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Triterpenesaponins are a major group of active components in natural productswith nonspecific antiviral activities, while T20 peptide (enfuvirtide),which contains a helix zone-binding domain (HBD), is a gp41-specificHIV-1 fusion inhibitor. In this paper, we report the design, synthesis,and structure–activity relationship (SAR) of a group of hybridmolecules in which bioactive triterpene sapogenins were covalentlyattached to the HBD-containing peptides via click chemistry. We foundthat either the triterpenes or peptide part alone showed weak activityagainst HIV-1 Env-mediated cell–cell fusion, while the hybridsgenerated a strong cooperative effect. Among them, P26–BApcexhibited anti-HIV-1 activity against both T20-sensitive and -resistantHIV-1 strains and improved pharmacokinetic properties. These resultssuggest that this scaffold design is a promising strategy for developingnew HIV-1 fusion inhibitors and possibly novel antiviral therapeuticsagainst other viruses with class I fusion proteins. [ABSTRACT FROM AUTHOR]

Low molecular weight heparins (LMWHs), depolymerized from unfractionated heparin (UFH), are widely used as anticoagulant drugs in clinic. The variable degradation methods result the different types of LMWHs, such as enoxaparin prepared by alkaline degradation following benzylation and nadroparin degraded by nitrous acid and subsequent reduction. They have different anticoagulant activities, molecular weight and special oligosaccharide sequences. Oligosaccharide analysis of the heparinase-catalyzed digestion products of heparin and LMWHs is an important way to explore the fine structural composition. In this work, a MHC-2D-LC-MS system using SAX followed by SEC and tandem to MS was applied to analyze the heparinase-products of LMWHs. 15 components of enoxaparin and 20 components of nadroparin were separated and unambiguously characterized with mass spectrum, including eight common disaccharides, and the special structural domains resistant to enzyme digestion which have the 3- O sulfated residue and/or characteristic terminal residues and the linkage region tetrasaccharides. • The platform of MHC 2D-LC linked to MS was applied to analyze digested LMWHs. • SAX is the first dimensional, SEC is the second dimensional chromatography. • 15 and 20 structural domains in enoxaparin and nadroparin were identified. • Common structural framework and special structural domains were observed. [ABSTRACT FROM AUTHOR]

[Display omitted] • Two fractions (A and C) of GAGs from porcine jejunum and duodenum were purified. • A is a heparan sulfate, and C is composed of β- d -GlcA-(1 → 4) and β- d -GalNAc-(1 → 3). • They possessed controllable anti-coagulant activity in vitro. • Additional sulfate modification enhanced the effect. To make full use of the porcine sources and develop better choice of novel GAGs as anti-coagulants, two fractions of GAGs from the porcine jejunum (A) and duodenum (C) have been separated & purified. The products were further sulfated to give B and D in order to test the influence of sulfate pattern on the bioactivity. The results showed that the relative molecular weight range of A was 3000–50,000 (Mw, g/mol), whereas C had an average molecular weight of 75,885 (Mw, g/mol). A was identified as a novel heparan sulfate through enzymatic hydrolysis analysis. C was a chondroitin like polysaccharide mainly composed of β - d -GlcA-(1 → 4) and β - d -GalNAc-(1 → 3). A possessed controllable anti-coagulant activity (7 IU/mg) in vitro. The activity of D almost achieved the same magnitude of A. This study demonstrated the anticoagulant potential of the polysaccharides, providing solid foundation for development of anti-coagulants from porcine intestine. [ABSTRACT FROM AUTHOR]

