Development of Photoswitchable Tethered Ligands that Target the µ-Opioid Receptor.

Converting known ligands into photoswitchable derivatives offers the opportunity to modulate compound structure with light and hence, biological activity. In doing so, these probes provide unique control when evaluating G-protein-coupled receptor (GPCR) mechanism and function. Further conversion of such compounds into covalent probes, known as photoswitchable tethered ligands (PTLs), offers additional advantages. These include localization of the PTLs to the receptor binding pocket. Covalent localization increases local ligand concentration, improves site selectivity and may improve the biological differences between the respective isomers. This work describes chemical, photophysical and biochemical characterizations of a variety of PTLs designed to target the µ-opioid receptor (µOR). These PTLs were modeled on fentanyl, with the lead disulfide-containing agonist found to covalently interact with a cysteine-enriched mutant of this medically-relevant receptor.

Fentanyl and the Fluorinated Fentanyl Derivative NFEPP Elicit Distinct Hydrogen-Bond Dynamics of the Opioid Receptor.

The development of safe therapeutics to manage pain is of central interest for biomedical applications. The fluorinated fentanyl derivative N-(3-fluoro-1-phenethylpiperidin-4-yl)-N-phenylpropionamide (NFEPP) is potentially a safer alternative to fentanyl because unlike fentanyl─which binds to the µ-opioid receptor (MOR) at both physiological and acidic pH─NFEPP might bind to the MOR only at acidic pH typical of inflamed tissue. Knowledge of the protonation-coupled dynamics of the receptor-drug interactions is thus required to understand the molecular mechanism by which receptor activation initiates cell signaling to silence pain. To this end, here we have carried out extensive atomistic simulations of the MOR in different protonation states, in the absence of opioid drugs, and in the presence of fentanyl vs NFEPP. We used graph-based analyses to characterize internal hydrogen-bond networks that could contribute to the activation of the MOR. We find that fentanyl and NFEPP prefer distinct binding poses and that, in their binding poses, fentanyl and NFEPP partake in distinct internal hydrogen-bond networks, leading to the cytoplasmic G-protein-binding region. Moreover, the protonation state of functionally important aspartic and histidine side chains impacts hydrogen-bond networks that extend throughout the receptor, such that the ligand-bound MOR presents at its cytoplasmic G-protein-binding side, a hydrogen-bonding environment where dynamics depend on whether fentanyl or NFEPP is bound, and on the protonation state of specific MOR groups. The exquisite sensitivity of the internal protein-water hydrogen-bond network to the protonation state and to details of the drug binding could enable the MOR to elicit distinct pH- and opioid-dependent responses at its cytoplasmic G-protein-binding site.

Identifying reliable ions for the statistical differentiation of structurally similar fentanyl analogs.

Definitive identification of fentanyl analogs based on mass spectral comparison is challenging given the high degree of structural and, hence, spectral similarity. To address this, a statistical method was previously developed in which two electron-ionization (EI) mass spectra are compared using the unequal variance t-test. Normalized intensities of corresponding ions are compared, testing the null hypothesis (H0 ) that the difference in intensity is equal to zero. If H0 is accepted at all m/z values, the two spectra are statistically equivalent at the specified confidence level. If H0 is not accepted at any m/z value, then there is a significant difference in intensity at that m/z value between the two spectra. In this work, the statistical comparison method is applied to distinguish EI spectra of valeryl fentanyl, isovaleryl fentanyl, and pivaloyl fentanyl. Spectra of the three analogs were collected over a 9-month period and at different concentrations. At the 99.9% confidence level, the spectra of corresponding isomers were statistically associated. Spectra of different isomers were statistically distinct, and ions responsible for discrimination were identified in each comparison. To account for inherent instrument variations, discriminating ions for each pairwise comparison were ranked based on the magnitude of the calculated t-statistic (tcalc ) value. For a given comparison, ions with higher tcalc values are those with the greatest difference in intensity between the two spectra and, therefore, are considered more reliable for discrimination. Using these methods, objective discrimination among the spectra was achieved and ions considered most reliable for discrimination of these isomers were identified.

