Structure of the Nanobody-Stabilized Active State of the Kappa Opioid Receptor.

The κ-opioid receptor (KOP) mediates the actions of opioids with hallucinogenic, dysphoric, and analgesic activities. The design of KOP analgesics devoid of hallucinatory and dysphoric effects has been hindered by an incomplete structural and mechanistic understanding of KOP agonist actions. Here, we provide a crystal structure of human KOP in complex with the potent epoxymorphinan opioid agonist MP1104 and an active-state-stabilizing nanobody. Comparisons between inactive- and active-state opioid receptor structures reveal substantial conformational changes in the binding pocket and intracellular and extracellular regions. Extensive structural analysis and experimental validation illuminate key residues that propagate larger-scale structural rearrangements and transducer binding that, collectively, elucidate the structural determinants of KOP pharmacology, function, and biased signaling. These molecular insights promise to accelerate the structure-guided design of safer and more effective κ-opioid receptor therapeutics.

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.

Discovery of sinomenine/8-Bis(benzylthio)octanoic acid hybrids as potential anti-leukemia drug candidate via mitochondrial pathway.

Traditional Chinese medicine Qingfengteng primarily acquired from the dried canes of Sinomenium acutum (Thunb.) Rehd. et Wils. var. cinereum Rehd. et Wils. and S. acutum (Thunb.) Rehd. et Wils. For the therapeutic treatment of rheumatism, acute arthritis, and rheumatoid arthritis based on Qingfengteng, sinomenine hydrochloride was recently made the principal active ingredient in various dosage forms. 8-Bis(benzylthio)octanoic acid (CPI-613) was an orphan medicine that the FDA and EMA approved orphan for the treatment of certain resistant malignancies. Its unique mode of action and minimal toxicity toward normal tissues made for an apt pharmacophore. In order to expand the field of sinomenine anticancer structures, sinomenine/8-Bis(benzylthio)octanoic acid derivatives were designed and synthesized. Among them, target hybrids e4 stood out for having notable cytotoxic effects against cancer cell lines, especially for K562 cells, with IC50 values of 2.45 µM and high safety. In-depth investigations demonstrated that e4 caused apoptosis by stopping the cell cycle at G1 phase, and doing so by altering the morphology of the nucleus and causing membrane potential of the in mitochondria to collapse. These results indicated e4 exerted an antiproliferative effect through apoptosis induction via mitochondrial pathway.

Design and synthesis of unique morphinan-type molecules: Their application to the search for the unexplored binding domain between opioid receptors and morphinan ligands.

The morphinan skeleton is valued in drug discovery for its beneficial physicochemical properties and is recognized as a crucial template for opioid receptor ligands. In morphinan derivatives, it is well-established that the nitrogen atom within the piperidine ring (D-ring) interacts with the amino acid residues of the opioid receptors. This interaction is recognized as one of the crucial pharmacophores between the morphinan molecule and the opioid receptors. Consequently, the structure-activity relationships (SAR) surrounding the D-ring are not well-studied, due to concerns that structural transformations around the nitrogen at the 17-position could disrupt this interaction. In this study, we found that our novel morphinan-type ligands with a side chain containing a heteroatom positioned above the d-ring have binding affinity for the opioid receptors. These novel skeletons could provide unique templates with the desired side chain above the D-ring in the morphinan skeleton, and thus, potentially advance the SAR studies of morphinan ligands with the opioid receptors.

Structure of the µ-opioid receptor-Gi protein complex.

The µ-opioid receptor (µOR) is a G-protein-coupled receptor (GPCR) and the target of most clinically and recreationally used opioids. The induced positive effects of analgesia and euphoria are mediated by µOR signalling through the adenylyl cyclase-inhibiting heterotrimeric G protein Gi. Here we present the 3.5 Å resolution cryo-electron microscopy structure of the µOR bound to the agonist peptide DAMGO and nucleotide-free Gi. DAMGO occupies the morphinan ligand pocket, with its N terminus interacting with conserved receptor residues and its C terminus engaging regions important for opioid-ligand selectivity. Comparison of the µOR-Gi complex to previously determined structures of other GPCRs bound to the stimulatory G protein Gs reveals differences in the position of transmembrane receptor helix 6 and in the interactions between the G protein α-subunit and the receptor core. Together, these results shed light on the structural features that contribute to the Gi protein-coupling specificity of the µOR.

