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Refereed/Peer-reviewed Discovery of sustainable and benign-by-design drugs to combat emerging health pandemics calls for new analytical technologies to explore the chemical and pharmacological properties of Nature’s unique chemical space. Here, we present a new analytical technology workflow, polypharmacology-labeled molecular networking (PLMN), where merged positive and negative ionization tandem mass spectrometry-based molecular networking is linked with data from polypharmacological high-resolution inhibition profiling for easy and fast identification of individual bioactive constituents in complex extracts. The crude extract of Eremophila rugosa was subjected to PLMN analysis for the identification of antihyperglycemic and antibacterial constituents. Visually easy-interpretable polypharmacology scores and polypharmacology pie charts as well as microfractionation variation scores of each node in the molecular network provided direct information about each constituent’s activity in the seven assays included in this proof-of-concept study. A total of 27 new noncanonical nerylneryl diphosphate-derived diterpenoids were identified. Serrulatane ferulate esters were shown to be associated with antihyperglycemic and antibacterial activities, including some showing synergistic activity with oxacillin in clinically relevant(epidemic) methicillin-resistant Staphylococcus aureus strains and some showing saddle-shaped binding to the active site of proteintyrosine phosphatase 1B. PLMN is scalable in the number and types of assays included and thus holds potential for a paradigm shift toward polypharmacological natural-products-based drug discovery.

Thirteen previously undescribed chromene meroterpenoids, capitachromenic acids A-M (3-6, 7a, 7b, 8a, 8b, 9a, 9b, 10a, 10b, and 11b), were identified from an ethyl acetate extract of Rhododendron capitatum, using dual high-resolution α-glucosidase and PTP1B inhibition profiling in combination with HPLC-PDA-HRMS-SPE-NMR. In addition, one known chromene meroterpenoid, daurichromenic acid (15), and its biosynthetic precursor, grifolic acid (12), two C-methylated flavanones, (2S)-5,7,4'-trihydroxy-8-methylflavanone (1) and farrerol (2), and two triterpenoids, oleanolic acid (14a) and ursolic acid (14b), were identified. New structures were elucidated by extensive 1D and 2D NMR analysis, and absolute configurations of new chromene meroterpenoids were assigned by analysis of their ECD spectra on the basis of the empirical chromane helicity rule and from Rh2(OCOCF3)4-induced ECD spectra by applying the bulkiness rule. Compounds 5, 9a, 9b, 12, and 15 showed α-glucosidase inhibitory activity with IC50 values ranging from 8.0 to 93.5 μM, while compounds 3, 5, 8b, 9a, 9b, 10b, 11b, 12, and 15 showed PTP1B inhibitory activity with IC50 values ranging from 2.5 to 68.1 μM.

Thirteen previously undescribed chromene meroterpenoids, capitachromenic acids A-M (

Background Naloxone, an opioid receptor antagonist, is used as a pharmacological tool to detect tonic endogenous activation of opioid receptors in experimental pain models. We describe a pharmacokinetic model linking naloxone pharmacokinetics to its main metabolite after high-dose naloxone infusion. Methods Eight healthy volunteers received a three-stage stepwise high-dose i.v. naloxone infusion (total dose 3.25 mg kg−1). Naloxone and naloxone-3-glucuronide (N3G) plasma concentrations were sampled from infusion onset to 334 min after infusion discontinuation. Pharmacokinetic analysis was performed using non-linear mixed effect models (NONMEM). The predictive performances of Dowling's and Yassen's models were evaluated, and target-controlled infusion simulations were performed. Results Three- and two-compartment disposition models with linear elimination kinetics described the naloxone and N3G concentration time-courses, respectively. Two covariate models were developed: simple (weight proportional) and complex (with the shallow peripheral volume of distribution linearly increasing with body weight). The median prediction error (MDPE) and wobble for Dowling's model were –32.5% and 33.4%, respectively. For Yassen's model, the MDPE and wobble were 1.2% and 19.9%, respectively. Conclusions A parent–metabolite pharmacokinetic model was developed for naloxone and N3G after high-dose naloxone infusion. No saturable pharmacokinetics were observed. Whereas Dowling's model was inaccurate and over-predicted naloxone concentrations, Yassen's model accurately predicted naloxone pharmacokinetics. The newly developed covariate models may be used for high-dose TCI-naloxone for experimental and clinical practice. Clinical trials registration NCT01992146.

Type 2 diabetes is a chronic metabolic disorder affecting millions of people worldwide, and new drug leads are urgently needed. The dried stem of Dendrobium officinale is used in China to alleviate the symptoms of diabetes, but investigations of its bioactive components have been limited. We have applied a simple, fast, and effective method, combining triple high-resolution profiling of radical scavenging and α-glucosidase and α-amylase inhibition with high-performance liquid chromatography - photodiode-array detection - high-resolution mass spectrometry - solid-phase extraction - nuclear magnetic resonance spectroscopy (HR-bioassay/HPLC-PDA-HRMS-SPE-NMR). Two new compounds, 3,4,4′-trihydroxy-5-methoxybibenzyl (5) and 3,4-dihydroxy-3′,4′,5-trimethoxybibenzyl (8a), along with fourteen known compounds, were identified. Twelve of these were associated with radical scavenging activity, six with α-glucosidase inhibition, and one with α-amylase inhibition. Compound 5, dendrocandin U, and 3,4-dihydroxy-4′,5-dimethoxybibenzyl (12) are reported as α-glucosidase inhibitors for the first time, and 12 is furthermore reported as an α-amylase inhibitor for the first time.

