Edward L. Stahl

Development of functionally selective, small molecule agonists at kappa opioid receptors

Background: Kappa opioid receptor (KOR) signaling may produce antinociception through G protein or dysphoria through βarrestin pathways. Results: Two highly selective, brain penetrant agonist scaffolds bias KOR signaling toward G protein coupling and produce antinociception in mice. Conclusion: Described are first-in-class small molecule agonists that bias KOR signaling through G proteins. Significance: Functionally selective KOR agonists can now be used in vivo. The kappa opioid receptor (KOR) is widely expressed in the CNS and can serve as a means to modulate pain perception, stress responses, and affective reward states. Therefore, the KOR has become a prominent drug discovery target toward treating pain, depression, and drug addiction. Agonists at KOR can promote G protein coupling and βarrestin2 recruitment as well as multiple downstream signaling pathways, including ERK1/2 MAPK activation. It has been suggested that the physiological effects of KOR activation result from different signaling cascades, with analgesia being G protein-mediated and dysphoria being mediated through βarrestin2 recruitment. Dysphoria associated with KOR activation limits the therapeutic potential in the use of KOR agonists as analgesics; therefore, it may be beneficial to develop KOR agonists that are biased toward G protein coupling and away from βarrestin2 recruitment. Here, we describe two classes of biased KOR agonists that potently activate G protein coupling but weakly recruit βarrestin2. These potent and functionally selective small molecule compounds may prove to be useful tools for refining the therapeutic potential of KOR-directed signaling in vivo.

Biased agonists of the kappa opioid receptor suppress pain and itch without causing sedation or dysphoria

Biased agonists of the kappa opioid receptor may relieve intractable itch without causing unwanted side effects. Itch relief from biased agonists Activating the kappa opioid receptor (KOR) can relieve itching that is not caused by allergic reactions. However, compounds that activate this receptor also cause unwanted side effects, such as dysphoria and sedation. KOR activation can trigger multiple downstream signaling pathways. Brust et al. characterized a biased agonist of this receptor that preferentially activated one downstream pathway over another. This agonist relieved itch in rodents without causing dysphoria or sedation. Thus, biased KOR agonists may provide a long-sought therapeutic option for intractable itch without the unwanted side effects. Agonists targeting the kappa opioid receptor (KOR) have been promising therapeutic candidates because of their efficacy for treating intractable itch and relieving pain. Unlike typical opioid narcotics, KOR agonists do not produce euphoria or lead to respiratory suppression or overdose. However, they do produce dysphoria and sedation, side effects that have precluded their clinical development as therapeutics. KOR signaling can be fine-tuned to preferentially activate certain pathways over others, such that agonists can bias signaling so that the receptor signals through G proteins rather than other effectors such as βarrestin2. We evaluated a newly developed G protein signaling–biased KOR agonist in preclinical models of pain, pruritis, sedation, dopamine regulation, and dysphoria. We found that triazole 1.1 retained the antinociceptive and antipruritic efficacies of a conventional KOR agonist, yet it did not induce sedation or reductions in dopamine release in mice, nor did it produce dysphoria as determined by intracranial self-stimulation in rats. These data demonstrated that biased agonists may be used to segregate physiological responses downstream of the receptor. Moreover, the findings suggest that biased KOR agonists may present a means to treat pain and intractable itch without the side effects of dysphoria and sedation and with reduced abuse potential.

A Novel Method for Analyzing Extremely Biased Agonism at G Protein–Coupled Receptors

Seven transmembrane receptors were originally named and characterized based on their ability to couple to heterotrimeric G proteins. The assortment of coupling partners for G protein–coupled receptors has subsequently expanded to include other effectors (most notably the βarrestins). This diversity of partners available to the receptor has prompted the pursuit of ligands that selectively activate only a subset of the available partners. A biased or functionally selective ligand may be able to distinguish between different active states of the receptor, and this would result in the preferential activation of one signaling cascade more than another. Although application of the “standard” operational model for analyzing ligand bias is useful and suitable in most cases, there are limitations that arise when the biased agonist fails to induce a significant response in one of the assays being compared. In this article, we describe a quantitative method for measuring ligand bias that is particularly useful for such cases of extreme bias. Using simulations and experimental evidence from several κ opioid receptor agonists, we illustrate a “competitive” model for quantitating the degree and direction of bias. By comparing the results obtained from the competitive model with the standard model, we demonstrate that the competitive model expands the potential for evaluating the bias of very partial agonists. We conclude the competitive model provides a useful mechanism for analyzing the bias of partial agonists that exhibit extreme bias.

