RESUMO
δ opioid receptors (DORs) hold potential as a target for neurologic and psychiatric disorders, yet no DOR agonist has proven efficacious in critical phase II clinical trials. The exact reasons for the failure to produce quality drug candidates for the DOR are unclear. However, it is known that certain DOR agonists can induce seizures and exhibit tachyphylaxis. Several studies have suggested that those adverse effects are more prevalent in delta agonists that share the (+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC80)/4-[(αR*)-α-((2S*,5R*)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxybenzyl]-N,N-diethylbenzamide chemotype. There is a need to find novel lead candidates for drug development that have improved pharmacological properties to differentiate them from the current failed delta agonists. Our objective in this study was to identify novel DOR agonists. We used a ß-arrestin assay to screen a small G-protein coupled receptors (GPCR)-focused chemical library. We identified a novel chemotype of DOR agonists that appears to bind to the orthosteric site based of docking and molecular dynamic simulation. The most potent agonist hit compound is selective for the DOR over a panel of 167 other GPCRs, is slightly biased toward G-protein signaling and has anti-allodynic efficacy in a complete Freund's adjuvant model of inflammatory pain in C57BL/6 male and female mice. The newly discovered chemotype contrasts with molecules like SNC80 that are highly efficacious ß-arrestin recruiters and may suggest this novel class of DOR agonists could be expanded on to develop a clinical candidate drug. SIGNIFICANCE STATEMENT: δ opioid receptors are a clinical target for various neurological disorders, including migraine and chronic pain. Many of the clinically tested delta opioid agonists share a single chemotype, which carries risks during drug development. Through a small-scale high-throughput screening assay, this study identified a novel δ opioid receptor agonist chemotype, which may serve as alternative for the current analgesic clinical candidates.
Assuntos
Receptores Opioides delta , Receptores Opioides delta/agonistas , Receptores Opioides delta/metabolismo , Animais , Camundongos , Masculino , Humanos , Compostos de Espiro/farmacologia , Compostos de Espiro/química , Piperazinas/farmacologia , Piperazinas/química , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Benzamidas/farmacologia , Benzamidas/química , Cricetulus , beta-Arrestinas/metabolismo , Células HEK293 , Células CHORESUMO
The δ-opioid receptor (δOR) holds great potential as a therapeutic target. Yet, clinical drug development, which has focused on δOR agonists that mimic the potent and selective tool compound SNC80 have largely failed. It has increasingly become apparent that the SNC80 scaffold carries with it potent and efficacious ß-arrestin recruitment. Here, we screened a relatively small (5120 molecules) physical drug library to identify δOR agonists that underrecruit ß-arrestin, as it has been suggested that compounds that efficaciously recruit ß-arrestin are proconvulsant. The screen identified a hit compound and further characterization using cellular binding and signaling assays revealed that this molecule (R995045, compound 1) exhibited ten-fold selectivity over µ- and κ-opioid receptors. Compound 1 represents a novel chemotype at the δOR. A subsequent characterization of fourteen analogs of compound 1, however did not identify a more potent δOR agonist. Computational modeling and in vitro characterization of compound 1 in the presence of the endogenous agonist leu-enkephalin suggest compound 1 may also bind allosterically and negatively modulate the potency of Leu-enkephalin to inhibit cAMP, acting as a 'NAM-agonist' in this assay. The potential physiological utility of such a class of compounds will need to be assessed in future in vivo assays.
