Pharmacological characterization of novel synthetic opioids: Isotonitazene, metonitazene, and piperidylthiambutene as potent μ-opioid receptor agonists

Luca, Maria Antonietta De; Tocco, Graziella; Mostallino, Rafaela; Laus, Antonio; Caria, Francesca; Musa, Aurora; Pintori, Nicholas; Ucha, Marcos; Poza, Celia; Ambrosio, Emilio; Chiara, G. Di; Castelli, Paola

doi:10.1016/j.neuropharm.2022.109263

Cited by 21 publications

(10 citation statements)

References 59 publications

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“…In that study, all nitazenes produced fully efficacious antinociception, robust locomotor stimulation, and hypothermia, with a rank order of potency correlated with EC 50 values for MOR-mediated inhibition of cAMP. In male Sprague–Dawley rats, De Luca et al showed that isotonitazene was more potent than metonitazene and fentanyl as an antinociceptive agent . Malcolm et al showed that N -desethyl isotonitazene produced powerful respiratory depression in mice that was more severe and longer-lasting than that produced by fentanyl .…”

Section: Discussionmentioning

confidence: 99%

“…In male Sprague−Dawley rats, De Luca et al showed that isotonitazene was more potent than metonitazene and fentanyl as an antinociceptive agent. 60 Malcolm et al showed that N-desethyl isotonitazene produced powerful respiratory depression in mice that was more severe and longer-lasting than that produced by fentanyl. 61 Interestingly, in this same study, nitazene analogs were reported to act as "super agonists" in assays measuring MOR-mediated inhibition of cAMP (% E max range 103−108% of DAMGO) and βarrestin-2 recruitment (% E max range 108−120% of DAMGO).…”

Section: Acsmentioning

confidence: 99%

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In Vitro Functional Profiling of Fentanyl and Nitazene Analogs at the μ-Opioid Receptor Reveals High Efficacy for Gi Protein Signaling

Tsai,

Chen,

Baumann

et al. 2024

ACS Chem. Neurosci.

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Novel synthetic opioids (NSOs), including both fentanyl and non-fentanyl analogs that act as μ-opioid receptor (MOR) agonists, are associated with serious intoxication and fatal overdose. Previous studies proposed that G-protein-biased MOR agonists are safer pain medications, while other evidence indicates that low intrinsic efficacy at MOR better explains the reduced opioid side effects. Here, we characterized the in vitro functional profiles of various NSOs at the MOR using adenylate cyclase inhibition and β-arrestin2 recruitment assays, in conjunction with the application of the receptor depletion approach. By fitting the concentration−response data to the operational model of agonism, we deduced the intrinsic efficacy and affinity for each opioid in the Gi protein signaling and β-arrestin2 recruitment pathways. Compared to the reference agonist [D-Ala 2 ,N-MePhe 4 ,Gly-ol 5 ]enkephalin, we found that several fentanyl analogs were more efficacious at inhibiting cAMP production, whereas all fentanyl analogs were less efficacious at recruiting β-arrestin2. In contrast, the non-fentanyl 2-benzylbenzimidazole (i.e., nitazene) analogs were highly efficacious and potent in both the cAMP and β-arrestin2 assays. Our findings suggest that the high intrinsic efficacy of the NSOs in Gi protein signaling is a common property that may underlie their high risk of intoxication and overdose, highlighting the limitation of using in vitro functional bias to predict the adverse effects of opioids. In addition, the extremely high potency of many NSOs now infiltrating illicit drug markets further contributes to the danger posed to public health.

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Section: Discussionmentioning

confidence: 99%

Section: Acsmentioning

confidence: 99%

In Vitro Functional Profiling of Fentanyl and Nitazene Analogs at the μ-Opioid Receptor Reveals High Efficacy for Gi Protein Signaling

Tsai,

Chen,

Baumann

et al. 2024

ACS Chem. Neurosci.

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“…In another recent study, the in vitro potencies of MTZ, ETZ, ITZ, PTZ, and BTZ at MOR were highly correlated with their in vivo potencies for hypothermia, increased locomotion, and antinociception, with some exhibiting higher potencies in vitro and in vivo than fentanyl (Glatfelter et al, 2023). Similarly, ITZ and MTZ exhibited very high in vitro affinity and potency at MOR, high in vivo antinociceptive potency, as well as high potency to increase dopamine in the nucleus accumbens (De Luca et al, 2022). DEI led to more potent anti-nociception and greater and longer lasting apnea and respiratory depression (Malcolm et al, 2023).…”

Section: Discussionmentioning

confidence: 99%

“…In early studies, using C6 rat glioma cells expressing the recombinant rat mu opioid receptor (rMOR), ETZ had very high affinity (0.017 nM) for the [ 3 H]sufentanil binding site, and high potency (1.12 nM) at stimulating [ 35 S]GTPγS binding; this is more than 10 fold higher affinity and potency than fentanyl (Emmerson et al, 1996). ITZ stimulated the release of dopamine in rat nucleus accumbens shell to a greater extent than an equivalent dose of fentanyl (De Luca et al, 2022). Using HEK-293T cells, an array of substituted nitazenes showed no bias toward either βarrestin (βarr) or Gi signaling pathways; ITZ was twice as potent as fentanyl at stimulating both pathways (Vandeputte et al, 2020;Vandeputte et al, 2021).…”