Structure–Activity Relationship Studies of Functionally Selective Kappa Opioid Receptor Agonists that Modulate ERK 1/2 Phosphorylation While Preserving G Protein Over βArrestin2 Signaling Bias

Kappa opioid receptor (KOR) modulation is a promising target for drug discovery efforts due to KOR involvement in pain, depression, and addiction behaviors. We recently reported a new class of triazole KOR agonists that displays significant bias toward G protein signaling over βarrestin2 recruitment; interestingly, these compounds also induce less activation of ERK1/2 map kinases than the balanced agonist, U69,593. We have identified structure-activity relationships around the triazole scaffold that allows for decreasing the bias for G protein signaling over ERK1/2 activation while maintaining the bias for G protein signaling over βarrestin2 recruitment. The development of novel compounds, with different downstream signaling outcomes, independent of G protein/βarrestin2 bias, provides a more diverse pharmacological toolset for use in defining complex KOR signaling and elucidating the significance of KOR-mediated signaling.

Novel Allosteric Effects of Amiodarone at the Muscarinic M5 Receptor

Allosteric sites on muscarinic receptors may present superior therapeutic targets for several central nervous system disorders, due to the potential of allosteric ligands to provide more selective modulation and to preserve the spatiotemporal patterning that is characteristic of synaptic transmission. We have found that the antiarrhythmic drug amiodarone interacts allosterically with M1 and M5 muscarinic receptors. At both M1 and M5, amiodarone was only able to partially inhibit the binding of the orthosteric antagonist [3H]N-methylscopolamine (NMS). In addition, amiodarone was able to alter the rate of dissociation of [3H]NMS from M1 and M5 receptors. These findings suggest that NMS and amiodarone are able to bind to the receptor simultaneously. The pharmacology of the effect on NMS dissociation demonstrated that amiodarone was not interacting at the “common” site at which gallamine, obidoxime, and many other muscarinic allosteric ligands are known to bind. In functional studies, amiodarone enhanced the ability of acetylcholine (at EC20) to activate the M5 receptor; however, under the same conditions, amiodarone did not enhance M1 activation. More detailed studies at M5 found that the effect of amiodarone was to enhance the efficacy of acetylcholine, without increasing its potency. This report describes the first demonstration of allosteric enhancement of efficacy at the M5 receptor, and the first demonstration of enhancement of efficacy but not potency at any muscarinic receptor. In summary, amiodarone has been shown to be a novel positive allosteric modulator of muscarinic receptors that is selective for the M5 subtype, relative to M1.

Molecular-Interaction and Signaling Profiles of AM3677, a Novel Covalent Agonist Selective for the Cannabinoid 1 Receptor

