Rational design and synthesis of Oreoch-2 analogues as efficient broad-spectrum antimicrobial peptides.

In recent years, bacterial resistance has risen sharply, which seriously endangers public health due to the abuse of antibiotics and the lack of new antibiotics. Therefore, there is an urgent need for new antimicrobial agents to combat multidrug-resistant (MDR) bacterial infections. In this paper, six Oreoch-2 analogues were rationally designed and efficiently synthesized by using the truncation strategy with Oreoch-2 as the lead compound. Evaluation of these analogues against a panel of Gram-positive and Gram-negative bacteria including MDR strains was performed. Among them, ZN-5 and ZN-6 were identified to be broad-spectrum effective analogues, which were superior to their parent peptide Oreoch-2. In addition, ZN-5 and ZN-6 had good stability to the physiological environment, and much higher selectivity to bacterial cells than to mammalian cells. Time-kill kinetics and transmission electron microscope (TEM) studies suggested that these analogues were typical bactericidal agents and quickly eliminated bacteria in a bactericidal mode by disrupting bacterial cell membrane. Moreover, ZN-5 and ZN-6 could inhibit biofilm formation of Staphylococcus aureus ATCC25923. Compared with their parent peptide Oreoch-2, ZN-5 and ZN-6 not only possessed shortened peptide chains, but also showed slightly improved antibacterial activity and greatly reduced hemolysis. This indicates that they are ideal lead compounds of antimicrobial peptides, which can be developed as substitutes for traditional antibiotics.

Synthesis and antibacterial activity of novel 3-O-arylalkylcarbamoyl-3-O-descladinosyl-9-O-(2-chlorobenzyl)oxime clarithromycin derivatives.

A novel series of 3-O-arylalkylcarbamoyl-3-O-descladinosyl-9-O-(2-chlorobenzyl)oxime clarithromycin derivatives, were designed, synthesized and evaluated for their in vitro antibacterial activity. These derivatives were found to have strong activity against susceptible and resistant bacteria strains. Among them, compounds 7a and 7q showed the most potent activity (0.125 µg/mL) against erythromycin-resistant S. pneumoniae expressing the mefA gene. Moreover, compounds 7f, 7i, 7p and 7z displayed remarkably improved activity (4 µg/mL) against penicillin-resistant S. aureus ATCC31007, and compounds 7a, 7b, 7f, 7p and 7z showed improved activity (8 µg/mL) against erythromycin-resistant S. pyogenes. In particular, compound 7z exhibited potent and balanced activity against the tested drug-susceptible and -resistant bacterial strains.

Substitution of terminal amide with 1H-1,2,3-triazole: Identification of unexpected class of potent antibacterial agents.

3-Methoxybenzamide (3-MBA) derivatives have been identified as novel class of potent antibacterial agents targeting the bacterial cell division protein FtsZ. As one of isosteres for the amide group, 1,2,3-triazole can mimic the topological and electronic features of the amide, which has gained increasing attention in drug discovery. Based on these considerations, we prepared a series of 1H-1,2,3-triazole-containing 3-MBA analogues via isosteric replacement of the terminal amide with triazole, which had increased antibacterial activity. This study demonstrated the possibility of developing the 1H-1,2,3-triazole group as a terminal amide-mimetic element which was capable of both keeping and modulating amide-related bioactivity. Surprisingly, a different action mode of these new 1H-1,2,3-triazole-containing analogues was observed, which could open new opportunities for the development of antibacterial agents.

Mechanically tunable, antibacterial and bioactive mussel adhesive protein/hyaluronic acid coacervates as bioadhesives.