ABSTRACT YQA-14 is a novel and selective dopamine D3 receptor antagonist, with potential for the treatment of drug addiction. However, earlier compounds in its structural class tend to have poor oral bioavailability. The objectives of this study were to characterize the preclinical absorption, distribution, metabolism and excretion (ADME) properties and pharmacokinetics (PK) of YQA-14, then to simulate the clinical PK of YQA-14 using a physiologically based pharmacokinetics (PBPK) model to assess the likelihood of developing YQA-14 as a clinical candidate. For human PK prediction, PBPK models were first built in preclinical species, rats and dogs, for validation purposes. The model was then modified by input of human in vitro ADME data obtained from in vitro studies. The study data showed that YQA-14 is a basic lipophilic compound, with rapid absorption ( Tmax ~ 1 h) in both rats and dogs. Liver microsomal clearances and in vivo clearances were moderate in rats and dogs consistent with the moderate bioavailability observed in both species. The PBPK models built for rats and dogs simulated the observed PK data well in both species. The PBPK model refined with human data predicted that YQA-14 would have a clearance of 8.0 ml/min/kg, a volume distribution of 1.7 l/kg and a bioavailability of 16.9%. These acceptable PK properties make YQA-14 an improved candidate for further research and development as a potential dopamine D3R antagonism for the treatment of drug addiction in the clinic. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Background: Diabetes is one of the most prevalent human metabolic diseases. Wound healing in diabetes is frequently impaired and treatment remains challenging. Sphingolipid metabolites play important roles in the regulation of glucose metabolism. SPK1 is the key enzyme in the sphingolipid metabolic pathway. S1P/SPK plays a pivotal role in the signalling pathways of diverse cellular processes including proliferation, differentiation, migration, apoptosis in diverse cell types. Methods: To investigate the role of sphingosine kinase 1 (SPK1) in skin injury, plasmids containing the SPK1 gene (pcDNA3-FLAG-SPK1) were applied to cutaneous wounds on a streptozotocin-induced diabetic rat model over a 21-day period. The wound area and rate of wound healing were determined. The histopathological features of the healed wounds were also observed, and SPK1 expression in the skin was detected by immunohistochemistry. Results: There was a significant decrease in wound area in diabetic rats treated with 125 and 60μg/wound pcDNA3-FLAG-SPK1 (P <0.001–0.01). The mean sizes of the wounds were 0.67±0.15cm2, 0.83±0.18cm2, and 1.09±0.23cm2 in both treated and diabetic control group at the 7th day post-treatment respectively. In addition, wound healing in diabetic rats of test group was accelerated. At the 7th day, the mean rates of healing were 73.2±5.7% and 66±7.3% in test group of 125 and 60μg/wound respectively, and 55.4±9.9% in diabetic control group (P <0.001–0.01). Histology revealed that tissue sections from the treated diabetic rats contained more granulation tissue and capillaries than that of the control rats. There was high SPK1 expression in the skin of the treated diabetic rats. Conclusions: SPK1 gene therapy may represent a novel approach to cutaneous wound healing. [ABSTRACT FROM AUTHOR]

Abstract: Uplift of the Tibetan Plateau is ultimately driven by the Cenozoic collision between the Indian and Eurasian plates and their continued convergence. One approach for studying the Tibetan Plateau uplift is to verify the paleoelevation changes from collision to present day. This is important for understanding both the tectonics and the climatic effects. The new high resolution palynological record of the uppermost Oligocene to the lowest Miocene strata from the Lunpola Basin indicates that the vegetation types during the latest Oligocene–earliest Miocene were dominated by mixed coniferous–broadleaved forests being different from the modern steppe vegetation. By using the Coexistence Approach to the fossil pollen records, after calibration the effects of temperature difference and the lapse rate, a maximum paleoelevation of 3190±100m asl was estimated in the Lunpola Basin in the latest Oligocene–earliest Miocene, being 1500 to 2000m lower compared with the previous oxygen isotope paleoelevation in the same region. [Copyright &y& Elsevier]

Highlights: [•] The LLOQ of this method is better than previous published methods. [•] The esterification derivatization was used to increase the HPLC retention time and ionization efficiency in this article. [•] Peramivir is a novel anti-influenza drug which was shown to be safe and have well activity. [Copyright &y& Elsevier]

