Multitarget µ-Opioid Receptor Agonists─Neuropeptide FF Receptor Antagonists Induce Potent Antinociception with Reduced Adverse Side Effects.

The design of bifunctional compounds is a promising approach toward the development of strong analgesics with reduced side effects. We here report the optimization of the previously published lead peptide KGFF09, which contains opioid receptor agonist and neuropeptide FF receptor antagonist pharmacophores and is shown to induce potent antinociception and reduced side effects. We evaluated the novel hybrid peptides for their in vitro activity at MOP, NPFFR1, and NPFFR2 and selected four of them (DP08/14/32/50) for assessment of their acute antinociceptive activity in mice. We further selected DP32 and DP50 and observed that their antinociceptive activity is mostly peripherally mediated; they produced no respiratory depression, no hyperalgesia, significantly less tolerance, and strongly attenuated withdrawal syndrome, as compared to morphine and the recently FDA-approved TRV130. Overall, these data suggest that MOP agonist/NPFF receptor antagonist hybrids might represent an interesting strategy to develop novel analgesics with reduced side effects.

Development of a Potential-Modulated Electrochemiluminescence Measurement System for Selective and Sensitive Determination of the Controlled Drug Codeine.

Codeine is a common analgesic drug that is a pro-drug of morphine. It also has a high risk of abuse as a recreational drug because of its extensive distribution as an OTC drug. Therefore, sensitive and selective screening methods for codeine are crucial in forensic analytical chemistry. To date, a commercial analytical kit has not been developed for dedicated codeine determination, and there is a need for an analytical method to quantify codeine in the field. In the present work, potential modulation was combined with electrochemiluminescence (ECL) for sensitive determination of codeine. The potential modulated technique involved applying a signal to electrodes by superimposing an AC potential on the DC potential. When tris(2,2'-bipyridine)ruthenium(II) ([Ru(bpy)3]2+) was used as an ECL emitter, ECL activity was confirmed for codeine. A detailed investigation of the electrochemical reaction mechanism suggested a characteristic ECL reaction mechanism involving electrochemical oxidation of the opioid framework. Besides the usual ECL reaction derived from the amine framework, selective detection of codeine was possible under the measurement conditions, with clear luminescence observed in an acidic solution. The sensitivity of codeine detection by potential modulated-ECL was one order of magnitude higher than that obtained with the conventional potential sweep method. The proposed method was applied to codeine determination in actual prescription medications and OTC drug samples. Codeine was selectively determined from other compounds in medications and showed good linearity with a low detection limit (150 ng mL-1).

Assessing Abuse-Deterrent formulations utilizing Ion-Exchange resin complexation processed via Twin-Screw granulation for improved safety and effectiveness.

Opioid misuse is a public health crisis in the United States. In response, the FDA has approved drug products with abuse-deterrent features to reduce the risk of prescription opioid abuse. Abuse-deterrent formulations (ADFs) typically employ physical or chemical barriers or incorporate agonist-antagonist combinations as mechanisms to deter misuse. This study aims to assess the impact of abuse-deterrent properties, specifically ion-exchange resin complexation as a chemical barrier, on a model drug, promethazine hydrochloride (PMZ) tablets. Various formulations were developed through twin-screw wet granulation (TSWG) followed by twin-screw melt granulation (TSMG). In the TSWG process, the drug interacts with the resin through an exchange reaction, forming a drug-resin complex. Additionally, the study explored factors influencing the complex formation between the drug and resin, using the drug loading status as an indicator. DSC and ATR studies were carried out to confirm the formation of the drug-resin complex. Subsequently, hot melt granulation was employed to create a matrix tablet incorporating Kollidon® SR and Kollicoat® MAE 100P, thereby enabling sustained release properties. The drug-resin complex embedded in the matrix effectively deters abuse through methods like smoking, snorting, or parenteral injection, unless the drug can be extracted. In order to assess this, solvent extraction studies were conducted using an FDA-recommended solvents, determining the potential for abuse. Further investigations involved dissolution tests in change-over media, confirming the extended-release properties of the formulation. Results from dissolution studies comparing the ground and intact tablets provided positive evidence of the formulation's effectiveness in deterring abuse. Finally, alcohol-induced dose-dumping studies were conducted in compliance with FDA guidelines, concluding that the formulation successfully mitigates dose dumping in the presence of alcohol.

