Oxetane Ring Formation in Taxol Biosynthesis Is Catalyzed by a Bifunctional Cytochrome P450 Enzyme.

Taxol is a potent drug used in various cancer treatments. Its complex structure has prompted extensive research into its biosynthesis. However, certain critical steps, such as the formation of the oxetane ring, which is essential for its activity, have remained unclear. Previous proposals suggested that oxetane formation follows the acetylation of taxadien-5α-ol. Here, we proposed that the oxetane ring is formed by cytochrome P450-mediated oxidation events that occur prior to C5 acetylation. To test this hypothesis, we analyzed the genomic and transcriptomic information for Taxus species to identify cytochrome P450 candidates and employed two independent systems, yeast (Saccharomyces cerevisiae) and plant (Nicotiana benthamiana), for their characterization. We revealed that a single enzyme, CYP725A4, catalyzes two successive epoxidation events, leading to the formation of the oxetane ring. We further showed that both taxa-4(5)-11(12)-diene (endotaxadiene) and taxa-4(20)-11(12)-diene (exotaxadiene) are precursors to the key intermediate, taxologenic oxetane, indicating the potential existence of multiple routes in the Taxol pathway. Thus, we unveiled a long-elusive step in Taxol biosynthesis.

Polypharmacology-Labeled Molecular Networking: An Analytical Technology Workflow for Accelerated Identification of Multiple Bioactive Constituents in Complex Extracts.

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 non-canonical 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 protein-tyrosine 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.

Investigation of the Mechanism and Chemistry Underlying Staphylococcus aureus' Ability to Inhibit Pseudomonas aeruginosa Growth In Vitro.

Pseudomonas aeruginosa inhibits or eradicates Staphylococcus aureus in most in vitro settings. Nonetheless, P. aeruginosa and S. aureus are commonly isolated from chronically infected, nonhealing wounds and lungs of people with cystic fibrosis (CF). Therefore, we hypothesized that S. aureus could protect itself from P. aeruginosa through glucose-derived metabolites, such as small organic acids, preventing it from being eradicated. This in vitro study demonstrated that S. aureus populations, in the presence of glucose, secrete one or more substances that efficiently eradicate P. aeruginosa in a concentration-dependent manner. These substances had a molecular mass lower than three kDa, were hydrophilic, heat- and proteinase-resistant, and demonstrated a pH-dependent effect. Nuclear magnetic resonance analysis identified acetoin, acetic acid, and oligopeptides or cyclic peptides in glucose-grown S. aureus supernatants. All the tested wild-type and clinical S. aureus strain inhibited P. aeruginosa growth. Thus, we proposed a model in which a cocktail of these compounds, produced by established S. aureus populations in glucose presence, facilitated these two species' coexistence in chronic infections. IMPORTANCE Chronic infections affect a growing part of the population and are associated with high societal and personal costs. Multiple bacterial species are often present in these infections, and multispecies infections are considered more severe than single-species infections. Staphylococcus aureus and Pseudomonas aeruginosa often coexist in chronic infections. However, the interactions between these two species and their coexistence in chronic infections are not fully understood. By exploring in vitro interactions, we found a novel S. aureus-mediated inhibition of P. aeruginosa, and we suggested a model of the coexistence of the two species in chronic infections. With this study, we enhanced our understanding of the pathogenesis of chronic multispecies infections, which is crucial to paving the way for developing improved treatment strategies.

Tripterygium wilfordii cytochrome P450s catalyze the methyl shift and epoxidations in the biosynthesis of triptonide.

The diterpenoid triepoxides triptolide and triptonide from Tripterygium wilfordii (thunder god wine) exhibit unique bioactivities with potential uses in disease treatment and as a non-hormonal male contraceptives. Here, we show that cytochrome P450s (CYPs) from the CYP71BE subfamily catalyze an unprecedented 18(4→3) methyl shift required for biosynthesis of the abeo-abietane core structure present in diterpenoid triepoxides and in several other plant diterpenoids. In combination with two CYPs of the CYP82D subfamily, four CYPs from T. wilfordii are shown to constitute the minimal set of biosynthetic genes that enables triptonide biosynthesis using Nicotiana benthamiana and Saccharomyces cerevisiae as heterologous hosts. In addition, co-expression of a specific T. wilfordii cytochrome b5 (Twcytb5-A) increases triptonide output more than 9-fold in S. cerevisiae and affords isolation and structure elucidation by NMR spectroscopic analyses of 18 diterpenoids, providing insights into the biosynthesis of diterpenoid triepoxides. Our findings pave the way for diterpenoid triepoxide production via fermentation.