Aconitine (AC) is a highly toxic alkaloid from bioactive plants of the genus Aconitum, some of which have been widely used as medicinal herbs for thousands of years. In this study, we systematically evaluated the potential role of P-glycoprotein (P-gp) in the mechanisms underlying the low and variable bioavailability of oral AC. First, the bidirectional transport of AC across Caco-2 and MDCKII-MDR1 cells was investigated. The efflux of AC across monolayers of these two cell lines was greater than its influx. Additionally, the P-gp inhibitors, verapamil and cyclosporin A, significantly decreased the efflux of AC. An in situ intestinal perfusion study in rats showed that verapamil co-perfusion caused a significant increase in the intestinal permeability of AC, from 0.22×10−5 to 2.85×10−5 cm/s. Then, the pharmacokinetic profile of orally administered AC with or without pre-treatment with verapamil was determined in rats. With pre-treatment of verapamil, the maximum plasma concentration (Cmax) of AC increased sharply, from 39.43 to 1490.7ng/ml. Accordingly, a 6.7-fold increase in the area under the plasma concentration–time curve (AUC0–12h) of AC was observed when co-administered with verapamil. In silico docking analyses suggested that AC and verapamil possess similar P-gp recognition mechanisms. This work demonstrated that P-gp is involved in limiting the intestinal absorption of AC and attenuating its toxicity to humans. Our data indicate that potential P-gp-mediated drug–drug interactions should be considered carefully in the clinical application of aconite and formulations containing AC. [ABSTRACT FROM AUTHOR]

Although the polysaccharide utilization loci (PULs) activated by pectin have been defined, due to the complex of side-chain structure, the degradative mechanisms still remain vague. Thus, we hypothesize that there may have other specific PULs to target pectin. Here, we characterize loci-encoded proteins expressed by Bacteroides thetaiotaomicron (BT) that are involved in the pectin capturing, importation, de-branching and degradation into monosaccharides. Totally, four PULs contain ten enzymes and four glycan binding proteins which including a novel surface enzyme and a surface glycan binding protein are identified. Notably, PUL2 and PUL3 have not been reported so far. Further, we show that the degradation products support the growth of other Bacteroides spp. and probiotics. In addition, genes involved in this process are conservative in other Bacteroides spp. Our results further highlight the contribution of Bacteroides spp. to metabolism the pectic network. • Four discrete PULs are involved in the degradation process and two of them are novel. • Five enzymes which confer pectin degradation are firstly described in this research. • Degradation products of pectin support the growth of other gut microbes. • Genes involved in this process are conservative in other gut Bacteroides spp. [ABSTRACT FROM AUTHOR]

Highlights: [•] The bidirectional transports of aconitine across Caco-2 cells were investigated. [•] The influx of aconitine was temperature-, pH- and Na+-dependent. [•] The uptake of aconitine may be mediated by a sodium-dependent glucose transporter. [•] The efflux of aconitine was temperature- and pH-dependent, but Na+-independent. [•] The active efflux of aconitine was mediated mainly by P-gp. [Copyright &y& Elsevier]

Thesmall molecule fusion inhibitors N-(4-carboxy-3-hydroxyphenyl)-2,5-dimethylpyrrole(NB-2) and N-(3-carboxy-4-hydroxyphenyl)-2,5-dimethylpyrrole(A12) target a hydrophobic pocket of HIV-1 gp41 and havemoderate anti-HIV-1 activity. In this paper, we report the design,synthesis, and structure–activity relationship of a group ofhybrid molecules in which the pocket-binding domain segment of theC34 peptide was replaced with NB-2 and A12derivatives.In addition, the synergistic effect between the small molecule andpeptide moieties was analyzed, and lead compounds with a novel scaffoldwere discovered. We found that either the nonpeptide or peptide partalone showed weak activity against HIV-1-mediated cell–cellfusion, but the conjugates properly generated a strong synergisticeffect. Among them, conjugates Aoc−βAla–P26 andNoc−βAla–P26 exhibited a low nanomolar IC50in the cell–cell fusion assay and effectively inhibitedT20-sensitive and -resistant HIV-1 strains. Furthermore, the new moleculesexhibited better stability against proteinase K digestion than T20and C34. [ABSTRACT FROM AUTHOR]