Morphinan Evolution: The Impact of Advances in Biochemistry and Molecular Biology.

Morphinan-based opioids, derived from natural alkaloids like morphine, codeine and thebaine, have long been pivotal in managing severe pain. However, their clinical utility is marred by significant side effects and high addiction potential. This review traces the evolution of the morphinan scaffold in light of advancements in biochemistry and molecular biology, which have expanded our understanding of opioid receptor pharmacology. We explore the development of semi-synthetic and synthetic morphinans, their receptor selectivity and the emergence of biased agonism as a strategy to dissociate analgesic properties from undesirable effects. By examining the molecular intricacies of opioid receptors and their signaling pathways, we highlight how receptor-type selectivity and signaling bias have informed the design of novel analgesics. This synthesis of historical and contemporary perspectives provides an overview of the morphinan landscape, underscoring the ongoing efforts to mitigate the problems facing opioids through smarter drug design. We also highlight that most morphinan derivatives show a preference for the G protein pathway, although detailed experimental comparisons are still necessary. This fact underscores the utility of the morphinan skeleton in future opioid drug discovery.

In vitro µ-opioid receptor activation potential of U10 and ß-U10, positional isomers of the synthetic opioid naphthyl U-47700.

New synthetic opioids (NSOs) with diverse chemical structures continue to appear on recreational drug markets worldwide. U-type opioids have become one of the largest groups of non-fentanyl-related NSOs. Starting in 2020, a previously unreported U-compound coined "ß-U10" (2-naphthyl U-47700; N-[2-(dimethylamino)cyclohexyl]-N-methylnaphthalene-2-carboxamide) was identified in Australia and the United States. ß-U10 is a positional isomer of α-U10 (1-naphthyl U-47700), more commonly known as "U10." Here, the first comparative in vitro pharmacological characterization of naphthyl U-47700 (U10 and ß-U10), together with the structural analogue U-47700 and fentanyl, is reported. Application of a cell-based µ-opioid receptor (MOR) activation (ß-arrestin 2 recruitment) assay demonstrated ß-U10 (EC50 = 348 nM; Emax = 150% vs. hydromorphone) to be less potent than U-47700 (EC50 = 116 nM; Emax = 154%) and fentanyl (EC50 = 9.35 nM; Emax = 146%) but considerably more active than the α-isomer (EC50 value in the µM range). For the latter, maximum receptor activation could not be reached at 100 µM. The difference in MOR activation potential for U10 and ß-U10 stresses the importance of (analytical) differentiation between closely related analytes. The emergence of ß-U10 on the recreational drug market is an example of the continuing emergence of non-fentanyl-related NSOs and further emphasizes the need to closely monitor fluctuations in the drug supply.

Design and structural validation of peptide-drug conjugate ligands of the kappa-opioid receptor.

Despite the increasing number of GPCR structures and recent advances in peptide design, the development of efficient technologies allowing rational design of high-affinity peptide ligands for single GPCRs remains an unmet challenge. Here, we develop a computational approach for designing conjugates of lariat-shaped macrocyclized peptides and a small molecule opioid ligand. We demonstrate its feasibility by discovering chemical scaffolds for the kappa-opioid receptor (KOR) with desired pharmacological activities. The designed De Novo Cyclic Peptide (DNCP)-ß-naloxamine (NalA) exhibit in vitro potent mixed KOR agonism/mu-opioid receptor (MOR) antagonism, nanomolar binding affinity, selectivity, and efficacy bias at KOR. Proof-of-concept in vivo efficacy studies demonstrate that DNCP-ß-NalA(1) induces a potent KOR-mediated antinociception in male mice. The high-resolution cryo-EM structure (2.6 Å) of the DNCP-ß-NalA-KOR-Gi1 complex and molecular dynamics simulations are harnessed to validate the computational design model. This reveals a network of residues in ECL2/3 and TM6/7 controlling the intrinsic efficacy of KOR. In general, our computational de novo platform overcomes extensive lead optimization encountered in ultra-large library docking and virtual small molecule screening campaigns and offers innovation for GPCR ligand discovery. This may drive the development of next-generation therapeutics for medical applications such as pain conditions.