Evaluation of polyanionic cyclodextrins as high affinity binding scaffolds for fentanyl.

Cyclodextrins (CDs) have been previously shown to display modest equilibrium binding affinities (Ka ~ 100-200 M-1) for the synthetic opioid analgesic fentanyl. In this work, we describe the synthesis of new CDs possessing extended thioalkylcarboxyl or thioalkylhydroxyl moieties and assess their binding affinity towards fentanyl hydrochloride. The optimal CD studied displays a remarkable affinity for the opioid of Ka = 66,500 M-1, the largest value reported for such an inclusion complex to date. One dimensional 1H Nuclear Magnetic Resonance (NMR) as well as Rotational Frame Overhauser Spectroscopy (2D-ROESY) experiments supported by molecular dynamics (MD) simulations suggest an unexpected binding behavior, with fentanyl able to bind the CD interior in one of two distinct orientations. Binding energies derived from the MD simulations work correlate strongly with NMR-derived affinities highlighting its utility as a predictive tool for CD candidate optimization. The performance of these host molecules portends their utility as platforms for medical countermeasures for opioid exposure, as biosensors, and in other forensic science applications.

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.

Structurally diverse fentanyl analogs yield differential locomotor activities in mice.

Synthetic narcotics have been implicated as the single greatest contributor to increases in opioid-related fatalities in recent years. This study evaluated the effects of nine fentanyl-related substances that have emerged in the recreational drug marketplace, and for which there are no existing or only limited in vivo data. Adult male Swiss Webster mice were administered fentanyl-related substances and their effects on locomotion as compared to MOR agonist standards were recorded. In locomotor activity tests, morphine (100, 180 mg/kg), buprenorphine (1, 10 mg/kg), fentanyl (1, 10 mg/kg), cyclopropylfentanyl (1, 10 mg/kg), cyclopentylfentanyl (10 mg/kg), (±)-cis-3-methylbutyrylfentanyl (0.1, 1, 10 mg/kg), ortho-methylacetylfentanyl (10 mg/kg), para-chloroisobutyrylfentanyl (100 mg/kg), ocfentanil (1, 10 mg/kg), and ortho-fluoroacrylfentanyl (0.1, 1, 10 mg/kg) elicited significant (p ≤ 0.05) dose-dependent increases in locomotion. However, 2,2,3,3-tetramethylcyclopropylfentanyl did not have any effects on locomotion, even when tested up to 100 mg/kg, and 4'-methylacetylfentanyl (10, 100 mg/kg) significantly decreased locomotion. The rank order of efficacy for stimulating locomotion (maximum effect as a % of fentanyl's maximum effect) for fentanyl-related substances relative to MOR agonist standards was cyclopropylfentanyl (108.84 ± 20.21) > fentanyl (100 ± 15.3) > ocfentanil (79.27 ± 16.92) > morphine (75.9 ± 14.5) > (±)-cis-3-methylbutyrylfentanyl (68.04 ± 10.08) > ortho-fluoroacrylfentanyl (63.56 ± 19.88) > cyclopentylfentanyl (56.46 ± 8.54) > para-chloroisobutyrylfentanyl (22.44 ± 8.51) > buprenorphine (11.26 ± 2.30) > ortho-methylacetylfentanyl (9.45 ± 2.92) > 2,2,3,3-tetramethylcyclopropylfentanyl (6.75 ± 1.43) > 4'-methylacetylfentanyl (3.47 ± 0.43). These findings extend in vivo results from previous reports documenting additional fentanyl related-related substances that stimulate locomotion similar to known abused opioids while also identifying some anomalies.

Analysis, identification and confirmation of synthetic opioids using chloroformate chemistry: Retrospective detection of fentanyl and acetylfentanyl in urine and plasma samples by EI-GC-MS and HR-LC-MS.