Biodiscoveries within the Australian plant genus Eremophila based on international and interdisciplinary collaboration: results and perspectives on outstanding ethical dilemmas.

In a cross-continental research initiative, including researchers working in Australia and Denmark, and based on joint external funding by a 3-year grant from the Novo Nordisk Foundation, we have used DNA sequencing, extensive chemical profiling and molecular networking analyses across the entire Eremophila genus to provide new knowledge on the presence of natural products and their bioactivities using polypharmocological screens. Sesquiterpenoids, diterpenoids and dimers of branched-chain fatty acids with previously unknown chemical structures were identified. The collection of plant material from the Eremophila genus was carried out according to a 'bioprospecting agreement' with the Government of Western Australia. We recognize that several Eremophila species hold immense cultural significance to Australia's First Peoples. In spite of our best intentions to ensure that new knowledge gained about the genus Eremophila and any potential future benefits are shared in an equitable manner, in accordance with the Nagoya Protocol, we encounter serious dilemmas and potential conflicts in making benefit sharing with Australia's First Peoples a reality.

An inverse association between plasma benzoxazinoid metabolites and PSA after rye intake in men with prostate cancer revealed with a new method.

Prostate cancer (PC) is a common cancer among men, and preventive strategies are warranted. Benzoxazinoids (BXs) in rye have shown potential against PC in vitro but human studies are lacking. The aim was to establish a quantitative method for analysis of BXs and investigate their plasma levels after a whole grain/bran rye vs refined wheat intervention, as well as exploring their association with PSA, in men with PC. A quantitative method for analysis of 22 BXs, including novel metabolites identified by mass spectrometry and NMR, was established, and applied to plasma samples from a randomized crossover study where patients with indolent PC (n = 17) consumed 485 g whole grain rye/rye bran or fiber supplemented refined wheat daily for 6 wk. Most BXs were significantly higher in plasma after rye (0.3-19.4 nmol/L in plasma) vs. refined wheat (0.05-2.9 nmol/L) intake. HBOA-glc, 2-HHPAA, HBOA-glcA, 2-HPAA-glcA were inversely correlated to PSA in plasma (p < 0.04). To conclude, BXs in plasma, including metabolites not previously analyzed, were quantified. BX metabolites were significantly higher after rye vs refined wheat consumption. Four BX-related metabolites were inversely associated with PSA, which merits further investigation.

Ancient Biosyntheses in an Oil Crop: Glucosinolate Profiles in Limnanthes alba and Its Relatives (Limnanthaceae, Brassicales).

The glucosinolate (GSL) profiles of four Limnanthaceae species, including the oil crop Limnanthes alba (meadowfoam), were investigated by an ultrahigh-performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UHPLC-QToF-MS/MS) analysis of desulfoGSLs after desulfation of native GSLs, supplemented by NMR of desulfated 2-hydroxy-2-methylpropylGSL and 3-methoxybenzylGSL. Leaves, roots, and seeds were investigated, providing an overview of biosynthetic capabilities in the genera Floerkea and Limnanthes. Methoxyl groups on benzylGSLs were in meta but not para positions; two 3,5-disubstituted benzylGSLs are tentatively proposed. 2-Hydroxy-2-methylpropylGSL was accompanied by an isomer that was not a previously reported GSL. The combined GSL profile of the family included GSLs derived from valine, leucine, isoleucine, phenylalanine, and tyrosine, and possibly methionine and tryptophan. Substituted indole GSLs and GSLs derived from chain-elongated amino acids or alanine were searched for but not detected. Hypothetic glycosides of GSLs were detected at low levels. Based on biochemical interpretation, we suggest biosynthetic schemes and gene families (CYP79C, GSOH) relevant for tailoring GSL profiles in Limnanthes crops.

Reversal of ABCG2/BCRP-Mediated Multidrug Resistance by 5,3',5'-Trihydroxy-3,6,7,4'-Tetramethoxyflavone Isolated from the Australian Desert Plant Eremophila galeata Chinnock.