Discovery of 7-Azanorbornane-Based Dual Agonists for the Delta and Kappa Opioid Receptors through an In Situ Screening Protocol.

In medicinal chemistry, the copper-catalyzed click reaction is used to prepare ligand candidates. This reaction is so clean that the bioactivities of the products can be determined without purification. Despite the advantages of this in situ screening protocol, the applicability of this method for transmembrane proteins has not been validated due to the incompatibility with copper catalysts. To address this point, we performed ligand screening for the µ, δ, and κ opioid receptors using this protocol. As we had previously reported the 7-azanorbornane skeleton as a privileged scaffold for the G protein-coupled receptors, we performed the click reactions between various 7-substituted 2-ethynyl-7-azanorbornanes and azides. Screening assays were performed without purification using the CellKeyTM system, and the putative hit compounds were re-synthesized and re-evaluated. Although the "hit" compounds for the µ and the δ receptors were totally inactive after purifications, three of the four "hits" for the κ receptor were true agonists for this receptor and also showed activities for the δ receptor. Although false positive/negative results exist as in other screening projects for soluble proteins, this in situ method is effective in identifying novel ligands for transmembrane proteins.

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

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.

Pharmacological and Physicochemical Properties Optimization for Dual-Target Dopamine D3 (D3R) and µ-Opioid (MOR) Receptor Ligands as Potentially Safer Analgesics.

A new generation of dual-target µ opioid receptor (MOR) agonist/dopamine D3 receptor (D3R) antagonist/partial agonists with optimized physicochemical properties was designed and synthesized. Combining in vitro cell-based on-target/off-target affinity screening, in silico computer-aided drug design, and BRET functional assays, we identified new structural scaffolds that achieved high affinity and agonist/antagonist potencies for MOR and D3R, respectively, improving the dopamine receptor subtype selectivity (e.g., D3R over D2R) and significantly enhancing central nervous system multiparameter optimization scores for predicted blood-brain barrier permeability. We identified the substituted trans-(2S,4R)-pyrrolidine and trans-phenylcyclopropyl amine as key dopaminergic moieties and tethered these to different opioid scaffolds, derived from the MOR agonists TRV130 (3) or loperamide (6). The lead compounds 46, 84, 114, and 121 have the potential of producing analgesic effects through MOR partial agonism with reduced opioid-misuse liability via D3R antagonism. Moreover, the peripherally limited derivatives could have therapeutic indications for inflammation and neuropathic pain.

Quantification of the benzimidazole opioid analog isotonitazene in human hair using liquid chromatography-tandem mass spectrometry.

Benzimidazole opioids were originally developed from the late 1950s to 1970s as analgesics for medical use, although a lot of them could not be approved as licit medicines because of their severe side effects and physical dependence. Such benzimidazole opioid analogs as abused drug, however, have recently been found in illicit drug markets throughout the world. Isotonitazene is one such benzimidazole opioids, whose analgesic potency can be as much as 500 times greater than that of morphine, according to previous animal studies. In line with this potency, a couple of hundred fatalities related to it were reported to date. In this study, a well validated method for the quantification of isotonitazene in human hair samples using liquid chromatography (LC)-tandem mass spectrometry (MS/MS) was established, and could be applied to authentic samples which were seized by the police security bureau. Isotonitazene concentrations in the seized hair averaged 6.11 pg/mg. The LLOQ and LOD of this method were 1.25 and 2.5 pg/mg, respectively; the calibration curve of the substance in hair samples showed a good linearity in the concentration range of 2.5-250 pg/mg (r > 0.999); the extraction recovery rates were 87.3-105% in the tested range; the inter- and intra-day precisions and accuracies (%biases) were not greater than 9.09% for each determination. Isotonitazene in human hair showed good stability at room temperature and under dark storage conditions for 30 days. As for matrix effect in hair samples, moderate ion suppression of target substances could be found. This is the first report for the analysis of isotonitazene in human hair samples.

