Inverse, protean, and ligand-selective agonism: matters of receptor conformation.

Concepts regarding the mechanisms by which drugs activate receptors to produce physiological response have progressed beyond considering the receptor as a simple on-off switch. Current evidence suggests that the idea that agonists produce only varying degrees of receptor activation is obsolete and must be reconciled with data to show that agonist efficacy has texture as well as magnitude. Thus, agonists can block system constitutive response (inverse agonists), behave as positive and inverse agonists on the same receptor (protean agonists), and differ in the stimulus pattern they produce in physiological systems (ligand-selective agonists). The molecular mechanism for this seemingly diverse array of activities is the same, namely, the selective microaffinity of ligands for different conformational states of the receptor. This paper reviews evidence for the existence of the various types of agonism and the potential therapeutic utility of different agonist types.-Kenakin, T. Inverse, protean, and ligand-selective agonism: matters of receptor conformation.     

Functionally different agonists induce distinct conformations in the G protein coupling domain of the beta 2 adrenergic receptor

G protein-coupled receptors represent the largest class of drug discovery targets. Drugs that activate G protein-coupled receptors are classified as either agonists or partial agonists. To study the mechanism whereby these different classes of activating ligands modulate receptor function, we directly monitored ligand-induced conformational changes in the G protein-coupling domain of the beta(2) adrenergic receptor. Fluorescence lifetime analysis of a reporter fluorophore covalently attached to this domain revealed that, in the absence of ligands, this domain oscillates around a single detectable conformation. Binding to an antagonist does not change this conformation but does reduce the flexibility of the domain. However, when the beta(2) adrenergic receptor is bound to a full agonist, the G protein coupling domain exists in two distinct conformations. Moreover, the conformations induced by a full agonist can be distinguished from those induced by partial agonists. These results provide new insight into the structural consequence of antagonist binding and the basis of agonism and partial agonism.     

Stereochemistry of glutamate receptor agonist efficacy: engineering a dual-specificity AMPA/kainate receptor

Upon agonist binding, the bilobate ligand-binding domains of the ionotropic glutamate receptors (iGluR) undergo a cleft closure whose magnitude correlates broadly with the efficacy of the agonist. AMPA (alpha-amino-5-methyl-3-hydroxy-4-isoxazolepropionic acid) and kainate are nonphysiological agonists that distinguish between subsets of iGluR. Kainate acts with low efficacy at AMPA receptors. Here we report that the structure-based mutation L651V converts the GluR4 AMPA receptor into a dual-specificity AMPA/kainate receptor fully activated by both agonists. To probe the stereochemical basis of partial agonism, we have also investigated the correlation between agonist efficacy and a series of vibrational and fluorescence spectroscopic signals of agonist binding to the corresponding wild-type and mutant GluR4 ligand-binding domains. Two signals track the extent of channel activation: the maximal change in intrinsic tryptophan fluorescence and the environment of the single non-disulfide bonded C426, which appears to probe the strength of interactions with the ligand alpha-amino group. Both of these signals arise from functional groups that are poised to detect changes in the extent of channel cleft closure and thus provide additional information about the coupling between conformational changes in the ligand-binding domain and activation of the intact receptor.     

Inverse agonism: more than reverting constitutively active receptor signaling.

Seven-transmembrane receptors constitute one of the major families of proteins encoded by the genome. This type of receptor is one of the most important targets of the pharmaceutical industry, and many of the drugs with significant therapeutic action have been shown to be inverse agonists. Concepts regarding the mechanisms by which ligands activate and inactivate receptors are thought to be far more complex that a simple on-off switch. For both drug design and pharmacology principles, it is important to understand the mechanisms by which these drugs achieve their effects. Recent studies have demonstrated intriguing actions of inverse agonists. They have been shown not only to block constitutive responses of receptors but also to activate and regulate seven-transmembrane receptor signaling and trafficking. The activation of pathways by inverse agonists was shown to occur mainly via G-protein-independent mechanisms. These findings emphasize the importance of inverse agonism as a principle of receptor regulation. In this paper, we will review the evidence supporting inverse agonist promoted signaling and trafficking.     

