Lipid II as a Target for Novel Antibiotics: Structural and Molecular Dynamics Studies

Russian Journal of Bioorganic Chemistry - The growing problem of antibiotic resistance in medicine is focusing attention on antimicrobial compounds targeting non-protein molecules, which have more...     

保护需要从意识转为行动

缺少对原因的准确判断和建设性的途径，人与野生动物和谐相处就总是一种空幻。如今，雪豹的命运再一次把这个问题摆在我们面前。请听一位国际雪豹基金会高层人士的见解。     

Insight into Invertebrate Defensin Mechanism of Action: OYSTER DEFENSINS INHIBIT PEPTIDOGLYCAN BIOSYNTHESIS BY BINDING TO LIPID II*

Three oyster defensin variants (Cg-Defh1, Cg-Defh2, and Cg-Defm) were produced as recombinant peptides and characterized in terms of activities and mechanism of action. In agreement with their spectrum of activity almost specifically directed against Gram-positive bacteria, oyster defensins were shown here to be specific inhibitors of a bacterial biosynthesis pathway rather than mere membrane-active agents. Indeed, at lethal concentrations, the three defensins did not compromise Staphylococcus aureus membrane integrity but inhibited the cell wall biosynthesis as indicated by the accumulation of the UDP-N-acetylmuramyl-pentapeptide cell wall precursor. In addition, a combination of antagonization assays, thin layer chromatography, and surface plasmon resonance measurements showed that oyster defensins bind almost irreversibly to the lipid II peptidoglycan precursor, thereby inhibiting the cell wall biosynthesis. To our knowledge, this is the first detailed analysis of the mechanism of action of antibacterial defensins produced by invertebrates. Interestingly, the three defensins, which were chosen as representative of the oyster defensin molecular diversity, bound differentially to lipid II. This correlated with their differential antibacterial activities. From our experimental data and the analysis of oyster defensin sequence diversity, we propose that oyster defensin activity results from selective forces that have conserved residues involved in lipid II binding and diversified residues at the surface of oyster defensins that could improve electrostatic interactions with the bacterial membranes.     

A New Approach for the Discovery of Antibiotics by Targeting Non-Multiplying Bacteria: A Novel Topical Antibiotic for Staphylococcal Infections

In a clinical infection, multiplying and non-multiplying bacteria co-exist. Antibiotics kill multiplying bacteria, but they are very inefficient at killing non-multipliers which leads to slow or partial death of the total target population of microbes in an infected tissue. This prolongs the duration of therapy, increases the emergence of resistance and so contributes to the short life span of antibiotics after they reach the market. Targeting non-multiplying bacteria from the onset of an antibiotic development program is a new concept. This paper describes the proof of principle for this concept, which has resulted in the development of the first antibiotic using this approach. The antibiotic, called HT61, is a small quinolone-derived compound with a molecular mass of about 400 Daltons, and is active against non-multiplying bacteria, including methicillin sensitive and resistant, as well as Panton-Valentine leukocidin-carrying Staphylococcus aureus. It also kills mupirocin resistant MRSA. The mechanism of action of the drug is depolarisation of the cell membrane and destruction of the cell wall. The speed of kill is within two hours. In comparison to the conventional antibiotics, HT61 kills non-multiplying cells more effectively, 6 logs versus less than one log for major marketed antibiotics. HT61 kills methicillin sensitive and resistant S. aureus in the murine skin bacterial colonization and infection models. No resistant phenotype was produced during 50 serial cultures over a one year period. The antibiotic caused no adverse affects after application to the skin of minipigs. Targeting non-multiplying bacteria using this method should be able to yield many new classes of antibiotic. These antibiotics may be able to reduce the rate of emergence of resistance, shorten the duration of therapy, and reduce relapse rates.     