Comparative neuropharmacology of structurally distinct non-fentanyl opioids that are appearing on recreational drug markets worldwide.

BACKGROUND: The emergence of novel synthetic opioids (NSOs) is contributing to the opioid overdose crisis. While fentanyl analogs have historically dominated the NSO market, a shift towards non-fentanyl compounds is now occurring. METHODS: Here, we examined the neuropharmacology of structurally distinct non-fentanyl NSOs, including U-47700, isotonitazene, brorphine, and N-desethyl isotonitazene, as compared to morphine and fentanyl. Compounds were tested in vitro using opioid receptor binding assays in rat brain tissue and by monitoring forskolin-stimulated cAMP accumulation in cells expressing the human mu-opioid receptor (MOR). Compounds were administered subcutaneously to male Sprague-Dawley rats, and hot plate antinociception, catalepsy score, and body temperature changes were measured. RESULTS: Receptor binding results revealed high MOR selectivity for all compounds, with MOR affinities comparable to those of morphine and fentanyl (i.e., nM). All drugs acted as full-efficacy MOR agonists in the cyclic AMP assay, but nitazene analogs had greater functional potencies (i.e., pM) compared to the other drugs (i.e., nM). When administered to rats, all compounds induced opioid-like antinociception, catalepsy, and body temperature changes, but nitazenes were the most potent. Similar to fentanyl, the nitazenes had faster onset and decline of in vivo effects when compared to morphine. In vivo potencies to induce antinociception and catalepsy (i.e., ED50s) correlated with in vitro functional potencies (i.e., EC50s) but not binding affinities (i.e., Kis) at MOR. CONCLUSIONS: Collectively, our findings indicate that non-fentanyl NSOs pose grave danger to those individuals who use opioids. Continued vigilance is needed to identify and characterize synthetic opioids as they emerge in clandestine drug markets.

Peripheralization Strategies Applied to Morphinans and Implications for Improved Treatment of Pain.

Opioids are considered the most effective analgesics for the treatment of moderate to severe acute and chronic pain. However, the inadequate benefit/risk ratio of currently available opioids, together with the current 'opioid crisis', warrant consideration on new opioid analgesic discovery strategies. Targeting peripheral opioid receptors as effective means of treating pain and avoiding the centrally mediated side effects represents a research area of substantial and continuous attention. Among clinically used analgesics, opioids from the class of morphinans (i.e., morphine and structurally related analogues) are of utmost clinical importance as analgesic drugs activating the mu-opioid receptor. In this review, we focus on peripheralization strategies applied to N-methylmorphinans to limit their ability to cross the blood-brain barrier, thus minimizing central exposure and the associated undesired side effects. Chemical modifications to the morphinan scaffold to increase hydrophilicity of known and new opioids, and nanocarrier-based approaches to selectively deliver opioids, such as morphine, to the peripheral tissue are discussed. The preclinical and clinical research activities have allowed for the characterization of a variety of compounds that show low central nervous system penetration, and therefore an improved side effect profile, yet maintaining the desired opioid-related antinociceptive activity. Such peripheral opioid analgesics may represent alternatives to presently available drugs for an efficient and safer pain therapy.

Discovery of a Potent Highly Biased MOR Partial Agonist among Diastereomeric C9-Hydroxyalkyl-5-phenylmorphans.