In this work a bioadhesive was developed based on coacervates composed of recombinant mussel adhesive protein (MAP) and dopamine grafted hyaluronic acid (HA). Dopamine profoundly affected rheological attributes of the coacervates, leading to reduced rigidity, enhanced chain flexibility, more sol-like and fluid character and higher tolerance against structural collapse. The coacervates were rendered flowability, injectability, and adaptability, benefiting convenient delivery and making good contact with the skin to provide firm sealing for wounds of various shape and depth. It is the first time reported that MAP/HA coacervates are inherently antibacterial with 100 % growth inhibition against Gram-positive bacteria Staphylococcus aureus and Gram-negative bacteria Escherichia coli. The antibacterial capability was disclosed to be positively related to catechol content. To further enhance the coacervates bioactivity, a small bioactive peptide thymosin was added and was revealed to promote fibroblasts migration. The coacervates hold great potential as practical bioadhesives both from the perspective of rheological properties and biological activities.

Protective effect of ultra low molecular weight heparin on glutamate-induced apoptosis in cortical cells.

PURPOSE: To investigate the effect of ultra low molecular weight heparin (ULMWH) on glutamate induced apoptosis in rat cortical cells and to explore the possible mechanisms. MATERIALS AND METHODS: Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was first analyzed with Hoechst 33258 and then confirmed by DNA fragmentation. The concentration of free intracellular calcium ([Ca(2+)](i)) was determined with fura-2/AM fluorometry. The expression of Bcl-2 family protein and caspase-3 were evaluated with Western blot. RESULTS: Typical apoptotic morphological change in rat cortical cells treated with 100 micromol/L glutamate for 24h was detected by Hoechst 33258 staining, which was then confirmed by the DNA ladder of agarose gel electrophoresis. The apoptotic rate of the glutamate treated cells was up to 33.21%, and 24 h of treatment with glutamate increased [Ca(2+)](i), down-regulated Bcl-2 expression, up-regulated Bax expression, and increased caspase-3 activation in rat cortical cells. Our research demonstrated that ULMWH pretreatment can prevent the glutamate-induced apoptosis, attenuate the increase of [Ca(2+)](i) not only in medium containing Ca(2+) but also in Ca(2+)-free medium, up-regulate the expression of Bcl-2, down-regulate the expression of Bax, and decrease caspase-3 activation. CONCLUSION: ULMWH has neuroprotective capacity to antagonize glutamate-induced apoptosis in cortical cells, through decrease of Ca(2+) release and modulation of apoptotic processes.

Design, synthesis and biological activity evaluation of novel 4-subtituted 2-naphthamide derivatives as AcrB inhibitors.

A novel series of 4-substituted 2-naphthamide derivatives were designed, synthesized and evaluated for their biological activity. In particular, the ability of the compounds to potentiate the action of antibiotics, to inhibit Nile Red efflux and to target AcrB specifically was investigated. The results indicated that most of the 4-substituted 2-naphthamide derivatives were able to synergize with the antibiotics tested, and inhibit Nile Red efflux by AcrB in the resistant phenotype. Subsequent exclusion of compounds with off target effects such as outer- or inner membrane permeabilization identified compounds 7c, 7g, 12c, 12i and 13g as efflux pump inhibitors (EPIs). Particularly, compounds 7c, 7g and 12i were found to be the most potent EPIs, which synergized with the two substrates tested at lower concentrations than that of parent A3, demonstrating an improvement in potency as compared to A3. Additionally, when the outer membrane of E. coli was permeabilized, compound 12c displayed a huge increase in efficacy and was able to synergize with erythromycin at a concentration that was 16 times lower than that of the parent A3. Hence we were able to design and synthesize compounds that displayed significant increase in efficacy as EPIs against AcrB.

The relationship between the structure of dermatan sulfate derivatives and their antithrombotic activities.