The cannabinoid 1 receptor (CB1R) is one of the most abundant G protein-coupled receptors (GPCRs) in the central nervous system. CB1R involvement in multiple physiological processes, especially neurotransmitter release and synaptic function, has made this GPCR a prime drug discovery target, and pharmacological CB1R activation has been demonstrated to be a tenable therapeutic modality. Accordingly, the design and profiling of novel, drug-like CB1R modulators to inform the receptors ligand-interaction landscape and molecular pharmacology constitute a prime contemporary research focus. For this purpose, we report utilization of AM3677, a designer endocannabinoid (anandamide) analogue derivatized with a reactive electrophilic isothiocyanate functionality, as a covalent, CB1R-selective chemical probe. The data demonstrate that reaction of AM3677 with a cysteine residue in transmembrane helix 6 of human CB1R (hCB1R), C6.47(355), is a key feature of AM3677s ligand-binding motif. Pharmacologically, AM3677 acts as a high-affinity, low-efficacy CB1R agonist that inhibits forskolin-stimulated cellular cAMP formation and stimulates CB1R coupling to G protein. AM3677 also induces CB1R endocytosis and irreversible receptor internalization. Computational docking suggests the importance of discrete hydrogen bonding and aromatic interactions as determinants of AM3677s topology within the ligand-binding pocket of active-state hCB1R. These results constitute the initial identification and characterization of a potent, high-affinity, hCB1R-selective covalent agonist with utility as a pharmacologically active, orthosteric-site probe for providing insight into structure-function correlates of ligand-induced CB1R activation and the molecular features of that activation by the native ligand, anandamide.

Allosteric Modulation of the M3 Muscarinic Receptor by Amiodarone and N-Ethylamiodarone: Application of the Four-Ligand Allosteric Two-State Model

We have reported previously that amiodarone interacts with muscarinic receptors via a novel allosteric site. This study presents mechanistic details on the nature of that interaction. Amiodarone enhanced the maximal level of agonist-stimulated release of arachidonic acid (AA) from Chinese hamster ovary cells that expressed M3 muscarinic receptors; this enhancement was observed for acetylcholine and for the partial agonist pilocarpine. A similar effect of amiodarone was observed when pilocarpine was used to stimulate inositol phosphate (IP) metabolism, but not when acetylcholine was used. Subsequent studies showed that the IP response exhibited a much larger receptor reserve than the AA response, and reduction of that reserve by receptor alkylation unmasked amiodarones enhancement of the maximal IP response to acetylcholine. Modulating the receptor reserve also revealed acetylcholines greater affinity (KA) for the conformation of the receptor that mediates the AA response. The amiodarone analog N-ethylamiodarone (NEA) did not alter maximal agonist response but merely reduced agonist potency (that is, it appeared to be an antagonist). However, the action of NEA could be clearly distinguished from the action of the orthosteric antagonist NMS. Demonstration of this point was facilitated by an elaboration of Halls allosteric two-state model; this new model represents a system composed of two ligands that compete with each other at the orthosteric site and two ligands that compete with each other at the allosteric site. In conclusion, amiodarone competes with NEA at a novel, extracellular, allosteric site to enhance the maximal stimulation evoked by acetylcholine and pilocarpine in two different responses.

Characterization of structurally novel G protein biased CB1 agonists: Implications for drug development

&NA; The human cannabinoid subtype 1 receptor (hCB1R) is highly expressed in the CNS and serves as a therapeutic target for endogenous ligands as well as plant‐derived and synthetic cannabinoids. Unfortunately, acute use of hCB1R agonists produces unwanted psychotropic effects and chronic administration results in development of tolerance and dependence, limiting the potential clinical use of these ligands. Studies in &bgr;‐arrestin knockout mice suggest that interaction of certain GPCRs, including &mgr;‐, &dgr;‐, &kgr;‐opioid and hCB1Rs, with &bgr;‐arrestins might be responsible for several adverse effects produced by agonists acting at these receptors. Indeed, agonists that bias opioid receptor activation toward G‐protein, relative to &bgr;‐arrestin signaling, produce less severe adverse effects. These observations indicate that therapeutic utility of agonists acting at hCB1Rs might be improved by development of G‐protein biased hCB1R agonists. Our laboratory recently reported a novel class of indole quinulidinone (IQD) compounds that bind cannabinoid receptors with relatively high affinity and act with varying efficacy. The purpose of this study was to determine whether agonists in this novel cannabinoid class exhibit ligand bias at hCB1 receptors. Our studies found that a novel IQD‐derived hCB1 receptor agonist PNR‐4‐20 elicits robust G protein‐dependent signaling, with transduction ratios similar to the non‐biased hCB1R agonist CP‐55,940. In marked contrast to CP‐55,940, PNR‐4‐20 produces little to no &bgr;‐arrestin 2 recruitment. Quantitative calculation of bias factors indicates that PNR‐4‐20 exhibits from 5.4‐fold to 29.5‐fold bias for G protein, relative to &bgr;‐arrestin 2 signaling (when compared to G protein activation or inhibition of forskolin‐stimulated cAMP accumulation, respectively). Importantly, as expected due to reduced &bgr;‐arrestin 2 recruitment, chronic exposure of cells to PNR‐4‐20 results in significantly less desensitization and down‐regulation of hCB1Rs compared to similar treatment with CP‐55,940. PNR‐4‐20 (i.p.) is active in the cannabinoid tetrad in mice and chronic treatment results in development of less persistent tolerance and no significant withdrawal signs when compared to animals repeatedly exposed to the non‐biased full agoinst JWH‐018 or &Dgr;9‐THC. Finally, studies of a structurally similar analog PNR‐ 4‐02 show that it is also a G protein biased hCB1R agonist. It is predicted that cannabinoid agonists that bias hCB1R activation toward G protein, relative to &bgr;‐arrestin 2 signaling, will produce fewer and less severe adverse effects both acutely and chronically. Graphical abstract Figure. No caption available.