Comparison of taurine- and glycine-induced conformational changes in the M2-M3 domain of the glycine receptor

In the ionotropic glutamate receptor, the global conformational changes induced by partial agonists are smaller than those induced by full agonists. However, in the pentameric ligand-gated ion channel receptor family, the structural basis of partial agonism is not understood. This study investigated whether full and partial agonists induce different conformation changes in the glycine receptor chloride channel (GlyR). A substituted cysteine accessibility analysis demonstrated previously that glycine binding induced an increase in surface accessibility of all residues from Arg(271) to Lys(276) in the M2-M3 domain of the homomeric alpha1 GlyR. Here we compare the surface accessibility changes induced by the full agonist, glycine, and the partial agonist, taurine. In GlyRs incorporating the A272C, S273C, L274C, or P275C mutation, the reaction rate of the cysteine-specific compound, methanethiosulfonate ethyltrimethylammonium, depended on how strongly the receptors were activated but was agonist-independent. Reaction rates could not be compared in the R271C and K276C mutant GlyRs because methanethiosulfonate ethyltrimethylammonium did not modify the extremely small currents induced by saturating taurine or equivalent low glycine concentrations. The results indicate that bound taurine and glycine molecules impose identical conformational changes to the M2-M3 domain. We therefore conclude that the higher efficacy of glycine is due to an increased ability to stabilize a common activated configuration.     

Direct optical recording of intrinsic efficacy at a G protein-coupled receptor

Full and partial agonists activate receptors to varying degrees, presumably by inducing full or partial conformational changes in the receptor protein. Varying degrees of partial agonism corresponding to varying intrinsic efficacies have been demonstrated for many compounds acting at G-protein-coupled receptors, but a method to determine intrinsic efficacies directly at the receptor level has so far been lacking. Here we describe a method that allows the direct monitoring of agonist-induced conformational changes in G-protein-coupled receptors. The cyan (CFP) and yellow (YFP) variants of the green fluorescent protein were fused to the receptors. This resulted in fluorescence resonance energy transfer (FRET) between the CFP- and YFP-moieties. The extent of FRET was reduced in the presence of an agonist. The FRET signal strictly followed agonist occupancy of the receptor. Using the alpha(2)-adrenergic receptor as a model system, the full agonist noradrenaline produced a full signal, the partial agonist clonidine produced only a partial signal, and the antagonist phentolamine had no effect. Thus, optical recording of the agonist-induced conformational change in a G-protein-coupled receptor allows the direct analysis of the intrinsic efficacies of agonists.     

β2-Adrenergic ion-channel coupled receptors as conformational motion detectors

Ion Channel-Coupled Receptors (ICCRs) are artificial proteins comprised of a G protein-coupled receptor and a fused ion channel, engineered to couple channel gating to ligand binding. These novel biological objects have potential use in drug screening and functional characterization, in addition to providing new tools in the synthetic biology repertoire as synthetic K(+)-selective ligand-gated channels. The ICCR concept was previously validated with fusion proteins between the K(+) channel Kir6.2 and muscarinic M(2) or dopaminergic D(2) receptors. Here, we extend the concept to the distinct, longer β(2)-adrenergic receptor which, unlike M(2) and D(2) receptors, displayed barely detectable surface expression in our Xenopus oocyte expression system and did not couple to Kir6.2 when unmodified. Here, we show that a Kir6.2-binding protein, the N-terminal transmembrane domain of the sulfonylurea receptor, can greatly increase plasma membrane expression of β(2) constructs. We then demonstrate how engineering of both receptor and channel can produce β(2)-Kir6.2 ICCRs. Specifically, removal of 62-72 residues from the cytoplasmic C-terminus of the receptor was required to enable coupling, suggesting that ligand-dependent conformational changes do not efficiently propagate to the distal C-terminus. Characterization of the β(2) ICCRs demonstrated that full and partial agonists had the same coupling efficacy, that an inverse agonist had no effect and that the stabilizing mutation E122 W reduced agonist-induced coupling efficacy without affecting affinity. Because the ICCRs are expected to report motions of the receptor C-terminus, these results provide novel insights into the conformational dynamics of the β(2) receptor.     