Targeting Ferroptosis as a Novel Approach to Alleviate Aortic Dissection

A variety of programmed cell death types have been shown to participate in the loss of smooth muscle cells (SMCs) during the development of aortic dissection (AD), but it is still largely unclear whether ferroptosis is involved in the development of AD. In the present study, we found that the expression of key ferroptosis regulatory proteins, solute carrier family 7 member 11 (SLC7A11), ferroptosis suppressor protein 1 (FSP1) and glutathione peroxidase 4 (GPX4) were downregulated in aortas of Stanford type A AD (TAAD) patients, and liproxstatin-1, a specific inhibitor of ferroptosis, obviously abolished the β-aminopropionitrile (BAPN)-induced development and rupture of AD in mice. Furthermore, the expression of methyltransferase-like 3 (METTL3), a major methyltransferase of RNA m⁶A, was remarkably upregulated in the aortas of TAAD patients, and the protein levels of METTL3 were negatively correlated with SLC7A11 and FSP1 levels in human aortas. Overexpression of METTL3 in human aortic SMCs (HASMCs) inhibited, while METTL3 knockdown promoted SLC7A11 and FSP1 expression. More importantly, overexpression of METTL3 facilitated imidazole ketone erastin- and cystine deprivation-induced ferroptosis, while knockdown of METTL3 repressed ferroptosis of HASMCs. Overexpression of either SLC7A11 or FSP1 largely abrogated the effect of METTL3 on HASMC ferroptosis. Therefore, we have revealed that ferroptosis is a critical cause of AD in both humans and mice and that METTL3 promotes ferroptosis of HASMCs by inhibiting the expression of SLC7A11 and FSP1. Thus, targeting ferroptosis or m⁶A RNA methylation is a potential novel strategy for the treatment of AD.     

Functional amyloid: Turning swords into plowshares

Evidence is growing at an increasing-pace that amyloid fibers are not just the result of aberrant protein folding associated with neurodegenerative diseases, but are widespread in nature for beneficial reasons. Amyloid is an attractive building material because its robust design and simple repetitive structure make for very durable and metabolically cheap material. But this requires that the production of amyloid be put under firm control. This appears to involve the use of four to five chaperones that are expressed under the control of the same promoter as the amyloid proteins. Significant progress has been made in deciphering this process in E. coli''s csg operon, also found in Salmonella. Recently, we have discovered a new and unrelated operon (fap) responsible for amyloid production in Pseudomonas, which also confers biofilm-forming properties to E. coli. Intriguingly, this operon shares a number of features with csg, namely two homologous proteins (one of which, FapC, has been shown to be directly involved in amyloid build-up) and a small number of auxiliary proteins. However, FapC seems to be less economically structured than its E. coli counterpart, with a smaller number of repeats and very large and variable linker regions. Furthermore, the putative chaperones are not homologous to their csg counterparts and have intriguing homologies to proteins with other functions. These findings suggest that controlled amyloid production has arisen on many independent occasions due to the usefulness of the product and offers the potential for intriguing insights into how nature disarms and reconstructs a potentially very dangerous weapon.Key words: Pseudomonas, Fap, curli, chaperone, recycling, detection, amyloid operation

When proteins make a move, of which we don''t approveWhat is it that always intervenes?Urea and SDS, they have their place I guess,But first: send chaperones!Let strand meet friend,And till their pleats at handThey''ve got to be protected,All their folds respectedTill a state we like can be selected!

Fondly adapted from Tom Lehrer “Send the Marines.”     

靶向白血病干细胞作为治疗的新策略

白血病干细胞被认为是白血病发生、发展及治疗后复发的根源,具有其特异的性状特征,以及自我更新、增殖和分化等能力.近年来以白血病干细胞作为治疗靶点已成为白血病治疗的一种新策略,目前的研究主要针对白血病干细胞表面分子、细胞内信号通路及与微环境的相互作用这三方面展开.对白血病干细胞的进一步认识将有助于提供新的更为有效的治疗靶点.     

Transglycosylation Catalyzed by Almond β-glucosidase and Cloned Pichia etchellsii β-glucosidase II using Glycosylasparagine Mimetics as Novel Acceptors

The stability of almond β-glucosidase in five different organic media was evaluated. After 1 hour of incubation at 30°C, the enzyme retained 95, 91, 81, 74 and 56% relative activity in aqueous solutions [30% (v/v)] of dioxane, DMSO, DMF, acetone and acetonitrile, respectively. Transglucosylation involving p-nitrophenyl β-D-glucopyranoside as donor and β-1-N-acetamido-D-glucopyranose, which is a glycosylasparagine mimic, as acceptor was explored under different reaction conditions using almond βglucosidase and cloned Pichia etchellsii β-glucosidase II. The yield of disaccharides obtained in both reactions turned out to be 3%. Both enzymes catalyzed the formation of (1→3)- as well as (1→6)- regioisomeric disaccharides, the former being the major product in cloned β-glucosidase II reaction while the latter predominated in the almond enzyme catalyzed reaction. Use of β-1-N-acetamido-D-mannopyranose and β-1-N-acetamido-2-acetamido-2-deoxy-D-glucopyranose as acceptors in almond β-glucosidase catalyzed reactions, however, did not afford any disaccharide products revealing the high acceptor specificity of this enzyme.     