Approaches to Assess Biased Signaling at the CB1R Receptor

G protein-coupled receptors, such as the cannabinoid type 1 receptor (CB1R), have been shown to interact with multiple binding partners to transmit signals. In both transfected cell systems and in endogenously expressing cell lines, CB1R signaling has been described as multifaceted. The question remains as to how this highly widely expressed receptor signals in a given cell at a given time in vivo. The concept of functional selectivity, or biased agonism, describes the ability of an agonist to engage the receptor in a manner that preferentially engages certain signaling interactions (e.g., G proteins) over others (e.g., β-arrestins), presumably by stabilizing certain receptor conformations. There is growing interest in using such properties of ligands to direct signaling downstream of CB1R toward desirable therapeutic outcomes and to avoid adverse side effects. While it is not currently clear what pathways should be engaged and which should be avoided, the development of biased agonist tool compounds will aid in answering these questions. In this chapter, we discuss the approaches and caveats to assessing biased agonism at the CB1R.

G protein signaling–biased agonism at the κ-opioid receptor is maintained in striatal neurons

Triazole 1.1 is a G protein–biased agonist of the κ-opioid receptor in striatal neurons. Biasing pain treatment against side effects Stimulating opioid receptors with targeted agonists can treat pain, but such drugs often cause unwanted and even dangerous side effects because they induce multiple intracellular pathways downstream of the receptor. Studies in cell lines and mouse models have proposed that so-called “biased” agonists, those that activate only antinociceptive G protein signaling, may lessen pain and itch without causing side effects. Ho et al. confirm this potential for targeting the κ-opioid receptor (KOR) by showing the biochemical and physiological effects of biased KOR agonists specifically on striatal neurons in mice. These findings indicate that cell culture–based predictions of biased KOR agonists may hold true in vivo and therefore may be a better way to treat pain in patients. Biased agonists of G protein–coupled receptors may present a means to refine receptor signaling in a way that separates side effects from therapeutic properties. Several studies have shown that agonists that activate the κ-opioid receptor (KOR) in a manner that favors G protein coupling over β-arrestin2 recruitment in cell culture may represent a means to treat pain and itch while avoiding sedation and dysphoria. Although it is attractive to speculate that the bias between G protein signaling and β-arrestin2 recruitment is the reason for these divergent behaviors, little evidence has emerged to show that these signaling pathways diverge in the neuronal environment. We further explored the influence of cellular context on biased agonism at KOR ligand–directed signaling toward G protein pathways over β-arrestin–dependent pathways and found that this bias persists in striatal neurons. These findings advance our understanding of how a G protein–biased agonist signal differs between cell lines and primary neurons, demonstrate that measuring [35S]GTPγS binding and the regulation of adenylyl cyclase activity are not necessarily orthogonal assays in cell lines, and emphasize the contributions of the environment to assessing biased agonism.