Electron Impact Gas Chromatography-Mass Spectrometry (EI-GC-MS) and High Resolution Liquid Chromatography-Mass Spectrometry (HR-LC-MS) have been used in the analysis of products arising from the trichloroethoxycarbonylation of fentanyl and acetylfentanyl in urine and plasma matrices. The method involves the initial extraction of both synthetic opioids separately from the matrices followed by detection of the unique products that arise from their reaction with 2,2,2-trichloroethoxycarbonyl chloride (Troc-Cl), namely Troc-norfentanyl and Troc-noracetylfentanyl. The optimized protocol was successfully evaluated for its efficacy at detecting these species formed from fentanyl and acetylfentanyl when present at low and high levels in urine (fentanyl: 5 and 10 ng/mL and acetylfentanyl: 20 and 100 ng/mL) and plasma (fentanyl: 10 and 20 ng/mL and acetylfentanyl: 50 and 200 ng/mL), values that reflect levels reported in overdose victims. The HR-LC-MS method's LOQ (limit of quantitation) for the Troc-norfentanyl and Troc-noracetylfentanyl products was determined to be ~10 ng/mL for both species. Even though the superiority in the detection of these species by HR-LC-MS over EI-GC-MS, the latter method proved to be important in the detection of the second product from the reaction, namely 2-phenylethyl chloride that is crucial in the determination of the original opioid. This observation highlights the importance of using complimentary analytical techniques in the analysis of a sample, whether biological or environmental in nature. The method herein serves as a complementary, qualitative confirmation for the presence of a fentanyl in collected urine, plasma and by extension other biological samples amenable to the common extraction procedures described for opioid analysis. More importantly, the method's main strength comes from its ability to react with unknown fentanyls to yield products that can be not only detected by EI-GC-MS and HR-LC-MS but can then be used to retrospectively identify an unknown fentanyl.

Predicting the structure of unexplored novel fentanyl analogues by deep learning model.

Fentanyl and its analogues are psychoactive substances and the concern of fentanyl abuse has been existed in decades. Because the structure of fentanyl is easy to be modified, criminals may synthesize new fentanyl analogues to avoid supervision. The drug supervision is based on the structure matching to the database and too few kinds of fentanyl analogues are included in the database, so it is necessary to find out more potential fentanyl analogues and expand the sample space of fentanyl analogues. In this study, we introduced two deep generative models (SeqGAN and MolGPT) to generate potential fentanyl analogues, and a total of 11 041 valid molecules were obtained. The results showed that not only can we generate molecules with similar property distribution of original data, but the generated molecules also contain potential fentanyl analogues that are not pretty similar to any of original data. Ten molecules based on the rules of fentanyl analogues were selected for NMR, MS and IR validation. The results indicated that these molecules are all unreported fentanyl analogues. Furthermore, this study is the first to apply the deep learning to the generation of fentanyl analogues, greatly expands the exploring space of fentanyl analogues and provides help for the supervision of fentanyl.

Photochromic Fentanyl Derivatives for Controlled µ-Opioid Receptor Activation.

Photoswitchable ligands as biological tools provide an opportunity to explore the kinetics and dynamics of the clinically relevant µ-opioid receptor. These ligands can potentially activate or deactivate the receptor when desired by using light. Spatial and temporal control of biological activity allows for application in a diverse range of biological investigations. Photoswitchable ligands have been developed in this work, modelled on the known agonist fentanyl, with the aim of expanding the current "toolbox" of fentanyl photoswitchable ligands. In doing so, ligands have been developed that change geometry (isomerize) upon exposure to light, with varying photophysical and biochemical properties. This variation in properties could be valuable in further studying the functional significance of the µ-opioid receptor.

Fentanyl Structure as a Scaffold for Opioid/Non-Opioid Multitarget Analgesics.