Graphene oxide quantum dots-loaded sinomenine hydrochloride nanocomplexes for effective treatment of rheumatoid arthritis via inducing macrophage repolarization and arresting abnormal proliferation of fibroblast-like synoviocytes.

The characteristic features of the rheumatoid arthritis (RA) microenvironment are synovial inflammation and hyperplasia. Therefore, there is a growing interest in developing a suitable therapeutic strategy for RA that targets the synovial macrophages and fibroblast-like synoviocytes (FLSs). In this study, we used graphene oxide quantum dots (GOQDs) for loading anti-arthritic sinomenine hydrochloride (SIN). By combining with hyaluronic acid (HA)-inserted hybrid membrane (RFM), we successfully constructed a new nanodrug system named HA@RFM@GP@SIN NPs for target therapy of inflammatory articular lesions. Mechanistic studies showed that this nanomedicine system was effective against RA by facilitating the transition of M1 to M2 macrophages and inhibiting the abnormal proliferation of FLSs in vitro. In vivo therapeutic potential investigation demonstrated its effects on macrophage polarization and synovial hyperplasia, ultimately preventing cartilage destruction and bone erosion in the preclinical models of adjuvant-induced arthritis and collagen-induced arthritis in rats. Metabolomics indicated that the anti-arthritic effects of HA@RFM@GP@SIN NPs were mainly associated with the regulation of steroid hormone biosynthesis, ovarian steroidogenesis, tryptophan metabolism, and tyrosine metabolism. More notably, transcriptomic analyses revealed that HA@RFM@GP@SIN NPs suppressed the cell cycle pathway while inducing the cell apoptosis pathway. Furthermore, protein validation revealed that HA@RFM@GP@SIN NPs disrupted the excessive growth of RAFLS by interfering with the PI3K/Akt/SGK/FoxO signaling cascade, resulting in a decline in cyclin B1 expression and the arrest of the G2 phase. Additionally, considering the favorable biocompatibility and biosafety, these multifunctional nanoparticles offer a promising therapeutic approach for patients with RA.

A rapid and sensitive LC-MS/MS method for quantifying oxycodone, noroxycodone, oxymorphone and noroxymorphone in human plasma to support pharmacokinetic drug interaction studies of oxycodone.

A sensitive and reliable LC-MS/MS method was developed and validated for the quantification of oxycodone and metabolites in human plasma. The method has a runtime of 6 min and a sensitivity of 0.1 µg/L for all analytes. Sample preparation consisted of protein precipitation. Separation was performed on a Kinetix biphenyl column (2.1 × 100 mm, 1.7 µm), using ammonium formate 5 mm in 0.1% aqueous formic acid and methanol LC-MS grade 100% in gradient elution at a flow rate of 0.4 ml/min. Detection was performed in multiple reaction monitoring mode using positive electrospray ionization. The method was linear over the calibration range of 0.1-25.0 µg/L for oxycodone, noroxycodone and noroxymorphone and 0.1-5.0 µg/L for oxymorphone. The method demonstrated good performance in terms of intra- and interday accuracy (86.5-110.3%) and precision (CV 1.7-9.3%). The criteria for the matrix effect were met (CV < 15%) except for noroxymorphone, for which an additional method was applied to compensate for the matrix effect. Whole blood samples were stable for 4 h at room temperature. Plasma samples were stable for 24 h at room temperature and 3 months at -20°C. Furthermore, the method was successfully applied in a pharmacokinetic drug interaction study of oxycodone and enzalutamide in patients with prostate cancer.

Functional Activity of Enantiomeric Oximes and Diastereomeric Amines and Cyano Substituents at C9 in 3-Hydroxy-N-phenethyl-5-phenylmorphans.