Mechanisms of inverse agonism of antipsychotic drugs at the D(2) dopamine receptor: use of a mutant D(2) dopamine receptor that adopts the activated conformation

The antipsychotic drugs have been shown to be inverse agonists at the D(2) dopamine receptor. We have examined the mechanism of this inverse agonism by making mutations in residue T343 in the base of the sixth transmembrane spanning region of the receptor. T343R, T343S and T343K mutant D(2) dopamine receptors were made and the T343R mutant characterized in detail. The T343R mutant D(2) dopamine receptor exhibits properties of a receptor that resides more in the activated state, namely increased agonist binding affinity (independent of G-protein coupling and dependent on agonist efficacy), increased agonist potency in functional tests (adenylyl cyclase inhibition) and increased inverse agonist effects. The binding of agonists to the mutant receptor also shows sensitivity to sodium ions, unlike the native receptor, so that isomerization of the receptor to its inactive state may be driven by sodium ions. The binding of inverse agonists to the receptor is, however, unaffected by the mutation. We conclude that inverse agonism at this receptor is not achieved by the inverse agonist binding preferentially to the non-activated state of the receptor over the activated state. Rather the inverse agonist appears to bind to all forms of the receptor but then renders the receptor inactive.     

Microscopic kinetics and energetics distinguish GABA(A) receptor agonists from antagonists

Although agonists and competitive antagonists presumably occupy overlapping binding sites on ligand-gated channels, these interactions cannot be identical because agonists cause channel opening whereas antagonists do not. One explanation is that only agonist binding performs enough work on the receptor to cause the conformational changes that lead to gating. This idea is supported by agonist binding rates at GABA(A) and nicotinic acetylcholine receptors that are slower than expected for a diffusion-limited process, suggesting that agonist binding involves an energy-requiring event. This hypothesis predicts that competitive antagonist binding should require less activation energy than agonist binding. To test this idea, we developed a novel deconvolution-based method to compare binding and unbinding kinetics of GABA(A) receptor agonists and antagonists in outside-out patches from rat hippocampal neurons. Agonist and antagonist unbinding rates were steeply correlated with affinity. Unlike the agonists, three of the four antagonists tested had binding rates that were fast, independent of affinity, and could be accounted for by diffusion- and dehydration-limited processes. In contrast, agonist binding involved additional energy-requiring steps, consistent with the idea that channel gating is initiated by agonist-triggered movements within the ligand binding site. Antagonist binding does not appear to produce such movements, and may in fact prevent them.     

Partial agonism: mechanisms based on ligand-receptor interactions and on stimulus-response coupling

Substances eliciting, at very high concentrations, a lower maximal response of a particular biological system than a defined standard, are defined as partial agonists. The convention rests on the definition of a standard substance that achieves a 'full' maximal response; partial agonism being, therefore, relative. Various mechanisms lie behind this phenomenon: 1. Receptor-related mechanisms: the agonist-receptor complex exists in several conformational states from which only one, or only a few, activate the cell signaling pathway. This may occur when the receptor itself, or the agonist, exists in multiple states (e.g., in the form of enantiomers or stereoisomers), or when the agonist-receptor complex changes its conformation (receptor switch: two-state model of receptor activation). Furthermore, a steric hindrance by a 'wrong-way binding' of a part of the agonist's molecules may prevent the full 'correct' occupancy of receptors. 2. Mechanisms based on the efficacy of the stimulus-response coupling. The efficacy is then proportional to the sum of probabilities that receptors in individual states activate the cell-signaling pathway. Doses (concentrations) eliciting the half maximal response (EC50), or similar response sensitivity parameters, are not included in the definition of partial agonism. However, tight correlations exist between maximal response and EC50 in many, but not all, generic groups of agonistically acting substances. These relationships are frequently linear; intercepts and slopes of these 'E, KE plots' are characteristic for individual, putative mechanisms. Dose-response curves of partial agonists are akin to those obtained for a response to a full agonist after a stepwise partial inactivation of receptors by an irreversible inhibitor. Also, the E, KE plots obtained in these instances are similar to those of partial agonists. The receptor reserve, rather vaguely defined in early reports, is therefore closely linked to the phenomenon of partial agonism.     