One of the strategies in the search for safe and effective analgesic drugs is the design of multitarget analgesics. Such compounds are intended to have high affinity and activity at more than one molecular target involved in pain modulation. In the present contribution we summarize the attempts in which fentanyl or its substructures were used as a µ-opioid receptor pharmacophoric fragment and a scaffold to which fragments related to non-opioid receptors were attached. The non-opioid 'second' targets included proteins as diverse as imidazoline I2 binding sites, CB1 cannabinoid receptor, NK1 tachykinin receptor, D2 dopamine receptor, cyclooxygenases, fatty acid amide hydrolase and monoacylglycerol lipase and σ1 receptor. Reviewing the individual attempts, we outline the chemistry, the obtained pharmacological properties and structure-activity relationships. Finally, we discuss the possible directions for future work.

Proton Affinity Values of Fentanyl and Fentanyl Analogues Pertinent to Ambient Ionization and Detection.

Proton affinity is a major factor in the atmospheric pressure chemical ionization of illicit drugs. The detection of illicit drugs by mass spectrometry and ion mobility spectrometry relies on the analytes having greater proton affinities than background species. Evaluating proton affinities for fentanyl and its analogues is informative for predicting the likelihood of ionization in different environments and for optimizing the compounds' ionization and detection, such as through the addition of dopant chemicals. Herein, density functional theory was used to computationally determine the proton affinity and gas-phase basicity of 15 fentanyl compounds and several relevant molecules as a reference point. The range of proton affinities for the fentanyl compounds was from 1018 to 1078 kJ/mol. Fentanyl compounds with the higher proton affinity values appeared to form a bridge between the oxygen on the amide and the protonated nitrogen on the piperidine ring based on models and calculated bond distances. Experiments with fragmentation of proton-bound clusters using atmospheric flow tube-mass spectrometry (AFT-MS) provided estimates of relative proton affinities and showed proton affinity values of fentanyl compounds >1000 kJ/mol, which were consistent with the computational results. The high proton affinities of fentanyl compounds facilitate their detection by ambient ionization techniques in complex environments. The detection limits of the fentanyl compounds with AFT-MS are in the low femtogram range, which demonstrates the feasibility of trace vapor drug detection.

Respiratory depressant effects of fentanyl analogs are opioid receptor-mediated.

Opioid-related fatalities involving synthetic opioids have reached unprecedented levels. This study evaluated the respiratory depressant effects of seven fentanyl analogs that have either emerged in the illicit drug supply or been identified in toxicological analyses following fatal or non-fatal intoxications. Adult male Swiss Webster mice were administered fentanyl analogs (isobutyrylfentanyl, crotonylfentanyl, para-methoxyfentanyl, para-methoxybutyrylfentanyl, 3-furanylfentanyl, thiophenefentanyl, and benzodioxolefentanyl) and their effects on minute volume as compared to mu-opioid receptor (MOR) agonist standards (fentanyl, morphine, and buprenorphine) were measured using whole body plethysmography (WBP). All drugs elicited significant (p ≤ 0.05) hypoventilation relative to vehicle for at least one dose tested: morphine (1, 3.2, 10, 32 mg/kg), buprenorphine, (0.032, 0.1, 0.32, 1, 3.2 mg/kg), fentanyl (0.0032, 0.01, 0.032, 0.1, 1, 32 mg/kg), isobutyrylfentanyl (0.1, 0.32, 1, 3.2, 10 mg/kg), crotonylfentanyl (0.1, 0.32, 1, 3.2, 10 mg/kg), para-methoxyfentanyl (0.1, 0.32, 1, 3.2, 10 mg/kg), para-methoxybutyrylfentanyl (0.32, 1, 3.2, 10 mg/kg), 3-furanylfentanyl (0.1, 0.32, 1, 3.2, 10 mg/kg), thiophenefentanyl (1, 3.2, 10, 32, 100 mg/kg), and benzodioxolefentanyl (3.2, 10, 32, 100 mg/kg). The ED50 values for hypoventilation showed a rank order of potency as follows: fentanyl (ED50 = 0.96 mg/kg) > 3-furanylfentanyl (ED50 = 2.60 mg/kg) > crotonylfentanyl (ED50 = 2.72 mg/kg) > para-methoxyfentanyl (ED50 = 3.31 mg/kg) > buprenorphine (ED50 = 10.8 mg/kg) > isobutyrylfentanyl (ED50 = 13.5 mg/kg) > para-methoxybutyrylfentanyl (ED50 = 16.1 mg/kg) > thiophenefentanyl (ED50 = 18.0 mg/kg) > morphine (ED50 = 55.3 mg/kg) > benzodioxolefentanyl (ED50 = 10,168 mg/kg). A naloxone pretreatment (10 mg/kg) attenuated the hypoventilatory effects of all drugs. These results establish that the respiratory depressant effects of these fentanyl analogs are at least in part mediated by the MOR.

