DATHE M, LEUPOLD E, NIKOLENKO H, BEYERMANN M
Leibniz Institute of Molecular Pharmacology, Berlin, Germany
Background: The prerequisite for the successful application of all biological agents is the access to the site of action. Drug application to the brain is a particular challenge. One approach to overcome the blood brain barrier (BBB)-related limitations is the development of drug carriers and their modification with peptides to take advantage of physiology-based strategies.
We introduced a modified apoE-derived sequence, A2 (LRKLRKRLLR)2 as target recognizing and uptake mediating compound. The peptide exhibits characteristics of cell penetrating peptides (CPPs), comprises specific binding sites for the low density lipoprotein receptor (LDLr) and binds non-specifically to cell-surface heparan sulfate proteoglycans (HSPGs). These properties provide the potential for the activation of different cellular uptake modes. Introduction of two palmitoyl chains conferred detergent-like properties upon the cationic sequence. This lipopeptide allowed rapid and easy formation of different particulate systems.
Methods: Spectroscopic and calorimetric methods were used to characterise the particles and confocal laser scanning microscopy (CLSM) and fluorescence assisted cell sorting (FACS) were used to monitor cellular uptake.
Results: The peptide mediated efficient non-selective cellular uptake of liposomes into different cell lines such as endothelial cells of brain capillaries and large vessels. The uptake mode into capillary endothelial cells is endocytotic, but neither clathrin nor caveolae mediated. The LDLr does not play a role. Cell surface HSPGs are involved in the uptake process, providing an explanation for non-specificity and leading to the suggestion that electrostatic interactions between the carrier and cell membrane initiate macropinocytosis. In contrast, the cellular uptake of micelles is cell specific. P2A2 micelles are efficiently internalized into capillary endothelial cells whereas the uptake into endothelial cells of large vessels is low. The observations imply that on the various cell species different transport routes are activated and the properties of the particulate carrier, such as the size, surface density of the vector peptide, and peptide conformation influence the process.
Conclusion: Our studies lead to the conclusion that small micellar particulate structures with a high surface density of the cationic apoE vector peptide are highly favourable for the uptake into brain capillary endothelial cells.
Polymyxin B: how this Magic Bullet kills Gram-negative Bacteria?
DAUGELAVIČIUS R Vilnius University, Lithuania.
Background: Polycationic antimicrobial peptides are an important component of the innate defence of all species of life. These compounds are active against a broad range of bacterial strains, including antibiotic resistant isolates, and are synergistic with conventional antibiotics. The therapeutic use of a cationic antibiotic polymyxin B (PMB) was abandoned for a long time due to its undesirable side effects. However, the spread of resistance to currently used antibiotics has forced the reevaluation of PMB for clinical use. Using different substrates and inhibitors of energy metabolism we obtained information on the mechanism of PMB interaction with bacterial outer membrane (OM) as well as with the plasma membrane (PM).
Methods: An electrochemical monitoring of K+, Ca2+, H+, tetraphenylphosphonium (TPP+), and phenyldicarbaundecaborane (PCB-) ion fluxes across envelopes of E. coli and Pseudoalteromonas spp. cells was performed. In parallel, the cell binding of fluorescent compound dansylpolymyxin, OD of bacterial suspensions, ATP content of cells, and bactericidal activity of PMB were studied.
Results: Using different conditions of cell incubation, the OM permeabilizing activity of PMB was dissected from the PM depolarizing effects. These two stages were easily distinguishable in the presence of high concentrations of divalent cations and can be separated in time by 1-5 min interval. PMB-induced pores in bacterial envelope were registered, but the pore formation and depolarization of the PM were not obligatory for the dissipation of cell K+ gradient or the bactericidal action of this antibiotic. At conditions of increased ionic strength the dependence of membranotropic activity of PMB on metabolic state of the cells was discovered. Energization of the cells by glucose stimulated the binding of PMB to bacteria and the depolarizing activity of this antibiotic. Membranotropic effects of PMB were considerably stronger when all amount of the drug was added to the cell suspension at one stroke.