Porcine intestinal mucosa heparin was partially depolymerized by recombinant heparinase 1 (heparin lyase 1, originating from Flavobacterium heparinum and expressed in Escherichia coli) and then fractionated, leading to the isolation of 22 homogeneous oligosaccharides with sizes ranging from disaccharide to hexadecasaccharide. The purity of these oligosaccharides was determined by gel electrophoresis, strong anion exchange and reversed-phase ion-pairing high-performance liquid chromatography. The molecular mass of oligosaccharides was determined using electrospray ionization-mass spectrometry and their structures were elucidated using one- and two-dimensional nuclear magnetic resonance spectroscopy at 600 MHz. Five of the characterized oligosaccharides represent new compounds. The most prominent oligosaccharide comprises the common repeating unit of heparin, ΔUA2S-[-GlcNS6S-IdoA2S-]n-GlcNS6S, where ΔUA is 4-deoxy-α-l-threo-hex-4-eno-pyranosyluronic acid, GlcN is 2-deoxy-2-amino-d-glucopyranose, IdoA is l-idopyranosyluronic acid, S is sulfate and n = 0–7. A second prominent heparin oligosaccharide motif corresponds to ΔUA2S-[GlcNS6S-IdoA2S]n-GlcNS6S-IdoA-GlcNAc6S-GlcA-GlcNS3S6S (where n = 0–5 and GlcA is d-glucopyranosyluronic acid), a fragment of the antithrombin III binding site in heparin. The prominence of this second set of oligosaccharides and the absence of intact antithrombin III binding sites suggest that the -GlcNS3S6S-IdoA2S- linkage is particularly susceptible to heparinase 1. [ABSTRACT FROM AUTHOR]

Abstract: An oversulfated chondroitin sulfate (OSCS) was identified as a contaminant to pharmaceutical heparin and severe anaphylactoid reactions were ascribed to this contaminant. An examination of the biochemistry underlying both the anticoagulant activity and the toxic effects of oversulfated chondroitin sulfate was undertaken. This study demonstrates that the anticoagulant activity of this oversulfated chondroitin sulfate is primarily dependent on heparin cofactor II mediated inhibition of thrombin. Heparin and oversulfated chondroitin sulfate binding to coagulation, kinin–kallikrein and complement proteins were studied by surface plasmon resonance. While oversulfated chondroitin sulfate binds tightly to antithrombin III, unlike heparin, OSCS does not induce antithrombin III to undergo the conformational change required for its inactivation of thrombin and factor Xa. In contrast to heparin, oversulfated chondroitin sulfate tightly binds factor XIIa suggesting a biochemical mechanism for the factor XIIa-based enhancement of vasoactive bradykinin production. [Copyright &y& Elsevier]

Abstract: Peramivir is a novel influenza neuraminidase inhibitor. In this article, hydrophilic interaction chromatography coupled with tandem mass spectrometry was developed to determine peramivir in human plasma. The positive ion MRM mode was performed and the precursor to the product ion transitions of m/z 329→100 and 285→138 were used to measure peramivir and Ro 64-0802 (I.S.). Chromatographic separation was performed on an Amide-80 column with acetonitrile–water–formic acid (70:30:0.1, v/v/v, 0.5mL/min). The method was linear over a concentration range of 10–10,000ng/mL. The average inter-day/intra-day precision values were 3.7±1.8% and 4.3±1.8%, respectively, while the accuracy values were 97.0±4.8%. This method has been successfully applied to Phase I of clinical research of peramivir. [Copyright &y& Elsevier]

Recently, certain lots of heparin have been associated with an acute, rapid onset of serious side effects indicative of an allergic-type reaction. To identify potential causes for this sudden rise in side effects, we examined lots of heparin that correlated with adverse events using orthogonal high-resolution analytical techniques. Through detailed structural analysis, the contaminant was found to contain a disaccharide repeat unit of glucuronic acid linked β1→3 to a β-N-acetylgalactosamine. The disaccharide unit has an unusual sulfation pattern and is sulfated at the 2-O and 3-O positions of the glucuronic acid as well as at the 4-O and 6-O positions of the galactosamine. Given the nature of this contaminant, traditional screening tests cannot differentiate between affected and unaffected lots. Our analysis suggests effective screening methods that can be used to determine whether or not heparin lots contain the contaminant reported here. [ABSTRACT FROM AUTHOR]