The synthesis of stereochemically pure oximes, amines, saturated and unsaturated cyanomethyl compounds, and methylaminomethyl compounds at the C9 position in 3-hydroxy-N-phenethyl-5-phenylmorphans provided µ-opioid receptor (MOR) agonists with varied efficacy and potency. One of the most interesting compounds, (2-((1S,5R,9R)-5-(3-hydroxyphenyl)-2-phenethyl-2-azabicyclo[3.3.1]nonan-9-yl)acetonitrile), was found to be a potent partial MOR agonist (EC50 = 2.5 nM, %Emax = 89.6%), as determined in the forskolin-induced cAMP accumulation assay. Others ranged in potency and efficacy at the MOR, from nanomolar potency with a C9 cyanomethyl compound (EC50 = 0.85 nM) to its totally inactive diastereomer, and three compounds exhibited weak MOR antagonist activity (the primary amine 3, the secondary amine 8, and the cyanomethyl compound 41). Many of the compounds were fully efficacious; their efficacy and potency were affected by both the stereochemistry of the molecule and the specific C9 substituent. Most of the MOR agonists were selective in their receptor interactions, and only a few had δ-opioid receptor (DOR) or κ-opioid receptor (KOR) agonist activity. Only one compound, a C9-methylaminomethyl-substituted phenylmorphan, was moderately potent and fully efficacious as a KOR agonist (KOR EC50 = 18 nM (% Emax = 103%)).

C(21)-Di- and monofluorinated scaffold for thevinol/orvinol-based opioid receptor ligands.

Defluorination of the readily available 21,21,21-trifluorothevinone (7) with Mg + Me3SiCl allows the preparation of 21,21-difluorothevinone (10) and 21-fluorothevinone (11), which can be used as the starting compounds for syntheses of 21,21-difluoro- and 21-fluoro-substituted relatives of thevinols and orvinols. Taken together, thevinols and orvinols are well known to constitute a family of the highly potent 4,5α-epoxy-18,19-endo-(etheno/ethano)morphinan-type opioid receptor ligands. Alternatively, 10 and 18,19-dihydro-21,21-difluorothevinone (13) have been synthesized by the addition of Me3SiCHF2 to the carbonyl function of thevinal (12) and dihydrothevinal (18) followed by oxidation of the intermediate C(21)-difluorinated secondary alcohols. 21,21-Difluorothevinols were obtained both by the addition of RMgX or RLi to the 21,21-difluoroketones and by the addition of Me3SiCHF2 to the carbonyl function of the non-fluorinated 18,19-endo-(etheno/ethano)morphinan ketones. In general, these addition reactions have been shown to result in mixtures of the C(21)-epimeric alcohols. However, in some cases, the reactions proceeded with high stereoselectivity allowing the isolation of one of the epimeric alcohols by conventional crystallization. Preparations of the 21,21-difluorothevinols bearing an allyl, cyclopropylmethyl, or cyclobutylmethyl group at the N(17) nitrogen are also reported.

The atomistic level structure for the activated human κ-opioid receptor bound to the full Gi protein and the MP1104 agonist.

The kappa opioid receptor (κOR) is an important target for pain therapeutics to reduce depression and other harmful side effects of existing medications. The analgesic activity is mediated by κOR signaling through the adenylyl cyclase-inhibitory family of Gi protein. Here, we report the three-dimensional (3D) structure for the active state of human κOR complexed with both heterotrimeric Gi protein and MP1104 agonist. This structure resulted from long molecular dynamics (MD) and metadynamics (metaMD) simulations starting from the 3.1-Å X-ray structure of κOR-MP1104 after replacing the nanobody with the activated Gi protein and from the 3.5-Å cryo-EM structure of µOR-Gi complex after replacing the 168 missing residues. Using MD and metaMD we discovered interactions to the Gi protein with strong anchors to two intracellular loops and transmembrane helix 6 of the κOR. These anchors strengthen the binding, contributing to a contraction in the binding pocket but an expansion in the cytoplasmic region of κOR to accommodate G protein. These remarkable changes in κOR structure reveal that the anchors are essential for activation.

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.

A MOR Antagonist with High Potency and Antagonist Efficacy among Diastereomeric C9-Alkyl-Substituted N-Phenethyl-5-(3-hydroxy)phenylmorphans.