Mechanisms of agonism and inverse agonism at serotonin 5-HT1A receptors

Mechanisms of agonist and inverse agonist action at the serotonin 5-HT1A receptor have been studied using the modulation of guanosine 5'-O-(3-[35S]thiotriphosphate) ([35S]GTPgammaS) binding in membranes of Chinese hamster ovary (CHO) cells expressing the receptor (CHO-5-HTA1A cells). A range of agonists increased [35S]GTPgammaS binding with different potencies and to different maximal extents, whereas two compounds, methiothepin and spiperone, inhibited both agonist-stimulated and basal [5S]GTPgammaS binding, thus exhibiting inverse agonism. Potencies of agonists to stimulate [35S]GTPgammaS binding in membranes from CHO-5-HT1A cells were reduced by adding increasing concentrations of GDP to assays, whereas changes in sodium ion concentration did not affect agonist potency. The maximal effect of the agonists was increased by increasing sodium ion concentrations. The affinities of agonists in ligand binding assays were unaffected by changes in sodium ion concentration. Increasing GDP in the assays of the inverse agonists increased potency for spiperone to inhibit [35S]GTPgammaS binding and had no effect for methiothepin, in agreement with the sensitivity of these compounds to guanine nucleotides in ligand binding assays. Potencies for these inverse agonists were unaffected by changes in sodium ion concentration. These data were simulated using the extended ternary complex model. These simulations showed that the data obtained with agonists were consistent with these compounds achieving agonism by stabilising the ternary complex. For inverse agonists, the simulations showed that the mechanism for spiperone may be to stabilise forms of the receptor uncoupled from G proteins. Methiothepin, however, probably does not alter the equilibrium distribution of different receptor species; rather, this inverse agonist may stabilise an inactive form of the receptor that can still couple to G protein.     

Molecular dynamics simulations of the ligand-binding domain of an N-methyl-D-aspartate receptor

The mechanism of partial agonism at N-methyl-D-aspartate receptors is an unresolved issue, especially with respect to the role of protein dynamics. We have performed multiple molecular dynamics simulations (7 x 20 ns) to examine the behavior of the ligand-binding core of the NR1 subunit with a series of ligands. Our results show that water plays an important role in stabilizing different conformations of the core and how a closed cleft conformation of the protein might be stabilized in the absence of ligands. In the case of ligand-bound simulations with both full and partial agonists, we observed that ligands within the binding cleft may undergo distinct conformational changes, without grossly influencing the degree of cleft closure within the ligand-binding domain. In agreement with recently published crystallographic data, we also observe similar changes in backbone torsions corresponding to the hinge region between the two lobes for the partial agonist, D-cycloserine. This observation rationalizes the classification of D-cycloserine as a partial agonist and should provide a basis with which to predict partial agonism in this class of receptor by analyzing the behavior of these torsions with other potential ligands.     

Identification of an efficacy switch region in the ghrelin receptor responsible for interchange between agonism and inverse agonism

The carboxyamidated wFwLL peptide was used as a core ligand to probe the structural basis for agonism versus inverse agonism in the constitutively active ghrelin receptor. In the ligand, an efficacy switch could be built at the N terminus, as exemplified by AwFwLL, which functioned as a high potency agonist, whereas KwFwLL was an equally high potency inverse agonist. The wFw-containing peptides, agonists as well as inverse agonists, were affected by receptor mutations covering the whole main ligand-binding pocket with key interaction sites being an aromatic cluster in transmembrane (TM)-VI and -VII and residues on the opposing face of TM-III. Gain-of-function in respect of either increased agonist or inverse agonist potency or swap between high potency versions of these properties was obtained by substitutions at a number of positions covering a broad area of the binding pocket on TM-III, -IV, and -V. However, in particular, space-generating substitutions at position III:04 shifted the efficacy of the ligands from inverse agonism toward agonism, whereas similar substitutions at position III: 08, one helical turn below, shifted the efficacy from agonism toward inverse agonism. It is suggested that the relative position of the ligand in the binding pocket between this "efficacy shift region" on TM-III and the opposing aromatic cluster on TM-VI and TM-VII leads either to agonism, i.e. in a superficial binding mode, or it leads to inverse agonism, i.e. in a more profound binding mode. This relationship between different binding modes and opposite efficacy is in accordance with the Global Toggle Switch model for 7TM receptor activation.     

Repurposing FDA-approved drugs as FXR agonists: a structure based in silico pharmacological study

Farnesoid X receptor (FXR) modulates the expression of genes involved in lipid and carbohydrate homeostasis and inflammatory processes. This nuclear receptor is likely a tumor suppressor in several cancers, but its molecular mechanism of suppression is still under study. Several studies reported that FXR agonism increases the survival of colorectal, biliary tract, and liver cancer patients. In addition, FXR expression was shown to be down-regulated in many diseases such as obesity, irritable bowel syndrome, glomerular inflammation, diabetes, proteinuria, and ulcerative colitis. Therefore, development of novel FXR agonists may have significant potential in the prevention and treatment of these diseases. In this scenario, computer-aided drug design procedures can be resourcefully applied for the rapid identification of promising drug candidates. In the present study, we applied the molecular docking method in conjunction with molecular dynamics (MD) simulations to find out potential agonists for FXR based on structural similarity with the drug that is currently used as FXR agonist, obeticholic acid. Our results showed that alvimopan and montelukast could be used as potent FXR activators and outperform the binding affinity of obeticholic acid by forming stable conformation with the protein in silico. However, further investigational studies and validations of the selected drugs are essential to figure out their suitability for preclinical and clinical trials.     