Multidrug resistance (MDR) is a major challenge in cancer treatment, and the breast cancer resistance protein (BCRP) is an important target in the search for new MDR-reversing drugs. With the aim of discovering new potential BCRP inhibitors, the crude extract of leaves of Eremophila galeata, a plant endemic to Australia, was investigated for inhibitory activity of parental (HT29par) as well as BCRP-overexpressing HT29 colon cancer cells resistant to the chemotherapeutic SN-38 (i.e., HT29SN38 cells). This identified a fraction, eluted with 40% acetonitrile on a solid-phase extraction column, which showed weak growth-inhibitory activity on HT29SN38 cells when administered alone, but exhibited concentration-dependent growth inhibition when administered in combination with SN-38. The major constituent in this fraction was isolated and found to be 5,3',5'-trihydroxy-3,6,7,4'-tetramethoxyflavone (2), which at a concentration of 25 µg/mL potentiated the growth-inhibitory activity of SN-38 to a degree comparable to that of the known BCRP inhibitor Ko143 at 1 µM. A dye accumulation experiment suggested that 2 inhibits BCRP, and docking studies showed that 2 binds to the same BCRP site as SN-38. These results indicate that 2 acts synergistically with SN-38, with 2 being a BCRP efflux pump inhibitor while SN-38 inhibits topoisomerase-1.

4-Benzyloxylonchocarpin and Muracatanes A-C from Ranunculus muricatus L. and Their Biological Effects.

Ranunculus muricatus L. is a spiny fruit buttercup that is used in various traditional medicinal systems. In the current investigation of R. muricatus, the new chalcone 4-benzyloxylonchocarpin (1), the new anthraquinone muracatanes A (2), the new-to-nature anthraquinone muracatanes B (3), and the new naphthalene analog muracatanes C (4) were isolated, in addition to the three previously reported compounds, 4-methoxylonchocarpin (5), ß-sitosterol (6), and ß-sitosterol ß-D-glucopyranoside (7). Their structures were elucidated using 1D (1H and 13C) and 2D (COSY, HSQC, and HMBC) NMR spectroscopy and HR-ESI-MS. Chalcone 1 showed potent acetylcholinesterase inhibitory effects with Ki of 5.39 µM and Ki' of 3.54 µM, but none of the isolated compounds showed inhibitory activity towards butyrylcholinesterase. Anthraquinone 3 illustrated α-glucosidase inhibitory effects with IC50-values of 164.46 ± 83.04 µM. Compound 5 displayed moderate cytotoxic activity towards ovarian carcinoma (A2780, IC50 = 25.4 µM), colorectal adenocarcinoma (HT29, IC50 = 20.2 µM), breast cancer (MCF7, IC50 = 23.7 µM), and thyroid carcinoma (SW1736, IC50 = 26.2 µM) while it was inactive towards pharynx carcinoma (FaDu: IC50 > 30 µM).

Structure Elucidation and Absolute Configuration Determination of Nortriterpenoids from Picramnia glazioviana.

In this study, HPLC-PDA-HRMS-SPE-NMR data were used for initial analysis of the CH2Cl2 fraction of an EtOH extract of the leaves of Picramnia glazioviana. The HRMS, UV, and NMR data obtained from the HPLC-PDA-HRMS-SPE-NMR analysis were used to direct semipreparative HPLC isolation toward nortriterpenoids, which resulted in the isolation of 18 new and highly oxygenated nortriterpenoids (1-3, 5-10, 12-19, and 21), named picravianes C-T. Their structures were determined on the basis of analysis of UV, HRMS, and 2D NMR spectroscopic data, including determination of the relative configuration on the basis of coupling pattern analysis and nuclear Overhauser effect correlations. The absolute configurations of compounds 7, 9, 10, 14, 15, 17, 18, 19, and 21 were assigned using electronic circular dichroism data, and the cytotoxicity of compounds 6, 10, 14, 16, 17, 18, 19, and 21 was evaluated against MDA-MB-231 triple-negative breast cancer, SKBR-3 Her2-overexpressing breast cancer, and A549 lung cancer cells lines. The isolated compounds contain a hitherto undescribed modification of the terminal backbone and/or E-ring, and a possible biosynthetic pathway for their formation is proposed.