The 5-(3-hydroxy)phenylmorphan structural class of compounds are unlike the classical morphinans, 4,5-epoxymorphinans, and 6,7-benzomorphans, in that they have an equatorially oriented aromatic ring rather than the axial orientation of that ring found in the classical opioids. This modified and simplified opioid-like structure has been shown to retain antinociceptive activity, depending on its stereochemistry and substituents, and some of them have been found to be much more potent than morphine. A simple C9-hydroxy-5-(3-hydroxy)phenylmorphan enantiomer was found to be about 500 times more potent than morphine in vivo. We have previously examined C9-alkenyl and hydroxyalkyl substituents in the N-phenethyl-5-(3-hydroxy)phenylmorphan class of compounds. Comparable C9-alkyl (methyl through butyl) substituents, with their sets of diastereomers, have not been explored. All these compounds have now been synthesized to determine the effect chain-length and stereochemistry at the C9 position in the molecule might have on their interaction with opioid receptors. We now report the synthesis and in vitro activity of 16 compounds, the C9-methyl, ethyl, propyl, and butyl diastereomers, using the inhibition of forskolin-induced cAMP accumulation assay. Several potent (sub-nanomolar and nanomolar) MOR compounds were found to be selective agonists with varying efficacy. Of greatest interest, a selective MOR antagonist was discovered; it did not display any DOR or KOR agonist activity in vitro, was three times more potent than naltrexone, and was found to antagonize the EC90 of fentanyl at MOR to a greater extent than naltrexone.

Effect of removal of the 14-hydroxy group on the affinity of the 4,5-epoxymorphinan derivatives for orexin and opioid receptors.

To investigate the contribution of hydrogen bonding between the 14-hydroxy group and the 6-amide chain on the binding affinity of nalfurafine toward KOR and OX1R, we prepared the 14-H and 14-dehydrated nalfurafine and their five-membered D-ring nalfurafine (D-nor-nalfurafine) derivatives. The 14-H and 14-dehydrated nalfurafine derivatives showed almost the same affinity for KOR as nalfurafine and more potent affinity for OX1R. On the other hand, 14-H and 14-dehydrated D-nor-nalfurafine derivatives showed weak affinity for KOR and almost no affinity for OX1R. The conformational analyses suggested that the 6-amide chains of the nalfurafine derivatives are mainly oriented just at or downward from the C-ring, while those of the D-nor-nalfurafine derivatives were mainly oriented toward the upper side of the C-ring even in the absence of the 14-hydroxy group. We postulated that the ion-dipole interaction between the 6-amide and the 16-nitrogen might stabilize the upwardly oriented 6-amide group. These results suggested that the 14-hydroxy group and the ion-dipole interaction would play important roles in the orientation of the 6-amide group, which might control the affinity between KOR and OX1R.

Essential structure of orexin 1 receptor antagonist YNT-707: Conversion of the 16-cyclopropylmethyl group to the 16-sulfonamide group in d-nor-nalfurafine derivatives.

The five-membered D-ring nalfurafine (d-nor-nalfurafine) derivatives with a 16-sulfonamide group were synthesized. Conversion of the 16-cyclopropylmethyl group to the 16-benzenesulfonamide group in the d-nor-nalfurafine derivatives drastically improved the orexin 1 receptor (OX1R) antagonist activities. The intramolecular hydrogen bond between the 14-hydroxy and the 16-sulfonamide groups may play an important role in increasing the probability that the 6-amide group would be located at the lower side of the C-ring, leading to an active conformation for OX1R. The assay results and the conformational analyses of the 14-OH, 14-H, and 14-dehydrated d-nor-nalfurafine derivatives suggested that the 14- and 16-substituents of the d-nor-nalfurafine derivatives had a greater effect on the affinities for the OX1R than did the 14- and 17-substituents of nalfurafine derivatives.

Synthesis of unnatural morphinan compounds to induce itch-like behaviors in mice: Towards the development of MRGPRX2 selective ligands.

Mas-related G protein-coupled receptor X2 (MRGPRX2) mediates the itch response in neurons and is involved in atopic dermatitis (AD)-associated inflammation and itch. Potent and MRGPRX2-selective ligands are essential to an understanding of the detailed function of the receptor and to develop new therapeutic agents for its related diseases. (+)-TAN-67 (1), the enantiomer of the δ-opioid receptor (DOR) selective ligand (-)-TAN-67 (1), has been reported to activate MRGPRX2, although (+)-1 also interacts with DOR, which prevents investigators from interrogating the function of MRGPRX2. Here, we have succeeded in developing a novel unnatural morphinan compound (+)-2a by a transformation based on the structure of (+)-1, which removes the DOR binding affinity. (+)-2a activated both human MRGPRX2 and the mouse orthologue Mrgprb2 in in vitro experiments and induced itch-like behaviors in mice to the same extent as (+)-1. The (+)-2a-induced itch response in mice was suppressed by administration of the tripeptide QWF, an MRGPRX2/Mrgprb2 antagonist, or the antipruritic drug nalfurafine. Together, (+)-2a serves as a useful tool to elucidate the itch-related function/action of MRGPRX2 and its mouse orthologue Mrgprb2.