Type 2 diabetes is a chronic multifactorial disease affecting more than 425 million people worldwide, and new selective α-glucosidase inhibitors with fewer side effects are urgently needed. In this study, a crude ethyl acetate extract of Machilus litseifolia was fractionated by solid-phase extraction using C18 cartridges to give a fraction enriched in α-glucosidase inhibitors. Subsequent microfractionation and bioassaying of the eluate by high-performance liquid chromatography (HPLC) using a complementary pentafluorophenyl column allowed construction of a high-resolution α-glucosidase inhibition profile (biochromatogram). This was used to target high-performance liquid chromatography-photodiode array detection-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy (HPLC-PDA-HRMS-SPE-NMR) analysis toward α-glucosidase inhibitors. This led to the identification of 13 dicoumaroylated flavonol rhamnosides, of which seven (8, 10, 12a, 12b, 16, 17, and 18) are reported for the first time, and two lignans, of which one (5) is reported for the first time. IC50 values of isolated compounds toward α-glucosidase range from 5.9 to 35.3 μM, which is 8 to 91 times lower than the IC50 value of 266 μM measured for the reference compound acarbose.

BackgroundMedicinal plants are used to treat various disorders, including diabetes, globally in a range of formulations. While attention has mainly been on the aerial plant parts, there are only a few review studies to date that are focused on the natural constituents present in the plant roots with health benefits. Thus, the present study was performed to review in vivo studies investigating the antidiabetic potential of the natural compounds in plant roots.MethodsWe sorted relevant data in 2001–2019 from scientific databases and search engines, including Web of Knowledge, PubMed, ScienceDirect, Medline, Reaxys, and Google Scholar. The class of phytochemicals, plant families, major compounds, active constituents, effective dosages, type of extracts, time of experiments, and type of diabetic induction were described.ResultsIn our literature review, we found 104 plants with determined antidiabetic activity in their root extracts. The biosynthesis pathways and mechanism of actions of the most frequent class of compounds were also proposed. The results of this review indicated that flavonoids, phenolic compounds, alkaloids, and phytosteroids are the most abundant natural compounds in plant roots with antidiabetic activity. Phytochemicals in plant roots possess different mechanisms of action to control diabetes, including inhibition ofα-amylase andα-glucosidase enzymes, oxidative stress reduction, secretion of insulin, improvement of diabetic retinopathy/nephropathy, slow the starch digestion, and contribution against hyperglycemia.ConclusionThis review concludes that plant roots are a promising source of bioactive compounds which can be explored to develop against diabetes and diabetes-related complications.Graphical abstract

The physiological changes following Roux-en-Y gastric bypass (RYGB) surgery may impact drug release from mechanistically different controlled-release tablets, making generic substitution inappropriate. This study aimed to characterise the pharmacokinetic-pharmacodynamic relationships of oxycodone from a lipid-based and water-swellable controlled-release tablet in RYGB patients. Twenty RYGB patients received 10-mg oral solution oxycodone or 20-mg controlled-release (water-swellable or lipid-based) oxycodone in a three-way, randomised, semiblinded and cross-over study. Blood sampling and pupillary recordings were conducted over a 24-h period. A previously established pharmacokinetic-pharmacodynamic model of these three formulations in healthy volunteers was used in the analysis as a reference model. No differences in absorption kinetics were seen between controlled-release formulations in patients. However, the absorption lag time was 11.5 min in patients vs 14 min in healthy volunteers for controlled-release tablets (P

Oral controlled-release formulations are playing an ever-increasing role in opioid therapy; however, little is known about their influence on the relationship between pharmacokinetics and pharmacodynamics. The study aim was to characterize the pharmacokinetic-pharmacodynamics of two controlled-release tablet formulations and a liquid formulation of oxycodone in healthy, opioid-naïve volunteers, which can serve as a reference for future patient studies. A semi-double-blinded, three-way crossover study was conducted, with fifteen healthy volunteers receiving two differently designed 20 mg monophasic controlled-release oxycodone tablets and 10 mg oral solution oxycodone in a randomized order. Venous plasma concentrations and pupil diameter were determined pre-dose and 0.25, 0.5, 0.75, 1, 1.5, 2, 2.33, 2.66, 3, 3.33, 3.66, 4, 5, 6, 8, 12 and 24 hour post-dose. Oxycodone pharmacokinetics was best described by a two-compartment model with first-order absorption. The controlled-release formulations had an absorption lag of 0.23 hour and a slower absorption rate constant (k aCR = 0.19 hour -1 ) compared to the oral solution (k aSOL = 0.94 hour -1 ). Effects on pupil diameter were delayed relative to plasma (14 minutes half-life) for all formulations and were best described by a proportional E max model. The plasma concentration of oxycodone at half-maximum effect was lower in males (31.1 μg/L) compared to females (52.8 μg/L; P < .001). The absorption profile of controlled-release oxycodone formulations provided a prolonged onset and offset of action compared to oral solution oxycodone. The controlled-release formulations showed no differences in pharmacokinetic and pharmacodynamic parameters suggesting that both may be used interchangeably in human beings with normal gastrointestinal function.

The correct application of bio(iso)steric replacement, a potent tool for the design of optimized compounds, requires the continuous development of new isosters able to respond to specific target requirements. Among carboxylic acid isosters, as the hydroxylated pentatomic heterocyclic systems, the hydroxy-1,2,3-triazole represents one of the most versatile but less investigated. With the purpose to enlarge its bioisosteric application, we report the results of a study devoted to obtain potential biomimetics of the γ-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the central nervous system (CNS). A series of N1- and N2- functionalized 4-hydroxy-1,2,3-triazole analogues of the previous reported GABAAR ligands, including muscimol, 4-PIOL, and 4-PHP has been synthesized and characterized pharmacologically. Furthermore, this study led to development of straightforward chemical strategies directed to decorate the hydroxytriazole core scaffold, opening for further elaborative studies based on this system. The unsubstituted N1- and N2-piperidin-4-yl-4-hydroxy-1,2,3-triazole analogues (3a, 4a) of 4-PIOL and 4-PHP showed weak affinity (high to medium micromolar range), whereas substituting the 5-position of the triazole core with a 2-naphthylmethyl or 3,3-diphenylpropyl led to binding affinities in the low micromolar range. Based on electrostatic analysis and docking studies using a α1β2γ2 GABAAR homology model we were able to rationalize the observed divergence in SAR for the series of N1- and N2- piperidin-4-yl-4-hydroxy-1,2,3-triazole analogues, offering more detailed insight into the orthosteric GABAAR binding site.