Evaluation of the antidiabetic potential of Psidium guajava L. (Myrtaceae) using assays for α-glucosidase, α-amylase, muscle glucose uptake, liver glucose production, and triglyceride accumulation in adipocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Psidium guajava L. (Myrtaceae) leaves are used as an herbal antidiabetic remedy in several parts of the world. On Madagascar, both the bark and leaves are used for treatment of diabetes. MATERIALS AND METHODS: Dilution series of ethanolic extracts of P. guajava leaves and bark were used for determining inhibitory activities against yeast α-glucosidase and porcine α-amylase. Skeletal muscle glucose uptake was measured using 2-deoxy-D-(1-3H)-glucose in murine C2C12 skeletal muscle cells. Hepatic glucose-6-phosphatase activity in rat hepatoma H4IIE cells and triglyceride accumulation in murine 3T3-L1 adipocyte-like cells were assessed using Wako AutoKit Glucose assays and AdipoRed reagent, respectively. Cells were incubated for 18 h with the maximal non-toxic concentrations of the plant extracts determined by the lactate dehydrogenase cytotoxicity assay. RESULTS: Ethanolic extracts of P. guajava leaf and bark inhibited α-glucosidase with IC50 values of 1.0 ± 0.3 and 0.5 ± 0.01 µg/mL, respectively. In the α-amylase inhibition assay, the ethanolic extract of bark of P. guajava showed an IC50 value of 10.6 ± 0.4 µg/mL. None of the extracts were able to reduce glucose-6-phosphatase activity in rat hepatoma H4IIE cells. In contrast, P. guajava leaf extract significantly increased 2-deoxy-D-[1-3H]-glucose uptake in C2C12 muscle cells (161.4 ± 10.1%, p = 0.0015) in comparison to the dimethyl sulfoxide (DMSO) vehicle control, as did the reference compounds metformin (144.0 ± 7.7%, p = 0.0345) and insulin (141.5 ± 13.8%, p = 0.0495). Furthermore, P. guajava leaf and bark extracts, as well as the reference compound rosiglitazone, significantly enhanced triglyceride accumulation in 3T3-L1 cells (252.6 ± 14.2%, p < 0.0001, 211.1 ± 12.7%, p < 0.0001, and 201.1 ± 9.2%, p < 0.0001, respectively) to levels higher than the DMSO vehicle control. Moreover, P. guajava leaf extract significantly enhanced the triglyceride accumulation in 3T3-L1 cells compared to rosiglitazone. CONCLUSION: The results demonstrated that P. guajava leaf and bark extracts can be used as a natural source of α-glucosidase inhibitors. In addition, the bark extract of P. guajava was an effective α-amylase inhibitor. Moreover, P. guajava leaf extract improved glucose uptake in muscle cells, while both leaf and bark extracts enhanced the triglyceride content in adipocytes in culture. P. guajava leaf and bark extracts may thus hypothetically have future applications in the treatment of type 2 diabetes.

Integrating pathway elucidation with yeast engineering to produce polpunonic acid the precursor of the anti-obesity agent celastrol.

BACKGROUND: Celastrol is a promising anti-obesity agent that acts as a sensitizer of the protein hormone leptin. Despite its potent activity, a sustainable source of celastrol and celastrol derivatives for further pharmacological studies is lacking. RESULTS: To elucidate the celastrol biosynthetic pathway and reconstruct it in Saccharomyces cerevisiae, we mined a root-transcriptome of Tripterygium wilfordii and identified four oxidosqualene cyclases and 49 cytochrome P450s as candidates to be involved in the early steps of celastrol biosynthesis. Using functional screening of the candidate genes in Nicotiana benthamiana, TwOSC4 was characterized as a novel oxidosqualene cyclase that produces friedelin, the presumed triterpenoid backbone of celastrol. In addition, three P450s (CYP712K1, CYP712K2, and CYP712K3) that act downstream of TwOSC4 were found to effectively oxidize friedelin and form the likely celastrol biosynthesis intermediates 29-hydroxy-friedelin and polpunonic acid. To facilitate production of friedelin, the yeast strain AM254 was constructed by deleting UBC7, which afforded a fivefold increase in friedelin titer. This platform was further expanded with CYP712K1 to produce polpunonic acid and a method for the facile extraction of products from the yeast culture medium, resulting in polpunonic acid titers of 1.4 mg/L. CONCLUSION: Our study elucidates the early steps of celastrol biosynthesis and paves the way for future biotechnological production of this pharmacologically promising compound in engineered yeast strains.

Potential of Sorbus berry extracts for management of type 2 diabetes: Metabolomics investigation of 1H NMR spectra, α-amylase and α-glucosidase inhibitory activities, and in vivo anti-hyperglycaemic activity of S. norvegica.