All possible diastereomeric C9-hydroxymethyl-, hydroxyethyl-, and hydroxypropyl-substituted 5-phenylmorphans were synthesized to explore the three-dimensional space around the C9 substituent in our search for potent MOR partial agonists. These compounds were designed to lessen the lipophilicity observed with their C9-alkenyl substituted relatives. Many of the 12 diastereomers that were obtained were found to have nanomolar or subnanomolar potency in the forskolin-induced cAMP accumulation assay. Almost all these potent compounds were fully efficacious, and three of those chosen for in vivo evaluation, 15, 21, and 36, were all extremely G-protein biased; none of the three compounds recruited beta-arrestin2. Only one of the 12 diastereomers, 21 (3-((1S,5R,9R)-9-(2-hydroxyethyl)-2-phenethyl-2-azabicyclo[3.3.1]nonan-5-yl)phenol), was a MOR partial agonist with good, but not full, efficacy (Emax = 85%) and subnanomolar potency (EC50 = 0.91 nM) in the cAMP assay. It did not have any KOR agonist activity. This compound was unlike morphine in that it had a limited ventilatory effect in vivo. The activity of 21 could be related to one or more of three well-known theories that attempt to predict a dissociation of the desired analgesia from the undesirable opioid-like side-effects associated with clinically used opioids. In accordance with the theories, 21 was a potent MOR partial agonist, it was highly G-protein biased and did not attract beta-arrestin2, and it was found to have both MOR and DOR agonist activity. All the other diastereomers that were synthesized were either much less potent than 21 or had either too little or too much efficacy for our purposes. It was also noted that a C9-methoxymethyl compound with 1R,5S,9R stereochemistry (41) was more potent than the comparable C9-hydroxymethyl compound 11 (EC50 = 0.65 nM for 41 vs. 2.05 nM for 11). Both 41 and 11 were fully efficacious.

Detection, chemical analysis, and pharmacological characterization of dipyanone and other new synthetic opioids related to prescription drugs.

The emergence of structurally diverse new synthetic opioids (NSOs) has caused the opioid crisis to spiral to new depths. Little information is available about the pharmacology of most novel opioids when they first emerge. Here, using a ß-arrestin 2 recruitment assay, we investigated the in vitro µ-opioid receptor (MOR) activation potential of dipyanone, desmethylmoramide, and acetoxymethylketobemidone (O-AMKD) - recent NSOs that are structurally related to the prescription opioids methadone and ketobemidone. Our findings indicate that dipyanone (EC50=39.9 nM; Emax=155% vs. hydromorphone) is about equally active as methadone (EC50=50.3 nM; Emax=152%), whereas desmethylmoramide (EC50=1335 nM; Emax=126%) is considerably less active. A close structural analogue of ketobemidone (EC50=134 nM; Emax=156%) and methylketobemidone (EC50=335 nM; Emax=117%), O-AMKD showed a lower potency (EC50=1262 nM) and efficacy (Emax=109%). Evaluation of the opioid substitution product buprenorphine and its metabolite norbuprenorphine confirmed the increased in vitro efficacy of the latter. In addition to in vitro characterization, this report details the first identification and full chemical analysis of dipyanone in a seized powder, as well as a postmortem toxicology case from the USA involving the drug. Dipyanone was quantified in blood (370 ng/mL), in which it was detected alongside other NSOs (e.g., 2-methyl AP-237) and novel benzodiazepines (e.g., flualprazolam). While dipyanone is currently not commonly encountered in forensic samples worldwide, its emergence is worrisome and representative of the dynamic NSO market. Graphical Abstract.

Studies on diketopiperazine and dipeptide analogs as opioid receptor ligands.