Abstract: A simple, sensitive and reliable method was developed to determine simultaneously the concentrations of thienorphine and its metabolite thienorphine glucuronide conjugate in rat plasma by liquid chromatography–tandem mass spectrometry (LC–MS/MS). The metabolite was identified by MS: thienorphine glucuronide conjugate. Sample preparation involved protein precipitation with methanol. Analytes were separated on Finnigan BetaBasic-18 column (150mm×2.1mm i.d., 5μm) using methanol: water: formic acid (56:44:0.1, v/v/v) as mobile phase at a flow rate of 0.2ml/min. The method had a linear calibration curve over the concentration range of 0.1–50ng/ml for thienorphine and 2–1000ng/ml for thienorphine glucuronide conjugate, respectively. LOQ of thienorphine and thienorphine glucuronide conjugate was 0.1 and 2ng/ml, respectively. The intra- and inter-batch precisions were less than 12% and their recoveries were greater than 80%. Pharmacokinetic data of thienorphine and its metabolite thienorphine glucuronide conjugate obtained with this method following a single oral dose of 3mg/kg thienorphine to rats were also reported for the first time. [Copyright &y& Elsevier]

Low-molecular-weight polyguluronate sulfate (LPGS) was prepared as a heparinoid by chemical sulfation of polyguluronic acid (PG), which was isolated from the hydrolysate of alginate by pH-fractionated precipitation. The structure of LPGS was elucidated based on FT-IR and 2D NMR spectroscopy. Sulfation was demonstrated to occur at the C-2 and C-3 positions of the guluronic acid residues and the degree of substitution (DS) was 1.53 per monosaccharide residue. The weight-averaged molecular weight of LPGS was 11.4kDa determined by high performance gel permeation chromatography (HPGPC). The in vitro anticoagulant activity and in vivo anti-inflammatory activity of LPGS were evaluated. Results showed that LPGS significantly prolonged the whole blood clotting time and activated partial thrombinplastin time (APTT), and exhibited considerable anti-inflammatory activity in cotton pellet-induced granuloma in rats. [Copyright &y& Elsevier]

The pollen flux in coniferous and deciduous broadleaved mixed forest in the Changbai Mountains is presented in one year's experiments. The results indicate that arboreal pollen percentages are more than 65% and pollen flux is higher than 5000 grain·cm²·a-1, while less than 2% and lower than 1000 grain·cm²·a-1 for shrubby pollen, and less than 20% and lower than 3000 grain·cm²·a-1 for herbaceous pollen for most samples at different heights. The pollen assemblages are similar to those in the samples under 8 m height where Pinus and Quercus are dominant and followed by the few non-local pollen types, and Fraxinus percentages are high with a few non-local pollen types at 16 m to 32 m height as well as non-local pollen clearly increased at 40 m height. Comparisons between pollen assemblages and vegetation composition suggest that similarities are higher for pollen trap samples than for surface moss samples. [ABSTRACT FROM AUTHOR]

Abstract: A sensitive and specific high-performance liquid chromatography–tandem mass spectrometry method (LC/ESI/MS) was developed and validated for the identification and quantification of the novel lead compound of anticholinergic drug thiencynonate in rat plasma. The analytes were determined using positive electrospray ionization mass spectrometry in the selected reaction ion monitoring (SRM). The chromatography separation was on BetaBasic-18 column (150mm×2.1mm i.d., 3μm). The mobile phase was composed of methanol–water (70:30, v/v), containing 0.5‰ formic acid, which was pumped at a flow rate of 0.2ml/min. Phencynonate was selected as the internal standard (IS). Simultaneous MS detection of thiencynonate and IS was performed at m/z 364.4 (thiencynonate), m/z 358 (phencynonate), and the SRM of the two compounds were both at 156. Thiencynonate eluted at approximately 2.8min, phencynonate eluted at approximately 2.9min and no endogenous materials interfered with their measurement. Linearity was obtained over the concentration range of 1–100ng/ml in rat plasma. The lower limit of quantification (LLOQ) was reproducible at 1ng/ml in rat plasma. The precision measured was obtained from 2.47 to 9.28% in rat plasma. Extraction recoveries were in the range of 67.63–76.76% in plasma. This method was successfully applied to the identification and quantification of thiencynonate in pharmacokinetic studies. [Copyright &y& Elsevier]