A Novel Strategy Involved Anti-Oxidative Defense: The Conversion of NADH into NADPH by a Metabolic Network

The reduced nicotinamide adenine dinucleotide phosphate (NADPH) is pivotal to the cellular anti-oxidative defence strategies in most organisms. Although its production mediated by different enzyme systems has been relatively well-studied, metabolic networks dedicated to the biogenesis of NADPH have not been fully characterized. In this report, a metabolic pathway that promotes the conversion of reduced nicotinamide adenine dinucleotide (NADH), a pro-oxidant into NADPH has been uncovered in Pseudomonas fluorescens exposed to oxidative stress. Enzymes such as pyruvate carboxylase (PC), malic enzyme (ME), malate dehydrogenase (MDH), malate synthase (MS), and isocitrate lyase (ICL) that are involved in disparate metabolic modules, converged to create a metabolic network aimed at the transformation of NADH into NADPH. The downregulation of phosphoenol carboxykinase (PEPCK) and the upregulation of pyruvate kinase (PK) ensured that this metabolic cycle fixed NADH into NADPH to combat the oxidative stress triggered by the menadione insult. This is the first demonstration of a metabolic network invoked to generate NADPH from NADH, a process that may be very effective in combating oxidative stress as the increase of an anti-oxidant is coupled to the decrease of a pro-oxidant.     

Novel Eicosapentaenoic Acid-derived F3-isoprostanes as Biomarkers of Lipid Peroxidation