Gas Chromatography-Mass Spectrometry Analysis of Synthetic Opioids Belonging to the Fentanyl Class: A Review.

The rising number of deaths caused by fentanyl overdosing in the US due to the overwhelming illicit use of this synthetic opioid has started a global campaign to develop efficient ways to control its production and distribution as well as discovering efficient antidotes to mitigate its lethal effects. Another important vein of focused research established by various agencies lies in the development of efficient and practical protocols for the detection of this opioid and analogs thereof in various matrices, whether environmental or biological in nature, particularly in the field of gas chromatography-mass spectrometry (GC-MS). The following review will cover the literature dealing with the detection and identification of synthetic opioids belonging to the fentanyl class by GC-MS means and hyphenated versions of the technique. Detailed descriptions will be given for the GC-MS methods employed for the analysis of the opioid, starting with the nature of the extraction protocol employed prior to analysis to the actual findings presented by the cited reports. Great effort has gone into describing the methods involved in each paper in a detailed manner and these have been compiled by year in tables at the end of each section for the reader's convenience. Lastly, the review will end with concluding remarks about the state of GC-MS analysis with regards to these powerful opioids and what lies ahead for this analytical field.

Comparative analysis of vapor profiles of fentalogs and illicit fentanyl.

Availability of fentanyl is at a record high with 3138 kg of fentanyl and related substances being seized in 2019. Fentanyl's high toxicity makes a lethal dose for most mere milligrams. With such a high potency and a consistent rise of abuse, the chances of injury or death of frontline workers increase with every interaction. Development of a non-contact detection method for fentanyl would decrease the chances of a workplace mishap. To aid in the development of a non-contact detection method, target analytes in the vapor profile of fentanyl need to be identified. In order to achieve this goal, semi-quantitative headspace analysis of fentanyl analogs and confiscated fentanyl exhibits was accomplished using solid-phase microextraction and gas chromatography coupled with mass spectrometry (SPME-GC-MS). The vapor signatures of these samples were compared to a previously reported reference-grade fentanyl vapor signature to determine the target analyte(s) for fentanyl detection in the vapor phase. A total of 20 fentalogs and confiscated exhibits, with masses ranging from 2 to 19 mg, were sampled. N-Phenylpropanamide(NPPA) or N-phenethyl-4-piperidone(NPP) was identified as target analytes in 75% of these samples. This is a crucial component for the development of a non-contact detection method for fentanyl.

Improvements on a chemically contiguous hapten for a vaccine to address fentanyl-contaminated heroin.

Unintentional overdose deaths related to opioids and psychostimulants have increased in prevalence due to the adulteration of these drugs with fentanyl. Synergistic effects between illicit compounds and fentanyl cause aggravated respiratory depression, leading to inadvertent fatalities. Traditional small-molecule therapies implemented in the expanding opioid epidemic present numerous problems since they interact with the same opioid receptors in the brain as the abused drugs. In this study, we report an optimized dual hapten for use as an immunopharmacotherapeutic tool in order to develop antibodies capable of binding to fentanyl-contaminated heroin in the periphery, thus impeding the drugs' psychoactive effects on the central nervous system. This vaccine produced antibodies with nanomolar affinities and effectively blocked opioid analgesic effects elicited by adulterated heroin. These findings provide further insight into the development of chemically contiguous haptens for broad-spectrum immunopharmacotherapies against opioid use disorders.