The natural red food colorants carmine (E120) and carminic acid are currently produced from scale insects. The access to raw material is limited and current production is sensitive to fluctuation in weather conditions. A cheaper and more stable supply is therefore desirable. Here we present the first proof-of-concept of heterologous microbial production of carminic acid in Aspergillus nidulans by developing a semi-natural biosynthetic pathway. Formation of the tricyclic core of carminic acid is achieved via a two-step process wherein a plant type III polyketide synthase (PKS) forms a non-reduced linear octaketide, which subsequently is folded into the desired flavokermesic acid anthrone (FKA) structure by a cyclase and a aromatase from a bacterial type II PKS system. The formed FKA is oxidized to flavokermesic acid and kermesic acid, catalyzed by endogenous A. nidulans monooxygenases, and further converted to dcII and carminic acid by the Dactylopius coccus C-glucosyltransferase DcUGT2. The establishment of a functional biosynthetic carminic acid pathway in A. nidulans serves as an important step towards industrial-scale production of carminic acid via liquid-state fermentation using a microbial cell factory.

In this paper, quadruple high-resolution α-glucosidase/α-amylase/PTP1B/radical scavenging profiling combined with HPLC-HRMS-SPE-NMR were used for studying the polypharmacological properties of crude root bark extract of Morus alba L. This species is used as an anti-diabetic principle in many traditional treatment systems around the world, and the crude ethyl acetate extract of M. alba root bark was found to inhibit α-glucosidase, α-amylase and protein-tyrosine phosphatase 1B (PTP1B) with IC50 values of 1.70 ± 0.72, 5.16 ± 0.69, and 5.07 ± 0.68 μg/mL as well as showing radical scavenging activity equaling a TEAC value of (3.82 ± 0.14) × 104 mM per gram extract. Subsequent investigation of the crude extract using quadruple high-resolution α-glucosidase/α-amylase/PTP1B/radical scavenging profiling provided a quadruple biochromatogram that allowed direct correlation of the HPLC peaks with one or more of the tested bioactivities. This was used to target subsequent HPLC-HRMS-SPE-NMR analysis towards peaks representing bioactive analytes, and led to identification of a new Diels-Alder adduct named Moracenin E as well as a series of Diels-Alder adducts and isoprenylated flavonoids as potent α-glucosidase and α-amylase inhibitors with IC50 values in the range of 0.60–27.15 μM and 1.22–69.38 μM, respectively. In addition, these compounds and two 2-arylbenzofurans were found to be potent PTP1B inhibitors with IC50 values ranging from 4.04 to 21.67 μM. The high-resolution radical scavenging profile also revealed that almost all of the compounds possess radical scavenging activity. In conclusion the quadruple high-resolution profiling method presented here allowed a detailed profiling of individual constituents in crude root bark extract of M. alba, and the method provides a general tool for detailed mapping of bioactive constituents in polypharmacological herbal remedies.

Six species of Myrcia, commonly known as 'insulin plants' in Brazil, were assessed for their potential as functional foods. Thus, dual high-resolution α-glucosidase/PTP1B inhibition profiling and HPLC-HRMS-SPE-NMR analysis were used to identify the antidiabetic constituents of the crude ethyl acetate extract of M. rubella Cambess. A total of 31 compounds were identified, including seven α-glucosidase inhibitors with IC50 values between 0.6 and 22.4 μM: 4,5-dicaffeoylquinic acid, isoquercitrin, quercetin-3-O-β- d -glucuronide, kaempferol-3-O-(6″-galloyl)-β- d -glucopyranoside, quercetin-3-O-(6″-malonyl)-glucopyranoside, quercetin-3-O-(6″-(E)-feruloyl)-β- d -glucopyranoside, and quercetin-3-O-(2″-(E)-sinapoyl)-glucopyranoside as well as three strong PTP1B inhibitors with IC50 values between 0.4 and 3.1 μM: kaempferol-3-O-α- l -rhamnoside, astragalin, and arjunolic acid. These results show that M. rubella is a potential bifunctional food for management of type 2 diabetes.

Carminic acid, a glucosylated anthraquinone found in scale insects like Dactylopius coccus, has since ancient times been used as a red colorant in various applications. Here we show that a membrane-bound C-glucosyltransferase, isolated from D. coccus and designated DcUGT2, catalyzes the glucosylation of flavokermesic acid and kermesic acid into their respective C-glucosides dcII and carminic acid. DcUGT2 is predicted to be a type I integral endoplasmic reticulum (ER) membrane protein, containing a cleavable N-terminal signal peptide and a C-terminal transmembrane helix that anchors the protein to the ER, followed by a short cytoplasmic tail. DcUGT2 is found to be heavily glycosylated. Truncated DcUGT2 proteins synthesized in yeast indicate the presence of an internal ER-targeting signal. The cleavable N-terminal signal peptide is shown to be essential for the activity of DcUGT2, whereas the transmembrane helix/cytoplasmic domains, although important, are not crucial for its catalytic function., Carminic acid is a widely applied red colorant that is still harvested from insects because its biosynthesis is not fully understood. Here, the authors identify and characterize a membrane-bound C-glucosyltransferase catalyzing the final step during carminic acid biosynthesis.