Isoprostanes (iPs) are prostaglandin (PG) isomers generated by free radical-catalyzed peroxidation of polyunsaturated fatty acids (PUFAs). Urinary F₂-iPs, PGF_2α isomers derived from arachidonic acid (AA) are used as indices of lipid peroxidation in vivo. We now report the characterization of two major F₃-iPs, 5-epi-8,12-iso-iPF_3α-VI and 8,12-iso-iPF_3α-VI, derived from the ω-3 fatty acid, eicosapentaenoic acid (EPA). Although the potential therapeutic benefits of EPA receive much attention, a shift toward a diet rich in ω-3 PUFAs may also predispose to enhanced lipid peroxidation. Urinary 5-epi-8,12-iso-iPF_3α-VI and 8,12-iso-iPF_3α-VI are highly correlated and unaltered by cyclooxygenase inhibition in humans. Fish oil dose-dependently elevates urinary F₃-iPs in mice and a shift in dietary ω-3/ω-6 PUFAs is reflected by an increasing slope [m] of the line relating urinary 8, 12-iso-iPF_3α-VI and 8,12-iso-iPF_2α-VI. Administration of bacterial lipopolysaccharide evokes a reversible increase in both urinary 8,12-iso-iPF_3α-VI and 8,12-iso-iPF_2α-VI in humans on an ad lib diet. However, while excretion of the iPs is highly correlated (R² median = 0.8), [m] varies by an order of magnitude, reflecting marked inter-individual variability in the relative peroxidation of ω-3 versus ω-6 substrates. Clustered analysis of F₂- and F₃-iPs refines assessment of the oxidant stress response to an inflammatory stimulus in vivo by integrating variability in dietary intake of ω-3/ω-6 PUFAs.Isoprostanes (iPs),² a family of prostaglandin isomers, are generated initially in situ by free radical attack on polyunsaturated fatty acids (PUFAs) in cell membranes. There, they can be immunodetected and quantified by mass spectrometry (1). They are then cleaved by phospholipases (2), circulate in plasma, and are excreted in urine (3). F₂-iPs, isomers of PGF_2α (3), derived from peroxidation of arachidonic acid (AA), are the most studied species. F₂-iPs can be quantified in normal animal and human biological fluids and tissues, implying ongoing lipid peroxidation under physiological conditions, despite replete and diversified endogenous antioxidant defense systems (4). The measurement of urinary F₂-isoprostanes has been used to reflect lipid peroxidation noninvasively in several human diseases (5–8). In addition to their utility as markers of oxidant stress (OS), high concentrations of some F₂-iPs also possess biological activity in vitro, including bronchoconstriction (9), vasoconstriction (10), platelet aggregation (11, 12), and adhesion (13). These effects result from iPs acting as incidental ligands at prostaglandin receptors. It is unknown whether this capacity of individual iPs to ligate prostanoid receptors has relevance to the concentration of the multiple endogenous iP species likely to be formed simultaneously under conditions of oxidant stress in vivo.iPs analogous to the F₂-iPs may be formed from other fatty acid substrates (14–19), including the fish oil constituent, eicosapentaenoic acid (EPA) (20). Potentially beneficial effects of EPA consumption have been supported by a variety of epidemiological and interventional studies. EPA competes with AA for access to the cyclooxygenase enzymes (21), reducing production of AA-derived PGs (22, 23). This effect and/or substituted formation of EPA-derived PGs may explain the anti-inflammatory and cardioprotective effects ascribed to fish oils.The relatively unsaturated EPA and docosahexaenoic acid (DHA) in fatty fish are likely to be more susceptible to lipid peroxidation than AA, although it is unknown whether this might constrain beneficial effects derived from a shift in substrate-dependent enzymatic product formation. While F₂-iPs and F₃-iPs are excreted into urine in their original form, the F₄-iPs formed from DHA (Fig. 1) are at least partly metabolized to F₃-iPs before excretion (20, 24). Given the potential utility of noninvasive biomarkers of EPA and DHA peroxidation and our previous work with F₂-iPs (25, 26), we sought to determine whether members of group VI (Fig. 1B), 5-epi-8,12-iso-iPF_3α-VI and 8,12-iso-iPF_3α-VI (Fig. 1C) might be detectable in urine.Open in a separate windowFIGURE 1.F₃-iPs derived from EPA. A, formation and metabolism of isoprostanes. AA is more abundant than EPA and DHA in cell membranes obtained from individuals consuming a Western diet. Isoprostanes are formed from the corresponding PUFA substrate in situ following free radical attack. F₂-iPs and F₃-iPs are excreted into urine in their original form, but F₄-iPs are at least partly metabolized to F₃-iPs before excretion. (F₂-iPs: F₂-isoprostanes; F₃-iPs: F₃-isoprostanes; F₄-iPs: F₄-isoprostanes). B, six types of F₃-iPs. C, 5-epi-8,12-iso-iPF_3α-VI and 8,12-iso-iPF_3α-VI.     

Chitosan-Graft-Polyethylenimine/DNA Nanoparticles as Novel Non-Viral Gene Delivery Vectors Targeting Osteoarthritis

The development of safe and efficient gene carriers is the key to the clinical success of gene therapy. The present study was designed to develop and evaluate the chitosan-graft-polyethylenimine (CP)/DNA nanoparticles as novel non-viral gene vectors for gene therapy of osteoarthritis. The CP/DNA nanoparticles were produced through a complex coacervation of the cationic polymers with pEGFP after grafting chitosan (CS) with a low molecular weight (Mw) PEI (Mw = 1.8 kDa). Particle size and zeta potential were related to the weight ratio of CP:DNA, where decreases in nanoparticle size and increases in surface charge were observed as CP content increased. The buffering capacity of CP was significantly greater than that of CS. The transfection efficiency of CP/DNA nanoparticles was similar with that of the Lipofectamine™ 2000, and significantly higher than that of CS/DNA and PEI (25 kDa)/DNA nanoparticles. The transfection efficiency of the CP/DNA nanoparticles was dependent on the weight ratio of CP:DNA (w/w). The average cell viability after the treatment with CP/DNA nanoparticles was over 90% in both chondrocytes and synoviocytes, which was much higher than that of PEI (25 kDa)/DNA nanoparticles. The CP copolymers efficiently carried the pDNA inside chondrocytes and synoviocytes, and the pDNA was detected entering into nucleus. These results suggest that CP/DNA nanoparticles with improved transfection efficiency and low cytotoxicity might be a safe and efficient non-viral vector for gene delivery to both chondrocytes and synoviocytes.     