Hyperpolarisation of Mirfentanil by SABRE in the Presence of Heroin.

Mirfentanil, a fentanyl derivative that is a µ-opioid partial agonist, is hyperpolarised via Signal Amplification By Reversible Exchange (SABRE), a para-hydrogen-based technique. [Ir(IMes)(COD)Cl] (IMes=1,3-bis(2,4,6-trimethylphenyl)imidazole-2-ylidene, COD=cyclooctadiene) was employed as the polarisation transfer catalyst. Following polarisation transfer at 6.5 mT, the pyrazine-protons were enhanced by 78-fold (polarisation, P=0.04 %). The complex [Ir(IMes)(H)2 (mirfentanil)2 (MeOH)]+ is proposed to form based on the observation of two hydrides at δ -22.9 (trans to mirfentanil) and -24.7 (trans to methanol). In a mixture of mirfentanil and heroin, the former could be detected using SABRE at concentrations less than 1 % w/w. At the lowest concentration analyzed, the amount of mirfentanil present was 0.18 mg (812 µM) and produced a signal enhancement of -867-fold (P=0.42 %). following polarisation transfer at 6.5 mT.

Identification of human cytochrome P450 isozymes involved in the oxidative metabolism of carfentanil.

Carfentanil is an ultra-potent opioid with an analgesic potency 10,000 times that of morphine but has received little scientific investigation. In the present study, the human cytochrome P450 (CYP) isozymes catalyzing the oxidative metabolism of carfentanil were investigated. Using UHPLC-HRMS, Michaelis-Menten kinetics of formation for three major metabolites norcarfentanil (M1), pharmaceutical active metabolite 4-[(1-oxopropyl)phenylamino]-1-(2-hydroxyl-2-phenylethyl)-4-piperidinecarboxylic acid methyl ester (M11), and 4-[(1-oxopropyl)phenylamino]-1-(2-oxo-2-phenylethyl)-4-piperidinecarboxylic acid methyl ester (M15) were determined. Isozymes catalyzing the formation of the low abundant, highly active metabolite 1-[2-(2-hydroxylphenyl)ethyl]-4-[(1-oxopropyl)phenylamino]-4-piperidinecarboxylic acid methyl ester (M13) were also identified. Selective P450 inhibition studies with pooled human liver microsomes (HLMs) and recombinant CYP isozymes suggested that metabolites M1, M11, and M15 were predominantly formed by isozyme CYP3A5, followed by CYP3A4. Isozymes CYP2C8 and CYP2C9 also made contributions but to a much lesser extent. Highly potent metabolite M13 was predominantly formed by isozyme CYP2C9, followed by CYP2C8. These findings indicate that CYP3A5, CYP3A4, CYP2C8 and CYP2C9 play a major role in the transformation of carfentanil to M1 (norcarfentanil), M11, M13 and M15 through N-dealkylation of piperidine ring, hydroxylation of phenethyl group and ketone formation on phenethyl linker by human liver micrsomes.

The utility of visual appearance in predicting the composition of street opioids.

Background: With the emergence of unregulated fentanyl, people who use unregulated opioids are increasingly relying on appearance in an effort to ascertain the presence of fentanyl and level of drug potency. However, the utility of visual inspection to identify drug composition in the fentanyl era has not been assessed. Methods: We assessed client expectation, appearance, and composition of street drug samples being presented for drug checking. Results of a visual screening test were compared to fentanyl immunoassay strip testing. We calculated sensitivity, specificity and likelihood ratios (LR) to assess the accuracy of the common assumption that samples with a "pebbles" appearance contain fentanyl. Results: In total, of the 2502 unregulated opioid samples tested, 1820 (73.5%) appeared as "pebbles", of which 1729 (95.0%) tested positive for fentanyl for a sensitivity of 75.9% (95% Confidence Interval [CI]: 74.2-77.6) and specificity of 59.4% (95%CI: 57.5-61.3). Although, the odds of samples containing fentanyl was 4.60 (95%CI: 3.47-6.11) times higher among pebbles samples compared to non-pebble samples, the positive LR for pebbles to contain fentanyl was only 1.87 (CI: 1.59-2.19). The negative LR was more useful at 0.41 (95% CI: 0.36-0.46). Conclusions: A positive screening test for pebbles is not strongly enough associated to be used as a proxy for detecting fentanyl. While the absence of the appearance of pebbles does somewhat reduce the likelihood of fentanyl being present in a given sample, the high prevalence of fentanyl and fentanyl analogues in the drug supply and the risks of consumption are such that public health providers should routinely advise people who use unregulated opioids against solely relying on visual characteristics of drugs as a harm reduction strategy.