Assuntos
Receptores Opioides delta/agonistas , Regulação Alostérica/efeitos dos fármacos , Aminoácidos/química , Sítios de Ligação , AMP Cíclico/metabolismo , Encefalina Leucina/química , Encefalina Leucina/farmacologia , Células HEK293 , Humanos , Concentração Inibidora 50 , Simulação de Dinâmica Molecular , Receptores Opioides kappa/metabolismo , Receptores Opioides mu/metabolismo , beta-Arrestinas/metabolismoRESUMO
Background and Purpose: Mitragyna speciosa extract and kratom alkaloids decrease alcohol consumption in mice at least in part through actions at the δ-opioid receptor (δOR). However, the most potent opioidergic kratom alkaloid, 7-hydroxymitragynine, exhibits rewarding properties and hyperlocomotion presumably due to preferred affinity for the mu opioid receptor (µOR). We hypothesized that opioidergic kratom alkaloids like paynantheine and speciogynine with reduced µOR potency could provide a starting point for developing opioids with an improved therapeutic window to treat alcohol use disorder. Experimental Approach: We characterized paynantheine, speciociliatine, and four novel kratom-derived analogs for their ability to bind and activate δOR, µOR, and κOR. Select opioids were assessed in behavioral assays in male C57BL/6N WT and δOR knockout mice. Key Results: Paynantheine (10 mgâkg-1, i.p.) produced aversion in a limited conditioned place preference (CPP) paradigm but did not produce CPP with additional conditioning sessions. Paynantheine did not produce robust antinociception but did block morphine-induced antinociception and hyperlocomotion. Yet, at 10 and 30 mgâkg-1 doses (i.p.), paynantheine did not counteract morphine CPP. 7-hydroxypaynantheine and 7-hydroxyspeciogynine displayed potency at δOR but limited µOR potency relative to 7-hydroxymitragynine in vitro, and dose-dependently decreased voluntary alcohol consumption in WT but not δOR in KO mice. 7-hydroxyspeciogynine has a maximally tolerated dose of at least 10 mgâkg-1 (s.c.) at which it did not produce significant CPP neither alter general locomotion nor induce noticeable seizures. Conclusion and Implications: Derivatizing kratom alkaloids with the goal of enhancing δOR potency and reducing off-target effects could provide a pathway to develop novel lead compounds to treat alcohol use disorder with an improved therapeutic window.
RESUMO
µ-Opioid receptor agonists provide potent and effective acute analgesia; however, their therapeutic window narrows considerably upon repeated administration, such as required for treating chronic pain. In contrast, bifunctional µ/δ opioid agonists, such as the endogenous enkephalins, have potential for treating both acute and chronic pain. However, enkephalins recruit ß-arrestins, which correlate with certain adverse effects at µ- and δ-opioid receptors. Herein, we identify the C-terminus of Tyr-ψ[(Z)CF[double bond, length as m-dash]CH]-Gly-Leu-enkephalin, a stable enkephalin derivative, as a key site to regulate bias of both δ- and µ-opioid receptors. Using in vitro assays, substitution of the Leu5 carboxylate with amides (NHEt, NMe2, NCyPr) reduced ß-arrestin recruitment efficacy through both the δ-opioid and µ-opioid, while retaining affinity and cAMP potency. For this series, computational studies suggest key ligand-receptor interactions that might influence bias. These findings should enable the discovery of a range of tool compounds with previously unexplored biased µ/δ opioid agonist pharmacological profiles.
RESUMO
BACKGROUND AND PURPOSE: Mitragyna speciosa, more commonly known as kratom, is a plant that contains opioidergic alkaloids but is unregulated in most countries. Kratom is used in the self-medication of chronic pain and to reduce illicit and prescription opioid dependence. Kratom may be less dangerous than typical opioids because of the stronger preference of kratom alkaloids to induce receptor interaction with G proteins over ß-arrestin proteins. We hypothesized that kratom (alkaloids) can also reduce alcohol intake. EXPERIMENTAL APPROACH: We pharmacologically characterized kratom extracts, kratom alkaloids (mitragynine, 7-hydroxymitragynine, paynantheine, and speciogynine) and synthetic carfentanil-amide opioids for their ability to interact with G proteins and ß-arrestin at µ, δ, and κ opioid receptors in vitro. We used C57BL/6 mice to assess to which degree these opioids could reduce alcohol intake and whether they had rewarding properties. KEY RESULTS: Kratom alkaloids were strongly G protein-biased at all three opioid receptors and reduced alcohol intake, but kratom and 7-hydroxymitragynine were rewarding. Several results indicated a key role for δ opioid receptors, including that the synthetic carfentanil-amide opioid MP102-a G protein-biased agonist with modest selectivity for δ opioid receptors-reduced alcohol intake, whereas the G protein-biased µ opioid agonist TRV130 did not. CONCLUSION AND IMPLICATIONS: Our results suggest that kratom extracts can decrease alcohol intake but still carry significant risk upon prolonged use. Development of more δ opioid-selective synthetic opioids may provide a safer option than kratom to treat alcohol use disorder with fewer side effects.