Identification of the three-dimensional pharmacophore of κ-opioid receptor agonists

A selective κ-opioid receptor agonist might act as a powerful analgesic without the side effects of μ-opioid receptor-selective drugs such as morphine. The eight classes of known κ-opioid receptor agonists have different chemical structures, making it difficult to construct a pharmacophore model that takes them all into account. Here we propose a new three-dimensional pharmacophore model that encompasses the κ-activities of all classes, which utilizes conformational sampling of agonists by high-temperature molecular dynamics and pharmacophore extraction through a series of molecular superpositions.     

Distinct structural changes in a G protein-coupled receptor caused by different classes of agonist ligands

The activity of G protein-coupled receptors can be modulated by different classes of ligands, including agonists that promote receptor signaling and inverse agonists that reduce basal receptor activity. The conformational changes in receptor structure induced by different agonist ligands are not well understood at present. In this study, we employed an in situ disulfide cross-linking strategy to monitor ligand-induced conformational changes in a series of cysteine-substituted mutant M(3) muscarinic acetylcholine receptors. The observed disulfide cross-linking patterns indicated that muscarinic agonists trigger a separation of the N-terminal segment of the cytoplasmic tail (helix 8) from the cytoplasmic end of transmembrane domain I. In contrast, inverse muscarinic agonists were found to increase the proximity between these two receptor regions. These findings provide a structural basis for the opposing biological effects of muscarinic agonists and inverse agonists. This study also provides the first piece of direct structural information as to how the conformations induced by these two functionally different classes of ligands differ at the molecular level. Given the high degree of structural homology found among most G protein-coupled receptors, our findings should be of broad general relevance.     

Structural Mechanism of N-Methyl-D-Aspartate Receptor Type 1 Partial Agonism

N-methyl-D-aspartate (NMDA) receptors belong to a family of ionotropic glutamate receptors that contribute to the signal transmission in the central nervous system. NMDA receptors are heterotetramers that usually consist of two GluN1 and GluN2 monomers. The extracellular ligand-binding domain (LBD) of a monomer is comprised of discontinuous segments that form the functional domains D1 and D2. While the binding of a full agonist glycine to LBD of GluN1 is linked to cleft closure and subsequent ion-channel opening, partial agonists are known to activate the receptor only sub-maximally. Although the crystal structures of the LBD of related GluA2 receptor explain the mechanism for the partial agonism, structures of GluN1-LBD cannot distinguish the difference between full and partial agonists. It is, however, probable that the partial agonists of GluN1 alter the structure of the LBD in order to result in a different pharmacological response than seen with full agonists. In this study, we used molecular dynamics simulations to reveal an intermediate closure-stage for GluN1, which is unseen in crystal structures. According to our calculations, this intermediate closure is not a transient stage but an energetically stable conformation. Our results demonstrate that the partial agonist cannot exert firm GluN1-LBD closure, especially if there is even a small force that disrupts the LBD closure. Accordingly, this result suggests the importance of forces from the ion channel for the relationship between pharmacological response and the structure of the LBD of members of this receptor family.     

Allosteric agonist modulators

This article discusses a model to describe the effects of molecules that bind to a site on the receptor separate from that of the endogenous agonist to actively produce receptor signals (direct agonism). In addition, these molecules also modify the biological responses of the endogenous agonist (either potentiation or antagonism). The effects of such compounds in high-throughput screening assays are described as well as their effects on the dose-response curves to conventional agonists.     

Allosteric Agonist Modulators

This article discusses a model to describe the effects of molecules that bind to a site on the receptor separate from that of the endogenous agonist to actively produce receptor signals (direct agonism). In addition, these molecules also modify the biological responses of the endogenous agonist (either potentiation or antagonism). The effects of such compounds in high-throughput screening assays are described as well as their effects on the dose-response curves to conventional agonists.