How µ-opioid receptor recognizes fentanyl.

Roughly half of the drug overdose-related deaths in the United States are related to synthetic opioids represented by fentanyl which is a potent agonist of mu-opioid receptor (mOR). In recent years, X-ray crystal structures of mOR in complex with morphine derivatives have been determined; however, structural basis of mOR activation by fentanyl-like opioids remains lacking. Exploiting the X-ray structure of BU72-bound mOR and several molecular simulation techniques, we elucidated the detailed binding mechanism of fentanyl. Surprisingly, in addition to the salt-bridge binding mode common to morphinan opiates, fentanyl can move deeper and form a stable hydrogen bond with the conserved His2976.52, which has been suggested to modulate mOR's ligand affinity and pH dependence by previous mutagenesis experiments. Intriguingly, this secondary binding mode is only accessible when His2976.52 adopts a neutral HID tautomer. Alternative binding modes may represent a general mechanism in G protein-coupled receptor-ligand recognition.

Enhancing Nonfouling and Sensitivity of Surface-Enhanced Raman Scattering Substrates for Potent Drug Analysis in Blood Plasma via Fabrication of a Flexible Plasmonic Patch.

Surface-enhanced Raman scattering (SERS) is an ultrasensitive analytical technique, which is capable of providing high specificity; thus, it can be used for toxicological drug assay (detection and quantification). However, SERS-based drug analysis directly in human biofluids requires mitigation of fouling and nonspecificity effects that commonly appeared from unwanted adsorption of endogenous biomolecules present in biofluids (e.g., blood plasma and serum) onto the SERS substrate. Here, we report a bottom-up fabrication strategy to prepare ultrasensitive SERS substrates, first, by functionalizing chemically synthesized gold triangular nanoprisms (Au TNPs) with poly(ethylene glycol)-thiolate in the solid state to avoid protein fouling and second, by generating flexible plasmonic patches to enhance SERS sensitivity via the formation of high-intensity electromagnetic hot spots. Poly(ethylene glycol)-thiolate-functionalized Au TNPs in the form of flexible plasmonic patches show a twofold-improved signal-to-noise ratio in comparison to triethylamine (TEA)-passivated Au TNPs. Furthermore, the plasmonic patch displays a SERS enhancement factor of 4.5 ×107. Utilizing the Langmuir adsorption model, we determine the adsorption constant of drugs for two different surface ligands and observe that the drug molecules display stronger affinity for poly(ethylene glycol) ligands than TEA. Our density functional theory calculations unequivocally support the interaction between drug molecules and poly(ethylene glycol) moieties. Furthermore, the universality of the plasmonic patch for SERS-based drug detection is demonstrated for cocaine, JWH-018, and opioids (fentanyl, despropionyl fentanyl, and heroin) and binary mixture (trace amount of fentanyl in heroin) analyses. We demonstrate the applicability of flexible plasmonic patches for the selective assay of fentanyl at picogram/milliliter concentration levels from drug-of-abuse patients' blood plasma. The fentanyl concentration calculated in the patients' blood plasma from SERS analysis is in excellent agreement with the values determined using the paper spray ionization mass spectrometry technique. We believe that the flexible plasmonic patch fabrication strategy would be widely applicable to any plasmonic nanostructure for SERS-based chemical sensing for clinical toxicology and therapeutic drug monitoring.