To study the relationship between the structure of dermatan sulfate (DS) derivatives and their anti-thrombotic activities, DS-derived oligosaccharides (with different structures and relative molecular weight (M(r))) were prepared, and the effects of the DS-derived oligosaccharides on the activities of heparin cofactor II (HCII), activated protein C (APC), blood platelet, and vascular endothelial cells were studied. The major disaccharides of DS and polysulfated dermatan sulfate (PSDS) were IdoA-1-->3-GalNAc-4-OSO(3) and IdoA-2OSO(3)-1-->3-GalNAc4, 6-diOSO(3), respectively. The results showed that the consequence of the thrombotic inhibitory effects of DS and its derivatives were as follows: PSDS>low molecular weight polysulfated dermatan sulfate (LPSDS)>DS. Both DS and PSDS inhibited platelet aggregation in the concentration-dependent manner, and the IC(50) value of DS and PSDS is 12.7+/-1.3 and 28.6+/-0.9 mg/mL, respectively. DS oligosaccharides (DSOSs) and PSDS oligosaccharides (PSDSOSs) both significantly inhibited P-selectin expression on platelet surface (P<0.01), while DSOSs have no different effect compared with PSDSOSs. DSOSs and PSDSOSs significantly enhanced the activity of HCII in inhibiting thrombin in the plasma. The most active PSDSOS was PSDSOS(1) with M(r) of 4959, which enhanced the HCII activity by 91% (P<0.01). The experiments on APC activity showed that DS and its derivatives enhanced APC activity. The most active PSDSOS was PSDSOS(3) with M(r) of 2749, which enhanced the APC activity to 331+/-27% (P<0.01). DSOSs and PSDSOSs enhanced tissue plasminogen activator (t-PA) activity and reduced the plasminogen activator inhibitor (PAI) activity from cultured human umbilical vein endothelial cells (HUVEC), resulting in the ratio of t-PA/PAI going up. PSDSOSs which have the same M(r) as DSOSs produced more active effects in above assays, except for platelet aggregation.

Effects of dermatan sulfate derivatives on platelet surface P-selectin expression and protein C activity in blood of inflammatory bowel disease patients.

AIM: To investigate the effect of dermatan sulfate (DS) derivatives on platelet surface P-selectin expression and blood activated protein C (APC) activity in patients with inflammatory bowel disease (IBD), and to clarity the anti-inflammatory mechanism of DS derivatives. METHODS: Dermatan sulfate (DS) was sulfated with chlorosulfonic acid to prepare polysulfated dermatan sulfate (PSDS). The major disaccharides of DS and PSDS were determined by 1H nuclear magnetic resonance spectroscopy (1H-NMR) and 13C-NMR. Both DS and PSDS were depolymerized with hydrogen peroxide. The fragments were separated by gel filtration chromatography. The effects of DS derivatives on P-selectin expression were assayed by ELISA method, and blood APC activity was assayed by the synthetic chromogenic substrate method. RESULTS: The major disaccharides of DS and PSDS were IdoA-1-3-GalNAc-4-SO3 and IdoA-2SO3-1-3-GalNAc4, 6-diSO3, respectively. Compared with the adenosine diphosphate stimulated group and IBD control group, DS and its derivatives all had significant inhibitory effects on P-selectin expression (P<0.01), but there was no difference between DS-derived oligosaccharides (DSOSs) and PSDS-derived oligosaccharides (PSDSOSs). The experiments on APC activity showed that DS and its derivatives all enhanced APC activity. The most active DSOS was the one with a relative molecular weight (Mr) of 4,825, which enhanced the APC activity from 106.5+/-11.5% to 181.8+/-22.3% (P<0.01). With the decrease of Mr, the activity of DSOSs decreased gradually. The effect of PSDS on APC activity enhancement was more significant than that of DS, and the APC activity was raised to 205.2+/-22.1% (P<0.01). All the PSDSOSs were more active than DSOSs on the basis of comparable Mr. With the decrease of Mr, the activity of PSDSOSs increased gradually, and the most active PSDSOS was PSDSOS3 with Mr of 2,749, which enhanced the APC activity to 331.2+/-27.8% (P<0.01), then the activity of PSDSOSs decreased gradually. CONCLUSION: DS and its derivatives can significantly inhibit P-selectin expression on platelet surface, but the effect has no correlation with DS molecular mass and sulfation. The effect of DS or its derivatives on APC activity at molecular level involves complex mechanisms that depend on the molecular mass, the degree of sulfation, and the heterogeneous composition of DS. On the same molecular size, the higher the degree of DS sulfation, the more significant the effect on enhancing APC activity.