γ-hydroxybutyric acid (GHB) is a neuroactive substance with specific high-affinity binding sites. To facilitate target identification and ligand optimization, we herein report a comprehensive structure-affinity relationship study for novel ligands targeting these binding sites. A molecular hybridization strategy was used based on the conformationally restricted 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) and the linear GHB analog, trans-4-hydroxycrotonic acid (T-HCA). In general, all structural modifications performed on HOCPCA led to reduced affinity. In contrast, introduction of diaromatic substituents into the 4-position of T-HCA led to high-affinity analogs (medium nanomolar Ki) for the GHB high-affinity binding sites as the most high-affinity analogs reported to date. The SAR data formed the basis for a 3-dimensional pharmacophore model for GHB ligands, which identified molecular features important for high-affinity binding, with high predictive validity. These findings will be valuable in the f...

Objective: The spread of new psychoactive substances (NPS) has expanded rapidly in the last decade. The complexity of the pharmacological effects of NPS challenges the traditional treatment guidelines, and information of the emergence of new arrivals is valuable. Our knowledge on the actual range of recreational drugs used and NPS available in Denmark is limited as identification is possible only when consumers become patients in the healthcare system or through drug seizures. We aimed to detect classical recreational drugs and NPS in the urine of music festival attendees and evaluate if the use of NPS could have been predicted by comparing study data with drug seizure data from the previous year published by European and Danish health authorities.Methods: In a cross-sectional study, 44 urine samples were collected from three urinals at Roskilde Festival 2016—the largest Danish music festival. Two urinals were placed at music stages with late-night concerts, and one urinal was placed at a camp site. Samples were prepared using enzymatic hydrolysis followed by cationic and anionic solid phase extraction, and analysed using ultra performance liquid chromatography-high-resolution time-of-flight mass spectrometry (UPLC-HR-TOF-MS). Data were processed using an in-house library of 467 target substances, including legal and illegal drugs and metabolites. Urine drug-screening immunoassays were also evaluated and results were compared to UPLC-HR-TOF-MS results.Results: In total, 77 drugs, including metabolites, were qualitatively identified in the 44 urine samples. The recreational drugs identified were amphetamine (n = 30), cocaine (n = 44), MDA (n = 40), MDMA (n = 44), THC-COOH (n = 19) and ketamine (n = 17). No NPS were identified. Sample testing using the urine drug-screening immunoassays showed presence of cocaine (n = 27), methamphetamine/MDMA (n = 4), THC (n = 7), “Spice” (n = 7) and methylphenidate (n = 1). These discrepancies might be caused by differences in cut-off values between the analytical methods, limited specificity or cross-reactivity of the urine drug-screening immunoassays compared to UPLC-HR-TOFMS results.Conclusion: Widespread uses of classical recreational drugs were identified in pooled urine samples. The prevalence of NPS was not as comprehensive as expected based on the European and Danish health authorities reports on illegal drugs. Urine drug-screening immunoassays results are advised to be confirmed by chromatographic bioanalysis.

The worldwide increasing incidence of type 2 diabetes has fueled an intensified search for food and herbal remedies with preventive and/or therapeutic properties. Polygonum cuspidatum Siebold & Zucc. (Polygonaceae) is used as a functional food in Japan and South Korea, and it is also a well-known traditional antidiabetic herb used in China. In this study, dual high-resolution α-glucosidase and protein-tyrosine phosphatase 1B (PTP1B) inhibition profiling was used for the identification of individual antidiabetic constituents directly from the crude ethyl acetate extract and fractions of P. cuspidatum. Subsequent preparative-scale HPLC was used to isolate a series of α-glucosidase inhibitors, which after HPLC-HRMS and NMR analysis were identified as procyanidin B2 3,3″-O-digallate (3) and (−)-epicatechin gallate (5) with IC50 values of 0.42 ± 0.02 and 0.48 ± 0.0004 μM, respectively, as well as a series of stilbene analogues with IC50 value in the range from 6.05 ± 0.05 to 116.10 ± 2.04 μM. In addition, (tra...

A hyphenated procedure involving high-performance liquid chromatography, photodiode array detection, high-resolution mass spectrometry, solid-phase extraction, and nuclear magnetic resonance spectroscopy, i.e., HPLC-PDA-HRMS-SPE-NMR, has proven an effective technique for the identification of compounds in complex matrices. Most HPLC-PDA-HRMS-SPE-NMR investigations reported so far have relied on analytical-scale reversed-phase C18 columns for separation. Herein is reported the use of an analytical-scale pentafluorophenyl column as an orthogonal separation method following fractionation of a crude ethyl acetate extract of leaves of Coleonema album on a preparative-scale C18 column. This setup allowed the HPLC-PDA-HRMS-SPE-NMR analysis of 23 coumarins, including six new compounds, 8-O-β-d-glucopyranosyloxy-6-(2,3-dihydroxy-3-methylbut-1-yl)-7-methoxycoumarin (4), (Z)-6-(4-β-d-glucopyranosyloxy-3-methylbut-2-en-1-yl)-7-hydroxycoumarin (6), 6-(4-β-d-glucopyranosyloxy-3-methylbut-1-yl)-7-hydroxycoumarin (8), (Z...