CARP-1 Functional Mimetics: A Novel Class of Small Molecule Inhibitors of Medulloblastoma Cell Growth

Medulloblastomas (MBs) constitute an aggressive class of intracranial pediatric tumors. Current multimodality treatments for MBs include surgery, ionizing radiation, and chemotherapy. Toxic side effects of therapies coupled with high incidence of recurrence and the metastatic spread warrant development of more effective, less toxic therapies for this disease. CARP-1/CCAR1 is a peri-nuclear phospho-protein that is a co-activator of the cell cycle regulatory anaphase promoting complex/cyclosome (APC/C) E3 ligase. CARP-1 functional mimetics (CFMs) are a novel class of small molecule compounds that interfere with CARP-1 binding with APC/C subunit APC-2, and suppress growth of a variety of cancer cells in part by promoting apoptosis. Here we investigated MB growth inhibitory potential of the CFMs and found that CFM-4 inhibits growth of MB cells in part by inducing CARP-1 expression, promoting PARP cleavage, activating pro-apoptotic stress-activated protein kinases (SAPK) p38 and JNK, and apoptosis. Gene-array-based analysis of the CFM-4-treated Daoy MB cells indicated down-regulation of a number of key cell growth and metastasis-promoting genes including cell motility regulating small GTP binding protein p21Rac1, and extracellular matrix metallopeptidase (MMP)-10. Moreover, CFM-4 treatment stimulated expression of a number of molecules such as neurotrophin (NTF)3, and NF-κB signaling inhibitors ABIN1 and 2 proteins. Overexpression of NTF3 resulted in reduced MB cell viability while knock-down of NTF3 interfered with CFM-4-dependent loss of viability. CFMs also attenuated biological properties of the MB cells by blocking their abilities to migrate, form colonies in suspension, and invade through the matrix-coated membranes. Together our data support anti-MB properties of CFM-4, and provide a proof-of-concept basis for further development of CFMs as potential anti-cancer agents for MBs.     

ReviewIsoprostanes: Novel Bioactive Products of Lipid Peroxidation

Isoprostanes, are a novel group of prostaglandin-like compounds that are biosynthesised from esterified polyunsaturated fatty acid (PUFA) through a non-enzymatic free radical-catalysed reaction. Several of these compounds possess potent biological activity, as evidenced mainly through their pulmonary and renal vasoconstrictive effects, and have short half-lives. It has been shown that isoprostanes act as full or partial agonists through thromboxane receptors. Both human and experimental studies have indicated associations of isoprostanes and severe inflammatory conditions, ischemia-reperfusion, diabetes and atherosclerosis. Reports have shown that F²-isoprostanes are authentic biomarkers of lipid peroxidation and can be used as potential in vivo indicators of oxidant stress in various clinical conditions, as well as in evaluations of antioxidants or drugs for their free radical-scavenging properties.

Higher levels of F²-isoprostanes have been found in the normal human pregnancy compared to non-pregnancy, but their physiological role has not been well studied so far. Since bioactive F²-isoprostanes are continuously formed in various tissues and large amounts of these potent compounds are found unmetabolised in their free acid form in the urine in normal basal conditions with a wide inter-individual variation, their role in the regulation of normal physiological functions could be of further biological interest, but has yet to be disclosed. Their potent biological activity has attracted great attention among scientists, since these compounds are found in humans and animals in both physiological and pathological conditions and can be used as reliable biomarkers of lipid peroxidation.     

Antibiotics as growth promotants: mode of action

Recent concerns about the use of growth-promoting antibiotics in pig diets have renewed interest in the immunologic and growth-regulating functions of the gastrointestinal (GI) tract. The numerically dense and metabolically active microbiota ofthe pig GI tract represents a key focal point for such questions. The intestinal microbiota is viewed typically as a beneficial entity for the host. Intestinal bacteria provide both nutritional and defensive functions for their host. However, the host animal invests substantially in defensive efforts to first sequester gut microbes away from the epithelial surface, and second to quickly mount immune responses against those organisms that breach epithelial defenses. The impact of host responses to gut bacteria and their metabolic activities require special consideration when viewed in the context of pig production in which efficiency of animal growth is a primary objective. Here, we summarize the working hypothesis that antibiotics improve the efficiency of animal growth via their inhibition of the normal microbiota, leading to increased nutrient utilization and a reduction in the maintenance costs ofthe GI system. In addition, novel molecular ecology techniques are described that can serve as tools to uncover the relationship between intestinal microbiology and growth efficiency.