Abstract: A sensitive and specific high-performance liquid chromatographic assay with electrospray ionization mass spectrometry detection (LC-ESI-MS) has been developed and validated for the identification and quantification of the novel anticholinergic drug phencynonate in rat blood and urine. The sample pretreatment involves basification and iterative liquid–liquid extraction with ethyl ether–dichloromethane (2:1, v/v) solution, followed by LC separation and positive electrospray ionization mass spectrometry detection. The chromatography was on BetaBasic-18 column (150mm×2.1mm i.d., 3μm). The mobile phase was composed of methanol–water (85:15, v/v), containing 0.5‰ formic acid, which was pumped at a flow-rate of 0.2ml/min. Thiencynonate was selected as the internal standard (IS). Simultaneous MS detection of phencynonate and IS was performed at m/z 358.4 (phencynonate), m/z 364 (thiencynonate), and the selected reaction ion monitoring (SRM) of the two compounds was at 156. Phencynonate eluted at approximately 5.25min, thiencynonate eluted at approximately 5.10min and no endogenous materials interfered with their measurement. Linearity was obtained over the concentration range of 1–100ng/ml in rat blood and 1–500ng/ml in rat urine. The lower limit of quantification (LLOQ) was reproducible at 1ng/ml in both of rat blood and urine. The precision measured was obtained from 2.92 to 9.76% in rat blood and 4.17 to 9.76% in rat urine. Extraction recoveries were in the range of 69.57–79.49% in blood and 56.85–64.86% in urine. This method was successfully applied to the identification and quantification of phencynonate in pharmacokinetic studies. [Copyright &y& Elsevier]

• A method with MHC 2D-LC linked to MS was developed to analyze digested heparin. • SAX is the first dimensional, SEC is the second dimensional chromatography. • 8 common dp2, 8 enzyme resistant dp4 & a heparin–core protein link dp4 were observed. • Composition result implied their structure, activity and production properties. Heparin is a linear sulfated polysaccharide. It is composed of a repeating disaccharide unit with different sulfo patterns. The compositional analysis after heparin was decomposed to disaccharides and enzyme resistant domains is an important way to delve into its structure. Strong anion exchange (SAX) chromatography is commonly used for the compositional analysis due to its high resolution, stability and capability of quantitation. However, nonvolatile salt in mobile phase is not compatible with MS, then the structural domains cannot be identified without standards. Here, a new two-dimensional liquid chromatography system, multiple heart cut (MHC), was developed and linked to mass spectrometry (MS) directly to provide a comprehensive analysis of enzyme digested heparin. SAX was applied as the first dimensional chromatography, in which 17 peaks were observed and integrated in the digested heparin. Size-exclusion chromatography (SEC) was used as the second dimensional chromatography to desalt efficiently. Structural information of each component was then obtained with MS, including eight common disaccharides, eight enzyme resistant tetrasaccharides and a heparin–core protein linkage domain. The comparison of enzyme digested heparins obtained from different vendors using this system suggested their similar major structure and activity, but slightly different production processes. [ABSTRACT FROM AUTHOR]

Micelles with surface morpholino groups were stealthy at blood and normal tissue pH (7.4) due to the unprotonated hydrophilic morpholino groups on the surfaces. At tumor pH (＜7), the micelle surfaces were positively charged because of the protonation of the morpholino groups, which promoted the cellular uptake of the micelles. [ABSTRACT FROM AUTHOR]

• A 1−6 glucanase was firstly used to digest a β-glucan from H. erinaceus. • The oligosaccharides in the digested products were analyzed with HPAEC-MS. • The sequences of enzyme resistant oligosaccharides were elucidated with MS/MS. • A branch-on-branch structure of the β-glucan was estimated. A water-soluble β-glucan from fruiting body of Hericium erinaceus was obtained after water extraction, purification and fractionation. Analyses of monosaccharide composition, molecular weight and linkage mode demonstrated that it is a β-glucan with 1→3 and 1→6 linkage modes and a molecular weight of 13.3 kDa. An endo-1,6-β- d -glucanase was used to digest the β-glucan and the digested products over time were analyzed with a HPAEC-PAD-MS platform. The linkage mode of each glycosidic bond in the digested oligosaccharides were confirmed with MS/MS. At the end of digestion, enzyme resistant oligosaccharides were observed as several 1→6 linked glucoses with one or two 1→3 linked glucose residues. All these domains are more like constructional pieces from a branch-on-branch glucan, in which multiple 1→6 linked glucan chains are hooked through one or two 1→3 linked glucose residues. Averagely, there is a 1→3 linkage per six 1→6 linked glucoses in this branch-on-branch β-glucan. [ABSTRACT FROM AUTHOR]