The critical roles played by GABAA receptors as inhibitory regulators of excitation in the central nervous system has been known for many years. Aberrant GABAA receptor function and trafficking deficits have also been associated with several diseases including anxiety, depression, epilepsy, and insomnia. As a consequence, important drug groups such the benzodiazepines, barbiturates, and many general anesthetics, have become established as modulators of GABAA receptor activity. Nevertheless, there is much we do not understand about the roles and mechanisms of GABAA receptors at neural network and systems levels. It is therefore crucial to develop novel technologies and especially chemical entities that can interrogate GABAA receptor function in the nervous system. Here, we describe the chemistry and characterization of a novel set of 4-PIOL and 4-PHP analogues synthesized with the aim of developing a toolkit of drugs that can photo-inactivate GABAA receptors. Most of these new analogues show higher affinities/potencies compared to the respective lead compounds. This is indicative of cavernous areas being present near their binding sites that can be potentially associated with novel receptor interactions. The 4-PHP azide-analogue, 2d, possesses particularly impressive nanomolar affinity/potency, and is an effective UV-inducible photo-inhibitor of GABAA receptors with considerable potential for photo-control of GABAA receptor function in situ.

Medicinal plants have been widely used as (poly)pharmacological remedies and constitute a rich source for antidiabetic drug discovery. In the present study, forty medicinal plant samples collected in China were tested for inhibitory activity against α-glucosidase, α-amylase, and protein-tyrosine phosphatase 1B (PTP1B). Crude ethyl acetate extracts of Dioscorea bulbifera L., Boehmeria nivea Gaudich, Tinospora sagittata Gagnep. and Persicaria bistorta (L.) Samp. showed dual inhibitory activity towards α-glucosidase and PTP1B, and were chosen for further investigation. Subsequent dual high-resolution α-glucosidase/PTP1B profiling or triple high-resolution α-glucosidase/α-amylase/PTP1B profiling combined with HPLC-HRMS and NMR spectroscopy led to the identification of 28 metabolites with one or more bioactivities. Among these, three new phenanthrenes were identified from D. bulbifera, including one new biphenanthrene (10) exhibiting promising dual inhibitory activity towards α-glucosidase and PTP1B with IC50 values of 2.08 ± 0.19 and 3.36 ± 0.25 μM, respectively. Two triterpenoids and one fatty acid from B. nivea and T. sagittata as well as some commercially available fatty acids showed strong PTP1B inhibitory activity with IC50 values in the range of 4.89 ± 0.38 to 53.77 ± 4.20 μM.

Current methods for assessment of cellular uptake of cell-penetrating peptides (CPPs) often rely on detection of fluorophore-labeled CPPs. However, introduction of the fluorescent probe often confers changed physicochemical properties, so that the fluorophore-CPP conjugate may exhibit cytotoxic effects and membrane damage not exerted by the native CPP. In the present study, introduction of fluorine probes was investigated as an alternative to fluorophore labeling of a CPP, since this only confers minor changes to its overall physicochemical properties. The high sensitivity of 19 F NMR spectroscopy and the absence of background signals from naturally occurring fluorine enabled detection of internalized CPP. Also, degradation of fluorine-labeled peptides during exposure to Caco-2 cells could be followed by using 19 F NMR spectroscopy. In total, five fluorinated analogues of the model CPP penetratin were synthesized by using commercially available fluorinated amino acids as labels, including one analogue also carrying an N-terminal fluorophore. The apparent cellular uptake was considerably higher for the fluorophore-penetratin conjugate indicating that the fluorophore moiety promoted uptake of the peptide. The use of 19 F NMR spectroscopy enabled monitoring of the fate of the CPPs over time by establishing molar balances, and by verifying CPP integrity upon uptake. Thus, the NMR-based method offers several advantages over currently widespread methods relying on fluorescence detection. The present findings provide guidelines for improved labeling strategies for CPPs, thereby expanding the repertoire of analytical techniques available for studying degradation and uptake of CPPs.

Gabazine, a γ-aminobutyric acid type A (GABAA) receptor antagonist, has previously been reported to inhibit the binding of [3H]NCS-382, a representative ligand of the high-affinity binding site for the neuroactive substance γ-hydroxybutyric acid (GHB). We herein report a study on the structural determinants of gabazine for binding to (i) the orthosteric binding site of the GABAA receptor and (ii) the high-affinity GHB binding site. Expanding the structural diversity of available ligands for the high-affinity GHB binding sites, this study identified 2-(imidazo[1,2-b]pyridazin-2-yl)acetic acid as a novel ligand-scaffold leading to analogues with relatively high affinity (Ki 0.19–2.19 μM) and >50 times selectivity for the [3H]NCS-382 over [3H]muscimol binding sites. These results highlight that gabazine interacts with the high-affinity GHB and orthosteric GABAA receptor binding sites differently and that distinct analogues can be generated to select between them. To facilitate further in vivo studies, a prom...

Aim: High-dose administration of the μ-opioid receptor inverse agonist naloxone (NX), has previously been demonstrated to reinstate nocifensive behavior in the late phase of inflammatory injuries. However, no current analytical methods can provide pharmacokinetic insight into the pharmacodynamic response of high-dose administration of NX. Materials & methods: Based on protein precipitation using 50 μl human plasma, NX and naloxone-β-d-glucuronide (NXG) was analysed by UHPLC–MS/MS with 6 min cycle time. Results: A method for quantification of high-dose administered NX and NXG was developed and validated with intra- and interday precision and accuracy within ≤8.5% relative standard deviation (RSD) and -1.2–5.5% relative error (RE) for NX and ≤9.6% RSD and 0.6–6.5% RE for NXG. The method show excellent internal standard corrected matrix effects. Conclusion: A rapid UHPLC–MS/MS method was developed for quantification of NX and NXG in human plasma within 10–4000 ng/ml.