Assuntos
Alcoolismo , Mitragyna , Alcoolismo/tratamento farmacológico , Amidas , Analgésicos Opioides , Animais , Fentanila/análogos & derivados , Proteínas de Ligação ao GTP , Camundongos , Camundongos Endogâmicos C57BL , Alcaloides de Triptamina e SecologaninaRESUMO
As tool compounds to study cardiac ischemia, the endogenous δ-opioid receptors (δOR) agonist Leu5-enkephalin and the more metabolically stable synthetic peptide (d-Ala2, d-Leu5)-enkephalin are frequently employed. However, both peptides have similar pharmacological profiles that restrict detailed investigation of the cellular mechanism of the δOR's protective role during ischemic events. Thus, a need remains for δOR peptides with improved selectivity and unique signaling properties for investigating the specific roles for δOR signaling in cardiac ischemia. To this end, we explored substitution at the Phe4 position of Leu5-enkephalin for its ability to modulate receptor function and selectivity. Peptides were assessed for their affinity to bind to δORs and µ-opioid receptors (µORs) and potency to inhibit cAMP signaling and to recruit ß-arrestin 2. Additionally, peptide stability was measured in rat plasma. Substitution of the meta-position of Phe4 of Leu5-enkephalin provided high-affinity ligands with varying levels of selectivity and bias at both the δOR and µOR and improved peptide stability, while substitution with picoline derivatives produced lower-affinity ligands with G protein biases at both receptors. Overall, these favorable substitutions at the meta-position of Phe4 may be combined with other modifications to Leu5-enkephalin to deliver improved agonists with finely tuned potency, selectivity, bias and drug-like properties.
Assuntos
Encefalina Leucina/farmacologia , Receptores Opioides delta/metabolismo , Receptores Opioides mu/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Células CHO , Cricetulus , Encefalina Leucina/genética , Humanos , Fenilalanina , Receptores Opioides delta/agonistas , Receptores Opioides delta/genética , Receptores Opioides mu/agonistas , Receptores Opioides mu/genética , Transdução de Sinais/genéticaRESUMO
We report the selection of DNA-encoded small molecule libraries against protein targets within the cytosol and on the surface of live cells. The approach relies on generation of a covalent linkage of the DNA to protein targets by affinity labeling. This cross-linking event enables subsequent copurification by a tag on the recombinant protein. To access targets within cells, a cyclic cell-penetrating peptide is appended to DNA-encoded libraries for delivery across the cell membrane. As this approach assesses binding of DELs to targets in live cells, it provides a strategy for selection of DELs against challenging targets that cannot be expressed and purified as active.
Assuntos
Peptídeos Penetradores de Células/química , Proteínas/genética , Proteínas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Peptídeos Penetradores de Células/metabolismo , Reagentes de Ligações Cruzadas/química , Citosol/efeitos dos fármacos , Citosol/metabolismo , DNA/química , Fluoresceínas/química , Células HEK293 , Humanos , Lipídeos , Peptídeos Cíclicos/química , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Bibliotecas de Moléculas Pequenas/química , Tetra-Hidrofolato Desidrogenase/genética , Transfecção , Trimetoprima/farmacologiaRESUMO
Between 2000 and 2005 several studies revealed that morphine is more potent and exhibits fewer side effects in beta-arrestin 2 knockout mice. These findings spurred efforts to develop opioids that signal primarily via G protein activation and do not, or only very weakly, recruit beta-arrestin. Development of such molecules targeting the mu opioid receptor initially outpaced those targeting the kappa, delta and nociceptin opioid receptors, with the G protein-biased mu opioid agonist oliceridine/TRV130 having completed phase III clinical trials with improved therapeutic window to treat moderate-to-severe acute pain. Recently however, there has been a sharp increase in the development of novel G protein-biased kappa agonists. It is hypothesized that G protein-biased kappa agonists can reduce pain and itch, but exhibit fewer side effects, such as anhedonia and psychosis, that have thus far limited the clinical development of unbiased kappa opioid agonists. Here we summarize recently discovered G protein-biased kappa agonists, comparing structures, degree of signal bias and preclinical effects. We specifically reviewed nalfurafine, 22-thiocyanatosalvinorin A (RB-64), mesyl-salvinorin B, 2-(4-(furan-2-ylmethyl)-5-((4-methyl-3-(trifluoromethyl)benzyl)thio)-4H-1,2,4-triazol-3-yl)pyridine (triazole 1.1), 3-(2-((cyclopropylmethyl)(phenethyl)amino)ethyl)phenol (HS666), N-n-butyl-N-phenylethyl-N-3-hydroxyphenylethyl-amine (compound 5/BPHA), 6-guanidinonaltrindole (6'GNTI), and collybolide. These agonists encompass a variety of chemical scaffolds and range in both their potency and efficacy in terms of G protein signaling and beta-arrestin recruitment. Thus unsurprisingly, the behavioral responses reported for these agonists are not uniform. Yet, it is our conclusion that the kappa opioid field will benefit tremendously from future studies that compare several biased agonists and correlate the degree of signaling bias to a particular pharmacological response.