Morphinan derivatives with an oxabicyclo[3.2.1]octane structure as dual agonists toward δ and κ opioid receptors.

The κ opioid receptor (KOR) is one of the promising targets to develop analgesics lacking morphine like side effects. To seek a novel KOR agonist we designed 6-amide derivatives with an oxabicyclo[3.2.1]octane structure based on a proposed active conformation of a selective KOR agonist nalfurafine. All the synthesized compounds strongly bound to the KOR and some compound showed KOR selectivities. 6R-Amides were more potent and efficacious KOR agonists than the corresponding 6S-isomers. However, most 6-amide derivatives were partial KOR agonist. Conformational analyses of 6R- and 6S-amide derivatives and nalfurafine well accounted for the difference of KOR agonistic activities between two diastereomers. Surprisingly, the tested N-H amides were full δ opioid receptor (DOR) agonists. Among the tested compounds 7a with benzamide moiety was the most potent dual DOR/KOR agonist. On the other hand, 6S-phenylacetamide 8b was potent full DOR agonist with less efficacious agonist activity for the µ receptor and KOR. 6-Amide derivatives with an oxabicyclo[3.2.1]octane structure were expected to be a promising fundamental skeleton for the dual DOR/KOR agonists and/or selective DOR agonists.

Synthesis and Pharmacological Evaluation of Enantiopure N-Substituted Ortho-c Oxide-Bridged 5-Phenylmorphans.

The design of enantiopure stereoisomers of N-2-phenylcyclopropylmethyl-substituted ortho-c oxide-bridged phenylmorphans, the E and Z isomers of an N-cinnamyl moiety, and N-propyl enantiomers were based on combining the most potent oxide-bridged phenylmorphan (the ortho-c isomer) with the most potent N-substituent that we previously found with a 5-(3-hydroxy)phenylmorphan (i.e., N-2-phenylcyclopropyl methyl moieties, N-cinnamyl, and N-propyl substituents). The synthesis of the eight enantiopure N-2-phenylcyclopropylmethyl ortho-c oxide-bridged phenylmorphans and six additional enantiomers of the N-substituted ortho-c oxide-bridged phenylmorphans (N-E and Z-cinnamyl compounds, and N-propyl compounds) was accomplished. The synthesis started from common intermediates (3R,6aS,11aS)-10-methoxy-1,3,4,5,6,11a-hexahydro-2H-3,6a-methano-benzofuro[2,3-c]azocine (+)-6 and its enantiomer, (3S, 6aR, 11aR)-(-)-6, respectively. The enantiomers of ±-6 were obtained through salt formation with (S)-(+)- and (R)-(-)-p-methylmandelic acid, and the absolute configuration of the (R)-(-)-p-methylmandelate salt of (3S, 6aR, 11aR)-(-)-6 was determined by single-crystal X-ray analysis. The enantiomeric secondary amines were reacted with N-(2-phenylcyclopropyl)methyl derivatives, 2-(E)-cinnamyl bromide, and (Z)-3-phenylacrylic acid. These products led to all of the desired N-derivatives of the ortho-c oxide-bridged phenylmorphans. Their opioid receptor binding affinity was measured. The compounds with MOR affinity < 50 nM were examined for their functional activity in the forskolin-induced cAMP accumulation assay. Only the enantiomer of the N-phenethyl ortho-c oxide-bridged phenylmorphan ((-)-1), and only the (3S,6aR,11aR)-2-(((1S,2S)-2-phenylcyclopropyl)methyl)-1,3,4,5,6,11a-hexahydro-2H-3,6a-methanobenzofuro[2,3-c]azocin-10-ol isomer ((+)-17), and the N-phenylpropyl derivative ((-)-25) had opioid binding affinity < 50 nM. Both (-)-1 and (-)-25 were partial agonists in the cAMP assay, with the former showing high potency and low efficacy, and the latter with lower potency and less efficacy. Most interesting was the N-2-phenylcyclopropylmethyl (3S,6aR,11aR)-2-(1S,2S)-enantiomer ((+)-17). That compound had good MOR binding affinity (Ki = 11.9 nM) and was found to have naltrexone-like potency as a MOR antagonist (IC50 = 6.92 nM).