Given the heterogeneity within the γ-aminobutyric acid (GABA) receptor and transporter families, a detailed insight into the pharmacology is still relatively sparse. To enable studies of the physiological roles governed by specific receptor and transporter subtypes, a series of GABA analogues comprising five-membered nitrogen- and sulfur-containing heterocycles as amine bioisosteres were synthesized and pharmacologically characterized at native and selected recombinant GABAA receptors and GABA transporters. The dihydrothiazole and imidazoline analogues, 5-7, displayed moderate GAT activities and GABAA receptor binding affinities in the mid-nanomolar range ( Ki, 90-450 nM). Moreover, they exhibited full and equipotent agonist activity compared to GABA at GABAA-αβγ receptors but somewhat lower potency as partial agonists at the GABAA-ρ1 receptor. Stereoselectivity was observed for compounds 4 and 7 for the GABAA-αβγ receptors but not the GABAA-ρ1 receptor. This study illustrates how subtle differences in these novel amino GABA bioisosteres result in diverse pharmacological profiles in terms of selectivity and efficacy.

With the identification of novel antibiotics from nature being pivotal in the fight against human pathogenic bacteria, there is an urgent need for effective methodologies for expedited screening of crude extracts. Here we report the development and validation of a simple and dye-free antimicrobial assay in 96-well microplate format, for both determination of IC50 values and high-resolution inhibition profiling to allow pin-pointing of bioactive constituents directly from crude extracts. While commonly used antimicrobial assays visualize cell viability using dyes, the developed and validated assay conveniently uses OD600 measurements directly on the fermentation broth. The assay was validated with an investigation of the inhibitory activity of DMSO against Staphylococcus aureus, temperature robustness, interference by coloured crude extracts as well as inter-day reproducibility. The potential for high-resolution S. aureus growth inhibition profiling was evaluated on a crude extract of an inactive Alternaria sp., spiked with ciprofloxacin.

Crude chloroform, ethanol and acetone extracts of nineteen seaweed species were screened for their antioxidant and α-glucosidase inhibitory activity. Samples showing more than 60% α-glucosidase inhibitory activity, at a concentration of 1 mg/ml, were furthermore investigated using high-resolution α-glucosidase inhibition profiling combined with high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy (HR-bioassay/HPLC-HRMS-SPE-NMR). The results showed Ascophyllum nodosum and Fucus vesicolosus to be rich in antioxidants, equaling a Trolox equivalent antioxidant capacity of 135 and 108 mM Troloxmg(-1) extract, respectively. HR-bioassay/HPLC-HRMS-SPE-NMR showed the α-glucosidase inhibitory activity of A. nodosum, F. vesoculosus, Laminaria digitata, Laminaria japonica and Undaria pinnatifida to be caused by phlorotannins as well as fatty acids - with oleic acid, linoleic acid and eicosapentaenoic acid being the most potent with IC50 values of 0.069, 0.075 and 0.10 mM, respectively, and showing a mixed-type inhibition mode.

Type 2 diabetes (T2D) constituted 90% of the global 387 million diabetes cases in 2014. The enzyme protein-tyrosine phosphatase 1B (PTP1B) has been recognized as a therapeutic target for treatment of T2D and its adverse complications. With the aim of accelerating the investigation of complex natural sources, such as crude plant extracts, for potential PTP1B inhibitors, we have developed a bio-analytical platform combining high-resolution PTP1B inhibition profiling and high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy, i.e., HR-bioassay/HPLC-HRMS-SPE-NMR. Human recombinant PTP1B enzyme was used for the microplate-based PTP1B inhibition assay, which was optimized for pH and substrate concentration to be compatible with rate measurements within the 10 min incubation time. Subsequently, analytical-scale HPLC-based microfractionation followed by colorimetric microplate-based PTP1B bioassaying enabled construction of a high-resolution inhibition profile corresponding to the HPLC profile. The high-resolution PTP1B inhibition profiling was validated using an artificial mixture of known PTP1B inhibitors and non-inhibiting compounds as negative controls. Finally, a proof-of-concept study with a real sample was performed using crude ethyl acetate extract of the phytochemically hitherto unexplored plant Eremophila lucida. This led to the identification of the first viscidane type diterpene, i.e., 5-hydroxyviscida-3,14-dien-20-oic acid (9) as PTP1B inhibitor with an IC50 value of 42.0 ± 5.9 μM. In addition, a series of flavonoids, i.e., luteolin (1), dinatin (3a), tricin (3b), 3,6-dimethoxyapigenin (4), jaceidin (5), and cirsimaritin (6) as well as a cembrene diterpene, (3Z, 7E, 11Z)-15-hydroxycembra-3,7,11-trien-19-oic acid (8), were also identified for the first time from E. lucida.

Worldwide, 463 million people are affected by diabetes of which the majority is diagnosed with Type 2 Diabetes (T2D). T2D can ultimately lead to retinopathy, nephropathy, nerve damage, and amputation of the lower extremities. α-Glucosidase, responsible for converting starch to monosaccharides, is a key therapeutic target for the management of T2D. However, due to substantial side effects of currently marketed drugs, there is an urgent need for the discovery of new α-glucosidase inhibitors. In our ongoing efforts to identify novel α-glucosidase inhibitors from Nature, we are investigating the potential of endophytic filamentous fungi as sustainable sources of hits and/or leads for future antihyperglycemic drugs. Here we report one previously unreported xanthone (5) and two known xanthones (7 and 11) as α-glucosidase inhibitors, isolated from an endophytic Penicillium canescens, recovered from fruits of Juniperus polycarpos. The three xanthones 5, 7, and 11 showed inhibitory activities against α-glucosidase with IC50 values of 38.80 ± 1.01 μM, 32.32 ± 1.01 μM, and 75.20 ± 1.02 μM, respectively. Further pharmacological characterization revealed a mixed-mode inhibition for 5, a competitive inhibition for 7, while 11 acted as a non-competitive inhibitor.