Conclusions: 1. At low concentrations PMB compromises the barrier of the OM, while at higher concentrations it also depolarizes the PM by forming ion-permeable pores. 2. High ionic strength prevents the self-promoted entry of PMB into bacterialcells, though the ability to bind to the OM surface is not affected. 3. Suppression of energy metabolism of bacteria makes them more resistant to PMB.
Molecular Mechanism of Action and Cellular Consequences of the DNA Minor Groove Alkylating Agent S23906-1. LENGLET G1, DEPAUW S1, DAVID-CORDONNIER M-H1 1 INSERM U837-Centre de Recherches Jean-Pierre Aubert (JPARC), Team 4 "Molecular and Cellular Targeting for Cancer Treatment", Institut de Recherche sur le Cancer de Lille, Place de Verdun, F-59045 Lille cedex, France.
Background: Derived for the plant alkaloid acronycine, the drug candidate S23906-1 was synthetised on the basis of the stabilization of the natural reactive epoxide derivative of acronycine and addition a linearized phenyl group to increase its propensity to alkylate DNA. From a series of derivatives, S23906-1 was selected to enter in phase 1 clinical trial over its potent cytotoxic effects and its high antitumor potency on a wide variety of pre-clinical models.
Methods: The precise mechanism of action of S23906-1 over DNA was assessed using various biophysical, molecular and cellular approaches among which mass spectrometry, HPLC, EMSA, nuclease S1 cleavage, genomic DNA fluorescence studies, cytotoxicity measurements and transfection analysis.
Results: At the molecular level, S23906-1 alkylates guanine at the N2 position as does ET-743 (Trabectedin, Yondelis TM). By contrast to ET-743, S23906-1 does not stabilize the double helix of the DNA but induces a local opening of the DNA. At the cellular level, S23906-1 induces apoptosis in manner directly dependent on its DNA alkylation potency in correlation with double stand break formation in cells. In order to evaluate the implication of the local destabilization of the DNA helix on cellular toxicity, we identified, using a proteomic approach, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) as a protein specifically interacting with S23906-1 DNA adduct. GAPDH is a multifunctional protein implicated in glycolysis, DNA repair and apoptosis processes but also interacts with both double- (dsDNA) and single-stranded DNA (ssDNA). Using EMSA, no binding of GAPDH on ET743/DNA adduct was evidenced. The cellular consequences of GAPDH interaction with S23906-1/DNA adducts are evaluated using siRNA and transfection approaches.
Conclusions: S23906-1 is a potent DNA minor groove alkylating agent presenting particular molecular and cellular mechanisms of action contrasting with that of other DNA minor groove alkylating agents such as ET-743. Evidencing the role of specific DNA adduct recognition by cellular proteins would help understanding the link between the primary DNA modification and the resulting cell death process.
New neutralizing monoclonal antibodies from HIV-1 subtype C and CRF02_AG infected people. BALLA-JHAGJHOORSINGH S 1, CORTI D 2, LANZAVECCHIA A 2, DAVIS D 3 and The BMGF CAVD UCL Vaccine Discovery Consortium.
1. Institute of Tropical Medicine, Antwerp, Belgium; 2. Institute for Research in Biomedicine, Bellinzona, Switzerland; 3. Biomedical Primate Research Centre, Rijswijk, the Netherlands;
Background: The aim is to develop prophylactic human immunodeficiency virus vaccines targetting subtype C and circulating recombinant form CRF02_AG which are currently most prevalent. The recombinant prime, peptide boost immunization strategy has protected rhesus macaques against infection with a neutralization sensitive SHIV which, however, was constructed from a subtype B isolate albeit a variant of the immunogens. Protection correlated with activity in extended incubation phase neutralization assays against virus prepared in primary human cells. To develop this immunization strategy further, combinations of immunogens which can induce antibodies able to cross-neutralize fully heterologous isolates have to be identified. First, monoclonal antibodies with these properties need to be isolated. They can then be used to select recombinant glycoproteins and construct peptides.