Inhibitory effect of heparin-derived oligosaccharides on secretion of interleukin-4 and interleukin-5 from human peripheral blood T lymphocytes.

AIM: To investigate the inhibitory effect of heparin-derived oligosaccharides (Oligs) on secretion of interleukin-4 (IL-4) and interleukin-5 (IL-5) from human peripheral blood T lymphocytes (PBTLs). METHODS: Oligs were prepared by three different heparin depolymerization methods and separated by gel filtration chromatography. PBTLs from ten adult patients with allergic eosinophilic gastroenteritis were treated with phytahematoagglutinin (PHA) and Oligs. The supernatants from the cell culture of PBTLs were harvested and subjected to the determination of IL-4 and IL-5 contents by ELISA method. RESULTS: At the concentration of 5 microg/mL, Oligs with different Mr had different effects on the secretion of IL-4 and IL-5. The tetrasaccharide with Mr of 1,142, produced by depolymerizing heparin with hydrogen peroxide, had the strongest inhibitory effect on the secretion of IL-4. It decreased the IL-4 content from 375.6+/-39.2 ng/L (PHA group) to 12.5+/-5.7 ng/L (P<0.01). The hexasaccharide with Mr of 1,806, produced by depolymerizing heparin with beta-elimination method, had the strongest inhibitory effect on the secretion of IL-5. It decreased the IL-5 content from 289.2+/-33.4 ng/L (PHA group) to 22.0+/-5.2 ng/L (P<0.01). CONCLUSION: The inhibitory activity of Oligs on the secretion of IL-4 and IL-5 from human PBTLs closely depends on their molecular structure, and there may be an essential structure to act as an inhibitor. The most effective inhibitors of IL-4 and IL-5 secretion are tetrasaccharides and hexasaccharides, respectively.

Pharmacological effects and clinical applications of ultra low molecular weight heparins.

Heparin, one of the common anticoagulants, is clinically used to prevent and treat venous thromboembolism (VTE). Though it has been the drug of choice for many advanced medical and surgical procedures with a long history, the adverse events, such as bleeding, heparin-induced thrombocytopenia (HIT), allergic reactions, follow. Therefore, low molecular weight heparins (LMWHs) and ultra low molecular weight heparins (ULMWHs), with lower molecular weights, higher anti-FXa activity, longer half-life times and lower incidence of adverse events than unfractionated heparin (UFH), were researched and developed. Fondaparinux, a chemically synthesized ULMWH of pentasaccharide, has the same antithrombin III (AT-III)-binding sequence as found in UFH and LMWH. In addition, AVE5026 and RO-14, another two ULMWHs, are obtained by selective chemical depolymerization. In this paper, we review the preparation process, pharmacological effects and clinical applications of fondaparinux, AVE5026 and RO-14.

Mapping of low molecular weight heparins using reversed phase ion pair liquid chromatography-mass spectrometry.

Low molecular weight heparins (LMWHs) are structurally complex, highly sulfated and negatively charged, linear carbohydrate polymers prepared by chemical or enzymatic depolymerization of heparin. They are widely used as anticoagulant drugs possessing better bioavailability, longer half-life, and lower side effects than heparin. Comprehensive structure characterization of LMWHs is important for drug quality assurance, generic drug application, and new drug research and development. However, fully characterization of all oligosaccharide chains in LMWHs is not feasible for current available analytical technologies due to their structure complexity and heterogeneity. Fingerprinting profiling is an efficient way for LMWHs' characterization and comparison. In this work, we present a simple, sensitive, and powerful analytical approach for structural characterization of LMWHs. Two different LMWHs, enoxaparin and nadroparin, were analyzed using reversed phase ion pair electrospray ionization mass spectrometry (RPIP-ESI-MS). More than 200 components were identified, including major structures, minor structures, and process related impurities. This approach is robust for high resolution and complementary fingerprinting analysis of LMWHs.

