-OR in distinct ways, generating unique conformational ensembles that can be seen in its effects on the binding characteristics of both Nb39 and small-molecule allosteric modulators. It is possible that other factors, including differential rates of ligand dissociation could be confounding factors in Nb39 dissociation, but the fact that these differences are only seen for three ligands would argue against this. Although we have tested a number of structurally diverse µ-OR orthosteric ligands, and both full and partial agonists, it is feasible that each ligand generates distinct active conformations that are indistinguishable to Nb39, including as discussed above the possibility of β-arrestin preferring conformations. This is likely given evidence at the β2-adrenergic (Yao et al., 2006) and β1-adrenergic receptors (Warne et al., 2011) suggests partial agonist conformations differ from full agonist-bound conformations. Thus, it should be possible to develop selective nanobodies that recognize agonist-specific µ-OR* states. If true it is feasible that these different conformational states may contribute to the lower levels of cross-tolerance observed with methadone compared to other opioids in animal models (Neil, 1982; Posa et al., 2016), and the higher relative potency values for methadone than expected during opioid rotation in patients (Knotkova et al., 2009). Moreover, these different receptor states could explain the reported biased agonist nature of PMZ21(Manglik et al., 2016). However, this represents the most basic piece of the puzzle and in the whole cell many factors not present in rHDL particles, such as differential posttranslational processing of the receptor, and the presence of accessory proteins, as well as different membrane lipids could contribute to the ensemble of receptor conformations obtained.

The affinity of Nb39 for μ-OR (Huang et al., 2015) is low compared to Nb80 at the β2AR (Manglik et al., 2016). Thus, Nb39 does not bind receptor in the absence of agonist and so does not create agonist conformations of the receptor or force orthosteric agonist to bind to these unique conformations. Although it is possible that the agonist bound Nb39 conformation in the presence of Nb 39 could be different from the agonist and heterotrimerc G-protein-bound conformations, our findings using a mutated, soluble form of heterotrimeric G protein confirmed the biological relevance of our model system by demonstrating that Nb39 is a suitable surrogate for heterotrimeric G proteins which are the native ‘active-state sensors’ of R* states of GPCRs. However, for these assays protein tools such as nanobodies are preferred due to the challenges associated with using G-protein complexes made up of three subunits. These include selection of subunits, which themselves could cause differences in receptor binding (Remmers et al., 2000), inclusion or exclusion of nucleotide, selection and concentration of nucleotide, and nonspecific binding. Indeed, some native Gα subtypes and wild-type Gγ cannot be used as obligatory lipid groups (palmitoylation, geranyl-geranylation) require detergents which would disrupt the rHDL particles, and also could drive direct lipid-lipid association with the rHDL particles. This creates the possibility for multiphase association and dissociation kinetics which can make data analysis difficult, for example the K_d value may be an amalgamation of two distinct values derived from binding of G protein to receptor and to the rHDL discs. Moreover, heterotrimeric G-protein binding is not simply a bimolecular interaction between G protein and receptor but an enzymatic process in which nucleotide can dramatically influence rates and extents of binding. The extremely slow dissociation of heterotrimeric G protein makes the K_d calculated a ‘pseudo’ rate constant as the reaction is irreversible on the time scale of the assay in the absence of nucleotide.

In summary, we have described a novel method for the quantitative evaluation of efficacy of both orthosteric and allosteric ligands using purified µ-OR in rHDL particles. This biophysical technique is also able to identify ligands, in particular L-methadone and PZM21, that induce distinct conformations of µ-OR. Allosteric modulators of µ-OR are themselves capable of generating active conformations of µ-OR competent to bind Nb39 and of driving G-protein-independent high-affinity agonist binding. This technique is more sensitive than traditional measures of efficacy and is not reliant upon signal amplification. The methodology should be applicable to a wide variety of GPCRs.

All data generated and analyzed during the study are included in the manuscript and supporting files. Source files have been provided for Fig 3.

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Author details

1. Kathryn E Livingston

   1. Department of Pharmacology, University of Michigan Medical School, Ann Arbor, United States
   2. Edward F Domino Research Center, University of Michigan, Ann Arbor, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Methodology, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-8652-4045

2. Jacob P Mahoney

   1. Department of Pharmacology, University of Michigan Medical School, Ann Arbor, United States
   2. Edward F Domino Research Center, University of Michigan, Ann Arbor, United States

   Contribution

   Conceptualization, Writing—review and editing

   Competing interests

   No competing interests declared

3. Aashish Manglik

   Department of Pharmaceutical Chemistry, School of Pharmacy, University of California San Francisco, San Francisco, United States

   Contribution

   Resources, Visualization, Writing—review and editing

   Competing interests

   No competing interests declared

4. Roger K Sunahara

   Department of Pharmacology, University of California San Diego School of Medicine, La Jolla, United States

   Contribution

   Resources

   Competing interests

   No competing interests declared

5. John R Traynor

   1. Department of Pharmacology, University of Michigan Medical School, Ann Arbor, United States
   2. Edward F Domino Research Center, University of Michigan, Ann Arbor, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Methodology, Writing—original draft, Writing—review and editing

   For correspondence

   jtraynor@umich.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1849-8316

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