ETHNOPHARMACOLOGICAL RELEVANCE: Berries of Sorbus species have been used to treat type 2 diabetes in many regions in Europe. AIMS OF THE STUDY: To investigate the inhibitory activity of berry extract of Sorbus on the digestive enzymes α-amylase and α-glucosidase, two important targets for management of blood glucose for type 2 diabetics. Furthermore, to test the anti-hyperglycaemic potential of S. norvegica berry extract in vivo. MATERIALS AND METHODS: 70% acetone berry extracts of 16 Sorbus species were tested in vitro for inhibition of α-amylase and α-glucosidase. Single berry extracts were analysed by 1H-NMR spectroscopy and principal component analysis to evaluate the chemical profiles of the extracts. The anti-hyperglycaemic effect was evaluated in an oral starch tolerance test in STZ-treated C57BL/6 mice. RESULTS: The lowest IC50 values against α-amylase and α-glucosidase were obtained with the Sorbus species belonging to the subspecies Aria, which have simple leaves compared to pinnately compound leaves of the other Sorbus species. Species belonging to subspecies Aria grouped together and away from the other Sorbus species in the score plot, indicating a difference in chemistry. Both the carbohydrate- and polyphenol-fraction contributed to the enzyme inhibition. Extract of the most active species, S. norvegica, had anti-hyperglycaemic activity, at a level 36 times lower than clinically used acarbose, corresponding to a needed daily dose of 900 mg extract. CONCLUSIONS: Sorbus species of subspecies Aria have the potential to be used for management of type 2 diabetes.

Picraviane A and B: Nortriterpenes with limonoid-like skeletons containing a heptanolide E-ring system from Picramnia glazioviana.

Two highly oxygenated nortriterpenes, picraviane A and B, were isolated from the ethanolic extract of Picramnia glazioviana Engl. The structures were determined by analysis of HRMS and 2D NMR spectroscopic data. Single-crystal X-ray diffraction data was also obtained for picraviane B. The absolute configuration of both compounds were assigned by comparison of experimental and calculated vibrational and electronic circular dichroism spectroscopy, respectively. Picraviane A showed a moderate cytotoxic activity against the triple negative MDA-MB-231 breast cancer cell line. These compounds represent an undescribed class of natural products with limonoid-like skeletons containing a heptanolide as the E-ring.

Peptides from chicken processing by-product inhibit DPP-IV and promote cellular glucose uptake: potential ingredients for T2D management.

Inhibition of dipeptidyl peptidase IV (DPP-IV) and stimulation of muscle glucose uptake are two of the key strategies for management of type-2-diabetes (T2D). In the present study, four protein hydrolysates generated by enzymatic hydrolysis of chicken by-product, i.e., mechanical chicken deboning residue, were evaluated for their DPP-IV inhibitory activity as well as their effect on glucose uptake by skeletal muscle cells. The DPP-IV inhibitory assay was performed at two concentrations (1000 µg mL-1 and 10 µg mL-1) for the crude chicken protein hydrolysates. The hydrolysate with the highest DPP-IV inhibition was selected for preparative-scale fractionation using size-exclusion chromatography (SEC). The SEC fractions were tested for DPP-IV inhibitory activity as well as their effect on glucose uptake and metabolic activity of skeletal muscle cells. The muscle cells were treated with the SEC fractions and glucose uptake was measured based on luminescence detection of 2-deoxyglucose-6-phosphate (2DG6P). A fraction with peptides in the lower molecular weight range was shown to promote glucose uptake and to inhibit DPP-IV. Further chromatographic fractionation followed by inhibition assaying of the most potent SEC fraction led to isolation of five refined peptide fractions with more than 80% DPP-IV inhibition, which were subsequently analyzed with LC-HRMS/MS. This led to identification of 14 peptides as potential DPP-IV inhibitors from protein hydrolysates of mechanical chicken deboning residue.

Identification and characterization of a new antifungal peptide in fermented milk product containing bioprotective Lactobacillus cultures.