Using the structure of gliotoxin as a starting point, we have prepared two different chemotypes with selective affinity to the kappa opioid receptor (KOR). Using medicinal chemistry approaches and structure-activity relationship (SAR) studies, structural features required for the observed affinity were identified, and advanced molecules with favorable Multiparameter Optimization (MPO) and Ligand Lipophilicity (LLE) profiles were prepared. Using the Thermal Place Preference Test (TPPT), we have shown that compound2 blocks the antinociceptive effect of U50488, a known KOR agonist. Multiple reports suggest that modulation of KOR signaling is a promising therapeutic strategy in treating neuropathic pain (NP). As a proof-of-concept study, we tested compound 2 in a rat model of NP and recorded its ability to modulate sensory and emotional pain-related behaviors. Observed in vitro and in vivo results suggest that these ligands can be used to develop compounds with potential application as pain therapeutics.

Identification of the Putative Binding Site of a Benzimidazole Opioid (Etazene) and Its Metabolites at µ-Opioid Receptor: A Human Liver Microsomal Assay and Systematic Computational Study.

The synthetic benzimidazole opioid etazene (which has a 70-times higher analgesic activity than morphine), a recreational drug, has gained popularity as a novel psychoactive substance (NPS) on the illegal/darknet market; however, no experimental information is available at the molecular level on the binding mechanism and putative binding site of etazene and its metabolites at the µ-opioid receptor (MOR). In the present study, we investigated the metabolism of etazene in human liver microsomes using ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS). We also explored the possibilities of MOR activation by etazene and its metabolites by studying their binding mechanisms and interaction profiles at an active-state MOR model via molecular docking, binding free energy calculations, and all-atom molecular dynamics (MD) simulations. The putative metabolites of etazene were also predicted using the ADMET Predictor 10.1. The molecular docking studies and free energy calculations showed that etazene and its metabolites (M1, M2, and M5-M7) exhibited strong predicted binding affinity at MOR and showed overlapped binding orientation with MOR-bound agonist BU72, which was co-crystallized in the MOR X-ray crystal structure (PDB ID: 5C1M). MD also confirmed the stability of the MOR-etazene and MOR-M6 complexes. These results suggest that etazene and its metabolites may act as strong MOR agonists, highlighting the necessity of experimental validation. The insights from this study, such as key interactions between etazene and its metabolites and the MOR, will allow authorities to predict potential analogs and clarify the target-protein interactions associated with this illicit substance, granting advanced or rapid reactions to confiscating or banning potential emerging drugs.

Solving an Old Puzzle: Elucidation and Evaluation of the Binding Mode of Salvinorin A at the Kappa Opioid Receptor.

The natural product Salvinorin A (SalA) was the first nitrogen-lacking agonist discovered for the opioid receptors and exhibits high selectivity for the kappa opioid receptor (KOR) turning SalA into a promising analgesic to overcome the current opioid crisis. Since SalA's suffers from poor pharmacokinetic properties, particularly the absence of gastrointestinal bioavailability, fast metabolic inactivation, and subsequent short duration of action, the rational design of new tailored analogs with improved clinical usability is highly desired. Despite being known for decades, the binding mode of SalA within the KOR remains elusive as several conflicting binding modes of SalA were proposed hindering the rational design of new analgesics. In this study, we rationally determined the binding mode of SalA to the active state KOR by in silico experiments (docking, molecular dynamics simulations, dynophores) in the context of all available mutagenesis studies and structure-activity relationship (SAR) data. To the best of our knowledge, this is the first comprehensive evaluation of SalA's binding mode since the determination of the active state KOR crystal structure. SalA binds above the morphinan binding site with its furan pointing toward the intracellular core while the C2-acetoxy group is oriented toward the extracellular loop 2 (ECL2). SalA is solely stabilized within the binding pocket by hydrogen bonds (C210ECL2, Y3127.35, Y3137.36) and hydrophobic contacts (V1182.63, I1393.33, I2946.55, I3167.39). With the disruption of this interaction pattern or the establishment of additional interactions within the binding site, we were able to rationalize the experimental data for selected analogs. We surmise the C2-substituent interactions as important for SalA and its analogs to be experimentally active, albeit with moderate frequency within MD simulations of SalA. We further identified the non-conserved residues 2.63, 7.35, and 7.36 responsible for the KOR subtype selectivity of SalA. We are confident that the elucidation of the SalA binding mode will promote the understanding of KOR activation and facilitate the development of novel analgesics that are urgently needed.