• Enzymatic depolymerization kinetics of chemically-modified heparin was analyzed. • Low molecular weight chemically-modified heparins were prepared and characterized. • The order of chemical modification and HepI lysis affected the product MW distribution. • LMW partially 6- O -/ N -desulfated heparin suppressed bleomycin-induced cell apoptosis. Non-anticoagulant biological functions of heparin-based drugs have drawn increasing attention. However, the exploration into the non-anticoagulant activities of various low molecular weight heparins was associated with bleeding risks in clinical practice and often led to controversial conclusions due to the structural differences. In this study, we aimed to establish a process to produce a library of heparin derivatives with structural diversity and reduced/abolished anticoagulant activity through the combination of chemical modifications and enzymatic cleavage of heparins. The depolymerization characteristics of various selectively modified heparin derivatives by three heparinases were comprehensively analyzed. The order of periodate treatment and heparinase-I depolymerization was proved to significantly change the structural characteristics of the oligosaccharide products. Finally, among several heparin derivatives that screened in the bleomycin-induced cell apoptosis model, the low molecular weight partially 6- O- / N -desulfated heparins showed the strongest anti-apoptotic activities. This study provided a useful approach for future development of novel heparin-derivative medications. [ABSTRACT FROM AUTHOR]

• A FCS's structure was characterized with two bottom-up strategies. • The specificity of mild acid to hydrolyze fucose branches was investigated. • The backbone was systematically characterized after branches were removed over time. • Various structure domains were analyzed after FCS was chopped by catalytic oxidation. • The longest branch, linkage modes and sulfation patterns of FCS were confirmed. Two bottom-up strategies, disaccharide and oligosaccharide analyses, were applied to elucidate the structure of a fucosylated chondroitin sulfate (FCS). The FCS was hydrolyzed with mild acid. The remained part was digested with CS lyase for disaccharide analysis. The products from each step were analyzed and the results revealed that mild sulfuric acid mainly released sulfated fucose branches, but also affected some residues and sulfo-groups on the backbone. Over 140 oligosaccharide fragments were generated by catalytic oxidation and identified by HPSEC-MS, including sulfated fucose oligosaccharides exclusively from branches, sulfated backbone fragments, and junctional fragments. Based on the results provided by these two methods, the proposed backbone of the FCS is mainly composed of GlcA→GalNAc4S6S and GlcA→GalNAc6S, and the branch is mainly located at GalNAc. The longest branch observed is nonasaccharide, and most of the fucose on the branches are mono and/or di-sulfated. NMR results supported the conclusion. [ABSTRACT FROM AUTHOR]

Enfuvirtide (T20), is the first HIV fusion inhibitor approved for treatment of HIV/AIDS patients who fail to respond to the current antiretroviral drugs. However, its clinical application is limited because of short half-life, drug resistance and cross-reactivity with the preexisting antibodies in HIV-infected patients. Using an artificial peptide strategy, we designed a peptide with non-native protein sequence, AP3, which exhibited potent antiviral activity against a broad spectrum of HIV-1 strains, including those resistant to T20, and had remarkably longer in vivo half-life than T20. While the preexisting antibodies in HIV-infected patients significantly suppressed T20's antiviral activity, these antibodies neither recognized AP3, nor attenuated its anti-HIV-1 activity. Structurally different from T20, AP3 could fold into single-helix and interact with gp41 NHR. The two residues, Met and Thr, at the N-terminus of AP3 form a hook-like structure to stabilize interaction between AP3 and NHR helices. Therefore, AP3 has potential for further development as a new HIV fusion inhibitor with improved antiviral efficacy, resistance profile and pharmacological properties over enfuvirtide. Meanwhile, this study highlighted the advantages of artificially designed peptides, and confirmed that this strategy could be used in developing artificial peptide-based viral fusion inhibitors against HIV and other enveloped viruses. [ABSTRACT FROM AUTHOR]