Mechanistic Characterization of the Pharmacological Profile of HS-731, a Peripherally Acting Opioid Analgesic, at the µ-, δ-, κ-Opioid and Nociceptin Receptors.

Accumulated preclinical and clinical data show that peripheral restricted opioids provide pain relief with reduced side effects. The peripherally acting opioid analgesic HS-731 is a potent dual µ-/δ-opioid receptor (MOR/DOR) full agonist, and a weak, partial agonist at the κ-opioid receptor (KOR). However, its binding mode at the opioid receptors remains elusive. Here, we present a comprehensive in silico evaluation of HS-731 binding at all opioid receptors. We provide insights into dynamic interaction patterns explaining the different binding and activity of HS-731 on the opioid receptors. For this purpose, we conducted docking, performed molecular dynamics (MD) simulations and generated dynamic pharmacophores (dynophores). Our results highlight two residues important for HS-731 recognition at the classical opioid receptors (MOR, DOR and KOR), particular the conserved residue 5.39 (K) and the non-conserved residue 6.58 (MOR: K, DOR: W and KOR: E). Furthermore, we assume a salt bridge between the transmembrane helices (TM) 5 and 6 via K2275.39 and E2976.58 to be responsible for the partial agonism of HS-731 at the KOR. Additionally, we experimentally demonstrated the absence of affinity of HS-731 to the nociceptin/orphanin FQ peptide (NOP) receptor. We consider the morphinan phenol Y1303.33 responsible for this affinity lack. Y1303.33 points deep into the NOP receptor binding pocket preventing HS-731 binding to the orthosteric binding pocket. These findings provide significant structural insights into HS-731 interaction pattern with the opioid receptors that are important for understanding the pharmacology of this peripheral opioid analgesic.

In Silico Prediction of the Toxic Potential of Neuroprotective Bifunctional Molecules Based on Chiral N-Propargyl-1,2-amino Alcohol Derivatives.

N-Propargylamines are useful synthetic scaffolds for the synthesis of bioactive molecules, and in addition, they possess important pharmacological activities. We obtained several neuroprotective molecules, chiral 1,2-amino alcohols and 1,2-diamines, able to reduce by almost 70% the rotenone and oligomycin A-induced damage in SH-SY5Y cells. Furthermore, some molecules assessed also counteracted the toxicity evoked by the Ser/Thr phosphatase inhibitor okadaic acid. Before extrapolating these data to preclinical studies, we analyze the molecules through an in silico prediction system to detect carcinogenicity risk or other toxic effects. In light of these promising results, these molecules may be considered as a lead family of neuroprotective and relatively safe compounds.

Toward Continuous-Flow Hyperpolarisation of Metabolites via Heterogenous Catalysis, Side-Arm-Hydrogenation, and Membrane Dissolution of Parahydrogen.

Side-arm hydrogenation (SAH) by homogeneous catalysis has extended the reach of the parahydrogen enhanced NMR technique to key metabolites such as pyruvate. However, homogeneous hydrogenation requires rapid separation of the dissolved catalyst and purification of the hyperpolarised species with a purity sufficient for safe in-vivo use. An alternate approach is to employ heterogeneous hydrogenation in a continuous-flow reactor, where separation from the solid catalysts is straightforward. Using a TiO2 -nanorod supported Rh catalyst, we demonstrate continuous-flow parahydrogen enhanced NMR by heterogeneous hydrogenation of a model SAH precursor, propargyl acetate, at a flow rate of 1.5 mL/min. Parahydrogen gas was introduced into the flowing solution phase using a novel tube-in-tube membrane dissolution device. Without much optimization, proton NMR signal enhancements of up to 297 (relative to the thermal equilibrium signals) at 9.4 Tesla were shown to be feasible on allyl-acetate at a continuous total yield of 33 %. The results are compared to those obtained with the standard batch-mode technique of parahydrogen bubbling through a suspension of the same catalyst.