RESUMO
The impact that ß-arrestin proteins have on G protein-coupled receptor trafficking, signaling and physiological behavior has gained much appreciation over the past decade. A number of studies have attributed the side effects associated with the use of naturally occurring and synthetic opioids, such as respiratory depression and constipation, to excessive recruitment of ß-arrestin. These findings have led to the development of biased opioid small molecule agonists that do not recruit ß-arrestin, activating only the canonical G protein pathway. Similar G protein-biased small molecule opioids have been found to occur in nature, particularly within kratom, and opioids within salvia have served as a template for the synthesis of other G protein-biased opioids. Here, we present the first report of naturally occurring peptides that selectively activate G protein signaling pathways at δ opioid receptors, but with minimal ß-arrestin recruitment. Specifically, we find that rubiscolin peptides, which are produced as cleavage products of the plant protein rubisco, bind to and activate G protein signaling at δ opioid receptors. However, unlike the naturally occurring δ opioid peptides leu-enkephalin and deltorphin II, the rubiscolin peptides only very weakly recruit ß-arrestin 2 and have undetectable recruitment of ß-arrestin 1 at the δ opioid receptor.
Assuntos
Receptores Opioides delta/química , Receptores Opioides delta/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Animais , Células CHO , Cricetulus , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Encefalina Leucina/farmacologia , Modelos Moleculares , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Ensaio Radioligante , Receptores Opioides delta/genética , Ribulose-Bifosfato Carboxilase/síntese química , Ribulose-Bifosfato Carboxilase/química , Ribulose-Bifosfato Carboxilase/farmacologia , Transfecção , beta-Arrestina 2/genética , beta-Arrestina 2/metabolismoRESUMO
G protein-coupled receptors (GPCR) have a long history of being considered a prime target for drug development to treat a plethora of diseases and disorders. In fact in 1827, the first approved therapeutic in the United States was morphine, a drug that targets a GPCR, namely the mu opioid receptor. However, with the rise in biologics over the last two decades, the market share of small molecules targeting GPCRs has declined. Still, two phenomena concerning GPCR pharmacology, specifically heteromerization and biased signaling, have bolstered new interests in this particular class of drug targets. Heteromerization, the process by which two distinct GPCRs come together to form a unique signaling complex, has been demonstrated between many different GPCRs and has spurred efforts to discover heteromer selective drugs. Additionally, the discovery of biased signaling, a concept by which a GPCR can transduce intracellular signaling by favoring a specific pathway (e.g. G-protein) over another pathway (e.g. arrestin), has led to the development of signal-biased drugs with potentially fewer side effects. Our goal for this review is to highlight studies that have investigated the interplay of these two phenomena by providing an overview of the current literature describing instances where GPCR heteromers have distinct arrestin recruitment profiles when compared to the individual GPCRs, with a focus on those GPCRs expressed in the central nervous system. This article is part of the Special Issue entitled 'Receptor heteromers and their allosteric receptor-receptor interactions'.