Mold and yeast contamination constitutes a major problem in food commodities, including dairy products, hence new natural preventive measures are in high demand. The aim of the current study is to identify and characterize novel antifungal peptides produced by lactic acid bacteria (LAB) in sour cream. By the use of a newly developed image-based 96-well plate fungal growth inhibition assay targeting Debaryomyces hansenii, combined with a range of analytical tools comprising HPLC-high-resolution mass spectrometry, ultrahigh-performance liquid chromatography-Triple Quadrupole MS and nuclear magnetic resonance spectroscopy, we successfully identified a new antifungal peptide (DMPIQAFLLY; 1211 Da) in sour cream enriched with two bioprotective LAB strains. This peptide represents a fragment of casein, the most abundant protein in milk. Presumably, the proteolytic activity of these bioprotective strains results in the observed 4-fold higher concentration of the peptide during storage. Both bioprotective strains are able to generate this peptide in concentrations up to 0.4 µM, independently of the sour cream starter culture employed. The peptide attenuates the growth rate of D. hansenii at concentrations ≥35 µM, and results in smaller cells and more compact colonies. Hence, the peptide is likely contributing to the overall preserving effect of the investigated bioprotective LAB strains.

High-Resolution PTP1B Inhibition Profiling Combined with HPLC-HRMS-SPE-NMR for Identification of PTP1B Inhibitors from Miconia albicans.

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 µg/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-ß-d-glucopyranose (2), myricetin 3-O-α-l-rhamnopyranoside (3), quercetin 3-O-(2″-galloyl)-α-l-rhamnopyranoside (5), mearnsetin 3-O-α-l-rhamnopyranoside (6), and kaempferol 3-O-α-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 2α-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.

Quadruple high-resolution α-glucosidase/α-amylase/PTP1B/radical scavenging profiling combined with high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy for identification of antidiabetic constituents in crude root bark of Morus alba L.

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 ±â€¯0.72, 5.16 ±â€¯0.69, and 5.07 ±â€¯0.68 µg/mL as well as showing radical scavenging activity equaling a TEAC value of (3.82 ±â€¯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.

Stereochemistry as a determining factor for the effect of a cell-penetrating peptide on cellular viability and epithelial integrity.

Cell-penetrating peptides (CPPs) comprise efficient peptide-based delivery vectors. Owing to the inherent poor enzymatic stability of peptides, CPPs displaying partial or full replacement of l-amino acids with the corresponding d-amino acids might possess advantages as delivery vectors. Thus, the present study aims to elucidate the membrane- and metabolism-associated effects of l-Penetratin (l-PEN) and its corresponding all-d analog (d-PEN). These effects were investigated when exerted on hepatocellular (HepG2) or intestinal (Caco-2 and IEC-6) cell culture models. The head-to-head comparison of these enantiomeric CPPs included evaluation of their effects on cell viability and morphology, epithelial membrane integrity, and cellular ultrastructure. In all investigated cell models, a rapid decrease in cell viability, pronounced membrane perturbation and an altered ultrastructure were detected upon exposure to d-PEN. At equimolar concentrations, these observations were less pronounced or even absent for cells exposed to l-PEN. Both CPPs remained stable for at least 2 h during exposure to proliferating cells (cultured for 24 h), although d-PEN exhibited a longer half-life when compared with that of l-PEN when exposed to well-differentiated cell monolayers (cultured for 18-20 days). Thus, the stereochemistry of the CPP penetratin significantly influences its effects on cell viability and epithelial integrity when profiled against a panel of mammalian cells.

Fluorophore labeling of a cell-penetrating peptide induces differential effects on its cellular distribution and affects cell viability.

Cell-penetrating peptides constitute efficient delivery vectors, and studies of their uptake and mechanism of translocation typically involve fluorophore-labeled conjugates. In the present study, the influence of a number of specific fluorophores on the physico-chemical properties and uptake-related characteristics of penetratin were studied. An array of seven fluorophores belonging to distinct structural classes was examined, and the impact of fluorophore labeling on intracellular distribution and cytotoxicity was correlated to the physico-chemical properties of the conjugates. Exposure of several mammalian cell types to fluorophore-penetratin conjugates revealed a strong structure-dependent reduction in viability (1.5- to 20-fold lower IC50 values as compared to those of non-labeled penetratin). Also, the degree of less severe effects on membrane integrity, as well as intracellular distribution patterns differed among the conjugates. Overall, neutral hydrophobic fluorophores or negatively charged fluorophores conferred less cytotoxicity as compared to the effect exerted by positively charged, hydrophobic fluorophores. The latter conjugates, however, exhibited less membrane association and more clearly defined intracellular distribution patterns. Thus, selection of the appropriate flurophore is critical.