Plants are well-recognized sources of inhibitors for -glucosidase - a key target enzyme for management of type 2 diabetes. Recently, two advanced bioactivity-profiling techniques, i.e., ligand fishing and high-resolution inhibition profiling, have shown great promises for accelerating identification of -glucosidase inhibitors from complex plant extracts. Non-specific affinities and non-specific inhibitions are major sources of false positive hits from ligand fishing and highresolution inhibition profiling, respectively. In an attempt to minimize such false positive hits, we describe a new screening approach based on ligand fishing and high-resolution inhibition profiling for detection of high-affinity ligands and assessment of inhibitory activity, respectively. The complementary nature of ligand fishing and high-resolution inhibition profiling was explored to Manuscript for Talanta 3 identify-glucosidase inhibitory ligands from a complex mixture, and proof-of-concept was demonstrated with crude ethyl acetate extract of Ginkgo biloba. In addition to magnetic beads with a 3-carbon aliphatic linker, -glucosidase was immobilized on magnetic beads with a 21-carbon aliphatic linker; and the two different types of magnetic beads were compared for their hydrolytic activity and fishing efficiency.

Recent discovery of potent antibacterial antisense PNA-peptide conjugates encouraged development of a fast and efficient synthesis protocol that facilitates structure-activity studies. The use of an Fmoc/Boc protection scheme for both PNA monomers and amino acid building blocks in combination with microwave-assisted solid-phase synthesis proved to be a convenient procedure for continuous assembly of antisense PNA-peptide conjugates. A validated antisense PNA oligomer (CTCATACTCT; targeting mRNA of the acpP gene) was linked to N-terminally modified drosocin (i.e., RXR-PRPYSPRPTSHPRPIRV; X = aminohexanoic acid) or to a truncated Pip1 peptide (i.e., RXRRXR-IKILFQNRRMKWKK; X = aminohexanoic acid), and determination of the antibacterial effects of the resulting conjugates allowed assessment of the influence of different linkers as well as differences between the L- and D-forms of the peptides. The drosocin-derived compound without a linker moiety exhibited highest antibacterial activity against both wild-type Escherichia coli and Klebsiella pneumoniae (MICs in the range 2–4 μg/mL ∼ 0.3–0.7 μM), while analogues displaying an ethylene glycol (eg1) moiety or a polar maleimide linker also possessed activity toward wild-type K. pneumoniae (MICs of 4–8 μg/mL ∼ 0.6–1.3 μM). Against two colistin-resistant E. coli strains the linker-deficient compound proved most potent (with MICs in the range 2–4 μg/mL ∼ 0.3–0.7 μM). The truncated all-L Pip1 peptide had moderate inherent activity against E. coli, and this was unaltered or reduced upon conjugation to the antisense PNA oligomer. By contrast, this peptide was 8-fold less potent against K. pneumoniae, but in this case some PNA-peptide conjugates exhibited potent antisense activity (MICs of 2–8 μg/mL ∼ 0.3–1.2 μM). Most interestingly, the antibacterial activity of the D-form peptide itself was 2- to 16-fold higher than that of the L-form, even for the colistin- and tigecycline-resistant E. coli strains (MIC of 1–2 μg/mL ∼ 0.25–0.5 μM). Low activity was found for conjugates with a two-mismatch PNA sequence corroborating an antisense mode of action. Conjugates containing a D-form peptide were also significantly less active. In conclusion, we have designed and synthesized antisense PNA-drosocin conjugates with potent antibacterial activity against colistin- and tigecycline-resistant E. coli and K. pneumonia without concomitant hemolytic properties. In addition, a truncated D-form of Pip1 was identified as a peptide exhibiting potent activity against both wild-type and multidrug-resistant E. coli, P. aeruginosa, and A. baumannii (MICs within the range 1–4 μg/mL ∼ 0.25–1 μM) as well as toward wild-type Staphylococcus aureus (MIC of 2–4 μg/mL ∼ 0.5–1.0 μM).

Protein tyrosine phosphatase 1B (PTP1B) is an intracellular enzyme responsible for deactivation of the insulin receptor, and consequently acts as a negative regulator of insulin signal transduction. In recent years, PTP1B has become an important target for controlling insulin resistance and type 2 diabetes. In the present study, the ethyl acetate extract of leaves of Miconia albicans (IC50 = 4.92 &micro, g/mL) was assessed by high-resolution PTP1B inhibition profiling combined with HPLC-HRMS-SPE-NMR for identification of antidiabetic compounds. This disclosed eleven PTP1B inhibitors, including five polyphenolics: 1-O-(E)-caffeoyl-4,6-di-O-galloyl-&beta, d-glucopyranose (2), myricetin 3-O-&alpha, l-rhamnopyranoside (3), quercetin 3-O-(2&Prime, galloyl)-&alpha, l-rhamnopyranoside (5), mearnsetin 3-O-&alpha, l-rhamnopyranoside (6), and kaempferol 3-O-&alpha, l-arabinopyranoside (8) as well as eight triterpenoids: maslinic acid (13), 3-epi-sumaresinolic acid (14), sumaresinolic acid (15), 3-O-cis-p-coumaroyl maslinic acid (16), 3-O-trans-p-coumaroyl maslinic acid (17), 3-O-trans-p-coumaroyl 2&alpha, hydroxydulcioic acid (18), oleanolic acid (19), and ursolic acid (20). These results support the use of M. albicans as a traditional medicine with antidiabetic properties and its potential as a source of PTP1B inhibitors.