Section: Introductionmentioning

confidence: 99%

Pharmacologic Characterization of Substituted Nitazenes atμ,κ, andΔOpioid Receptors Suggests High Potential for Toxicity

Kozell,

Eshleman,

Wolfrum

et al. 2024

J Pharmacol Exp Ther

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The benzimidazole opioids (BO, substituted nitazenes) are highly potent MOR agonists with heroin-or fentanyl-like effects. These compounds have caused hospitalizations and fatal overdoses.We characterized the in vitro pharmacology and structure-activity relationships of 19 nitazenes with substitutions at three positions of the benzimidazole core. Affinities were assessed using agonist radioligand binding assays at human mu, kappa, and delta opioid receptors (MOR, KOR, and DOR, respectively) heterologously expressed in Chinese hamster ovary (CHO) cells. Notably, for MOR binding, nine substituted nitazenes had significantly higher affinities than fentanyl including Npyrrolidino etonitazene, N-pyrrilidino isonitazene, and N-desethyl isotonitazene; thirteen had subnanomolar affinities. Only metodesnitazene and flunitazene had significantly lower affinities than fentanyl. Affinities for the substituted nitazenes at KOR and DOR relative to MOR were 46-2580-and 180-1280-fold lower, respectively. Functional activities were assessed using [ 35 S]GTPγS binding assays. Four nitazenes had sub-nanomolar potencies at MOR: N-pyrrolidino etonitazene, Npyrrilidino isonitazene, N-pyrrilidino protonitazene and N-desethyl isotonitazene. Ten substituted nitazenes had significantly higher potencies than fentanyl. All tested nitazenes were full MOR agonists. Potencies at KOR and DOR relative to MOR were 7.3-7920-and 24-9400-fold lower, respectively. Thus, many of these compounds are high affinity/high potency MOR agonists with elevated potential to elicit toxicity and overdose at low doses. SIGNIFICANCE STATEMENT: The substituted nitazenes are a growing public health threat. The Although the 19 nitazenes tested vary in affinity, potency, and efficacy, a number are very high affinity, high potency, and high efficacy compoundshigher than fentanyl. Their pharmacology suggests high potential for harm.

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“…The following protocol was used to prepare the simulated systems. [35][36][37] The MOR and KOR models were built from the crystallographic structures of M. musculus MOR (PDB ID 6DDF) [23] and H. sapiens KOR (PDB ID 6VI4) coupled to their G-proteins. [24] The MOR and KOR were crystallized in the active state with the agonist [D-Ala 2 , N-MePhe 4 , Glyol]-enkephalin (for MOR) or the agonist (3R)-7-hydroxy-N-{(2S)-1-[(3R,4R)-4-(3-hydroxyphenyl)-3,4-dimethylpiperidin-1-yl]-3-methylbutan-2-yl}-1,2,3,4-tetrahydroisoquinoline-3-carboxamide (for KOR) occupying the binding site.…”

Section: Molecular Docking Methodsmentioning

confidence: 99%

In silico characterization of ligand–receptor interactions for U‐47700, N,N‐didesmethyl‐U‐47700, U‐50488 at mu‐ and kappa‐opioid receptors

Laus

Kumar

Caboni

et al. 2023

Archiv der Pharmazie

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The increasing misuse of novel synthetic opioids (NSOs) represents a serious public health concern. In this regard, U‐47700 (trans‐3,4‐dichloro‐N‐[2‐(dimethylamino)cyclohexyl]‐N‐methylbenzamide) and related “U‐compounds” emerged on recreational drug markets as synthetic substitutes for illicit heroin and constituents of counterfeit pain medications. While the pharmacology of U‐compounds has been investigated using in vitro and in vivo methods, there is still a lack of understanding about the details of ligand–receptor interactions at the molecular level. To this end, we have developed a molecular modeling protocol based on docking and molecular dynamics simulations to assess the nature of ligand–receptor interactions for U‐47700, N,N‐didesmethyl U‐47700, and U‐50488 at the mu‐opioid receptor (MOR) and kappa‐opioid receptor (KOR). The evaluation of ligand–receptor and ligand–receptor‐membrane interaction energies enabled the identification of subtle conformational shifts in the receptors induced by ligand binding. Interestingly, the removal of two key methyl groups from U‐47700, to form N,N‐didesmethyl U‐47700, caused a loss of hydrogen bond contact with tryptophan (Trp)229, which may underlie the lower interaction energy and reduced MOR affinity for the compound. Taken together, our results are consistent with the reported biological findings for U‐compounds and provide a molecular basis for the MOR selectivity of U‐47700 and KOR selectivity of U‐50488.

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Pharmacological characterization of novel synthetic opioids: Isotonitazene, metonitazene, and piperidylthiambutene as potent μ-opioid receptor agonists

Cited by 21 publications

References 59 publications

In Vitro Functional Profiling of Fentanyl and Nitazene Analogs at the μ-Opioid Receptor Reveals High Efficacy for Gi Protein Signaling

In Vitro Functional Profiling of Fentanyl and Nitazene Analogs at the μ-Opioid Receptor Reveals High Efficacy for Gi Protein Signaling

Pharmacologic Characterization of Substituted Nitazenes atμ,κ, andΔOpioid Receptors Suggests High Potential for Toxicity

In silico characterization of ligand–receptor interactions for U‐47700, N,N‐didesmethyl‐U‐47700, U‐50488 at mu‐ and kappa‐opioid receptors

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