Antagonistic effects of ultra-low-molecular-weight heparin on Aß25-35-induced apoptosis in cultured rat cortical neurons.

Low-molecular-weight heparin (LMWH) and ultra-low-molecular-weight heparin (ULMWH) are heparin's derivatives, having various pharmacological effects. The present study aims to investigate the effect of ULMWH on amyloid ß peptide (Aß25-35)-induced neurotoxicity in cultured rat cortical neurons, and LMWH was employed as a positive control agent. The neurons were incubated with Aß25-35 (35µM), Aß25-35 plus ULMWH (2, 10, 50 µg/ml) or LMWH (10 µg/ml) for 24h. The cell viability was assessed by MTT and LDH release. FITC-Annexin V/PI double staining, Hoechst 33258 staining, TUNEL and Western blotting for bcl-2 and caspase-3 were employed to measure the neuron apoptosis. Furthermore, the intracellular Ca(2+) concentration was measured by a fluorescent dye, Fura-2/AM. The results showed that ULMWH significantly increased cell viability and the protein expression levels of bcl-2 and decreased the LDH release, the number of apoptotic cells, the concentration of intracellular Ca(2+) and the protein expression levels of caspase-3 in cortical neurons, suggesting that ULMWH can obviously reduce Aß25-35-induced neurotoxic effects and might act as a potential agent for Alzheimer's disease.

Purification and properties of recombinant GST-heparinase III and optimization of cultivation conditions.

Heparinase III is an enzyme that specifically cleaves certain sequences of heparan sulfate. Previous reports showed that this enzyme expressed in Escherichia coli was highly prone to aggregation in inclusion bodies and lacks detectable biological activity. In this paper, we fused a glutathione-S-transferase (GST) tag to the N-terminus of heparinase III gene and expressed the fusion protein in Escherichia coli to develop an expression system of soluble heparinase III. As a result, approximately 80% of the fusion protein was soluble. The protein was then purified to near homogeneity via one-step affinity chromatography. A 199.4-fold purification was achieved and the purified enzyme had a specific activity of 101.7 IU/mg protein. This represented 32.3% recovery of the total activity of recombinant GST-heparinase III. The maximum enzyme production was achieved when bacteria were induced with 0.5 mmol/L isopropyl-beta-D-thiogalactoside at 15 degrees C for 12 h. The enzyme showed maximum activity at 30 degrees C and pH 7.5. And the enzyme activity was stimulated by 1 mmol/L Ca2+ and 150 mmol/L NaCl.

Antagonistic effects of ultra-low-molecular-weight heparin against cerebral ischemia/reperfusion injury in rats.

Heparin and low-molecular-weight heparin have long been proposed for stroke treatment. This study was conducted to demonstrate the antagonistic effects of ultra-low-molecular-weight heparin (ULMWH) on cerebral ischemic injury in rats and the mechanisms underlying the effects. Male Wistar rats were subjected to middle cerebral artery occlusion (MCAO) for 2h followed by reperfusion for 24h. ULMWH (0.5, 1 mg kg(-1), i.v.) was administered after the MCAO and reperfusion. Twenty-four hours after the reperfusion, neurological deficit scores, body weight and infarct volume were assessed. Spectrophotometric assay was used to determine the activity of superoxide dismutase (SOD) and content of malondialdehyde (MDA) of the brain. Furthermore, the intracellular Ca(2+) concentration ([Ca(2+)]i) was measured. The results showed that vein injection of ULMWH at doses of 0.5 and 1.0 mg kg(-1) exerted significant neuroprotective effects on rats with focal cerebral ischemic injury via significantly decreasing neurological deficit scores, increasing body weight, reducing the infarct volume. At the same time, ULMWH significantly decreased MDA content, and increased SOD activity in ischemic brain. Compared with model group, ULMWH decreased the intracellular calcium concentration remarkably. All these findings suggest that ultra-low-molecular-weight heparin might act as a neuroprotective agent useful in the treatment in focal cerebral ischemia.