In our ongoing efforts of finding natural fungicides to fight food and feed spoilage during production and storage, the antifungal potential of Ghanaian Uvaria chamae P. Beauv. was investigated, with emphasis on plant metabolites targeting the fungal plasma membrane (PM) H + -ATPase. Ethyl acetate extract of U. chamae was subjected to high-resolution fungal PM H + -ATPase inhibition screening followed by structural elucidation by high-performance liquid chromatography–high-resolution mass spectrometry–solid-phase extraction–nuclear magnetic resonance spectroscopy (HPLC–HRMS–SPE–NMR). This led to identification of a series of uncommon o -hydroxybenzylated flavanones and chalcones, i.e., chamanetin ( 8 ), isochamanetin ( 9 ), isouvaretin ( 10 ), uvaretin ( 11 ), dichamanetin ( 12 ), and diuvaretin ( 15 ). Preparative-scale isolation of the active metabolites allowed determination of IC 50 values for inhibition of the PM H + -ATPase, and growth inhibition of Saccharomyces cerevisiae and Candida albicans . These revealed a strong correlation between o -hydroxybenzyl substituents and PM H + -ATPase activity, with dichamanetin being the most potent compound, but showing moderate activity in the fungal growth inhibition assays.

Type 2 diabetes is a metabolic disorder affecting millions of people worldwide, and new drug leads or functional foods containing selective α-glucosidase inhibitors are needed. Crude extract of 24 plants were assessed for α-glucosidase inhibitory activity. Methanol extracts of Cinnamomum zeylanicum bark, Rheum rhabarbarum peel, and Rheum palmatum root and ethyl acetate extracts of C. zeylanicum bark, Allium ascalonicum peel, and R. palmatum root showed IC50 values below 20 μg/mL. Subsequently, high-resolution α-glucosidase profiling was used in combination with high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy for identification of metabolites responsible for the α-glucosidase inhibitory activity. Quercetin (1) and its dimer (2), trimer (3), and tetramer (4) were identified as main α-glucosidase inhibitors in A. ascalonicum peel, whereas (E)-piceatannol 3'-O-β-D-glucopyranoside (5), (E)-rhapontigenin 3'-O-β-D-glucopyranoside (6), (E)-piceatannol (8), and emodin (12) were identified as main α-glucosidase inhibitors in R. palmatum root.

The crude methanol extract of Pueraria lobata was investigated by dual high-resolution α-glucosidase inhibition and radical scavenging profiling combined with hyphenated HPLC-HRMS-SPE-NMR. Direct analysis of the crude extract without preceding purification was facilitated by combining chromatograms from two analytical-scale HPLC separations of 120 and 600 μg on-column, respectively. High-resolution α-glucosidase and radical scavenging profiles were obtained after microfractionation of the eluate in 96-well microplates. This allowed full bioactivity profiling of individual peaks in the HPLC chromatogram of the crude methanol extract. Subsequent HPLC-HRMS-SPE-NMR analysis allowed identification of 21 known compounds in addition to two new compounds, i.e., 3'-methoxydaidzein 8-C-[α-D-apiofuranosyl-(1→6)]-β-D-glucopyranoside and 6″-O-malonyl-3'-methoxydaidzin, as well as an unstable compound tentatively identified as 3'-de-O-methylpuerariafuran.

The chemical composition of the scale insect Dactylopius coccus was analyzed with the aim to discover newpossible intermediates in the biosynthesis of carminic acid. UPLC-DAD/HRMS analyses of fresh and dried insectsresulted in the identification of three novel carminic acid analogues and the verification of several previouslydescribed intermediates. Structural elucidation revealed that the three novel compounds were desoxyerythrolaccin-O-glucosyl (DE-O-Glcp), 5,6-didehydroxyerythrolaccin 3-O-β-D-glucopyranoside (DDE-3-O-Glcp),and flavokermesic acid anthrone (FKA). The finding of FKA in D. coccus provides solid evidence of a polyketide,rather than a shikimate, origin of coccid pigments. Based on the newly identified compounds, we present adetailed biosynthetic scheme that accounts for the formation of carminic acid (CA) in D. coccus and all describedcoccid pigments which share a flavokermesic acid (FK) core. Detection of coccid pigment intermediates inmembers of the Planococcus (mealybugs) and Pseudaulacaspis genera shows that the ability to form these pigmentsis taxonomically more widely spread than previously documented. The shared core-FK-biosyntheticpathway and wider taxonomic distribution suggests a common evolutionary origin for the trait in all coccid dyeproducing insect species.

Carminic acid is a C-glucosylated octaketide anthraquinone and the main constituent of the natural dye carmine (E120), possessing unique coloring, stability, and solubility properties. Despite being used since ancient times, longstanding efforts to elucidate its route of biosynthesis have been unsuccessful. Herein, a novel combination of enzymes derived from a plant (Aloe arborescens, Aa), a bacterium (Streptomyces sp. R1128, St), and an insect (Dactylopius coccus, Dc) that allows for the biosynthesis of the C-glucosylated anthraquinone, dcII, a precursor for carminic acid, is reported. The pathway, which consists of AaOKS, StZhuI, StZhuJ, and DcUGT2, presents an alternative biosynthetic approach for the production of polyketides by using a type III polyketide synthase (PKS) and tailoring enzymes originating from a type II PKS system. The current study showcases the power of using transient expression in Nicotiana benthamiana for efficient and rapid identification of functional biosynthetic pathways, including both soluble and membrane-bound enzymes.

A hyphenated procedure involving high-performance liquid chromatography, photodiode array detection, high-resolution mass spectrometry, solid-phase extraction, and nuclear magnetic resonance spectroscopy, i.e., HPLC-PDA-HRMS-SPE-NMR, has proven an effective technique for the identification of compounds in complex matrices. Most HPLC-PDA-HRMS-SPE-NMR investigations reported so far have relied on analytical-scale reversed-phase C