Insights into distinct signaling profiles of the µOR activated by diverse agonists.

Drugs targeting the µ-opioid receptor (µOR) are the most effective analgesics available but are also associated with fatal respiratory depression through a pathway that remains unclear. Here we investigated the mechanistic basis of action of lofentanil (LFT) and mitragynine pseudoindoxyl (MP), two µOR agonists with different safety profiles. LFT, one of the most lethal opioids, and MP, a kratom plant derivative with reduced respiratory depression in animal studies, exhibited markedly different efficacy profiles for G protein subtype activation and ß-arrestin recruitment. Cryo-EM structures of µOR-Gi1 complex with MP (2.5 Å) and LFT (3.2 Å) revealed that the two ligands engage distinct subpockets, and molecular dynamics simulations showed additional differences in the binding site that promote distinct active-state conformations on the intracellular side of the receptor where G proteins and ß-arrestins bind. These observations highlight how drugs engaging different parts of the µOR orthosteric pocket can lead to distinct signaling outcomes.

Analysis of highly potent synthetic opioid nitazene analogs and their positional isomers.

Four nitazenes (metonitazene, etonitazene, protonitazene, and isotonitazene), highly potent benzimidazole synthetic opioids, and their four nitro group positional isomers (isonitazenes) were synthesized and analyzed using infrared (IR) spectroscopy, gas chromatography/mass spectrometry (GC/MS), and liquid chromatography/mass spectrometry (LC/MS). In addition, the agonistic activity of all compounds at the human µ-opioid receptor was measured using a cell-based assay system. In the IR spectra, characteristic peaks for nitazenes and isonitazenes were observed. In GC/MS, all compounds were well separated on the chromatogram, although distinguishing nitazenes from the corresponding isonitazenes by electron ionization mass spectra was difficult. In LC/MS, all compounds were detected in both positive and negative modes of electrospray ionization. Characteristic fragment ions were observed in the product ion spectra of isonitazenes, enabling nitazenes to be distinguished from isonitazenes. All nitazenes tested demonstrated higher agonistic activity at the human µ-opioid receptors than the synthetic opioid fentanyl. The agonistic activities of isonitazenes were 11-35 times lower than those of the corresponding nitazenes. However, iso-etonitazene and iso-isotonitazene showed moderate activity similar to that of fentanyl, indicating that these drugs could cause poisoning at a comparable level as fentanyl, if these drugs are abused in the future.

Structure-based design of bitopic ligands for the µ-opioid receptor.

Mu-opioid receptor (µOR) agonists such as fentanyl have long been used for pain management, but are considered a major public health concern owing to their adverse side effects, including lethal overdose1. Here, in an effort to design safer therapeutic agents, we report an approach targeting a conserved sodium ion-binding site2 found in µOR3 and many other class A G-protein-coupled receptors with bitopic fentanyl derivatives that are functionalized via a linker with a positively charged guanidino group. Cryo-electron microscopy structures of the most potent bitopic ligands in complex with µOR highlight the key interactions between the guanidine of the ligands and the key Asp2.50 residue in the Na+ site. Two bitopics (C5 and C6 guano) maintain nanomolar potency and high efficacy at Gi subtypes and show strongly reduced arrestin recruitment-one (C6 guano) also shows the lowest Gz efficacy among the panel of µOR agonists, including partial and biased morphinan and fentanyl analogues. In mice, C6 guano displayed µOR-dependent antinociception with attenuated adverse effects, supporting the µOR sodium ion-binding site as a potential target for the design of safer analgesics. In general, our study suggests that bitopic ligands that engage the sodium ion-binding pocket in class A G-protein-coupled receptors can be designed to control their efficacy and functional selectivity profiles for Gi, Go and Gz subtypes and arrestins, thus modulating their in vivo pharmacology.