Methods: The subtypes of HIV-1 from infected patients attending the clinic in Antwerp were determined. Plasma from individuals infected with subtype C or CRF02_AG was mixed with dilutions of primary virus from a panel of HIV-1 isolates and incubated for 24 hours. Residual infectious virus was quantified following exposure of this mixture to phytohaemagglutinin transformed human peripheral blood mononuclear cells for one hour. The presence of infectious virus was determined by HIV-antigen ELISA of supernatants after 14 days’ culture. Reductions in infectious titre were calculated and expressed as neutralization indices. Memory B lymphocytes from individuals with cross-neutralizing antibodies were isolated, stimulated to divide and immortalized by Epstein-Barr virus. Dividing cultures were initially screened for antibodies binding to recombinant HIV-1 envelope gp140. Neutralizing activity of the monoclonal antibodies was subsequently determined in a standardized pseudovirus HOS.CD4-CCR5 based cell assay.
Results: Patients with plasma showing subtype-associated cross-neutralization were identified. So far, two antibodies with broad cross-neutralizing activity and a potency comparable to currently available human monoclonals have been isolated. Other antibodies have a more restricted range while recognising a variety of known epitopes on the external envelope glycoproteins although a subgroup bind to potentially novel neutralization epitopes.
Conclusions: New cross-neutralizing antibodies are available which can identify immunogens for use in recombinant prime, peptide boost immunization strategies against HIV-1 subtype C and CRF02_AG isolates.
Effect of antineoplastic agents on the surface properties of bacterial cells de CARVALHO CCCR IBB-Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Técnico, 1049-001 Lisbon, Portugal. Tel. +351218419594; Fax: +351218419062; E-mail: ccarvalho@ist.utl.pt
Background: The study of the effects of antineoplastic agents on bacteria is important to understand post-chemotherapy infections, generally only ascribed to immune suppression. Aims: 1) To evaluate the effect of drugs, such as mitomycin C and cyclophosphamide, on the viability and surface properties of bacteria. 2) To assess if the cells that survive exposure to these agents are also able to grow with antibiotics present.
Methods: Exponentially growing cells of Staphilococcus aureus, Rhodococcus erythropolis, Mycobacterium aurum and Escherichia coli were placed in medium containing 100, 50, 25, 12.5, 6.3, 3.1, 1.6 and 0.8 g/mL of mitomycin C (MC) or cyclophosphamide (CPP). The fatty acid composition of the cellular membranes was analysed by gas chromatography. Cell viability and membrane potential were determined by fluorescence microscopy and image analysis, using fluorescent dyes. Culturable cells were determined by the spread plate technique. Cell hydrophobicity was measured by the “microbial adhesion to hydrocarbon” test. The size of cell clusters and biofilm formation were determined by microscopy and image analysis.
Results: Both MC and CPP affected the fatty acid composition of the cellular membrane of all strains. A dose dependent increase in the degree of saturation of fatty acids occurred after exposure to MC. Both agents caused a dose dependent decrease in cell viability, part of the population presenting depolarised membranes. Cells exposed to MC produced biosurfactants, decreasing the medium surface tension and R. erythropolis cells produced significant amounts of exopolymeric substances on agar plates after 24h exposure to both agents. Cell clustering was promoted by increasing concentrations of these drugs. Cells that were able to grow after 24h exposure to each of the antineoplastic compounds, were also able to grow in the presence of 25 g/mL of chloramphenicol, suggesting cross-resistance between them.
Conclusions: 1) Bacterial cells are able to survive relatively high concentrations of both MC and CPP. 2) These cells can also grow in the presence of chloramphenicol. 3) Post-chemotherapy infections could be promoted by an adapted bacterial population after antineoplastic exposure.
The High Cytotoxicity of Cisplatin Nanocapsules in Ovarian Carcinoma Cells Depends on Uptake by Caveolae-Mediated Endocytosis HAMELERS IH, DE KROON AI Utrecht University, Utrecht, The Netherlands.
Background: Cisplatin is one of the most widely used agents in the treatment of solid tumors. However, its clinical use is limited by toxic side effects and by the occurrence of resistant tumor cell sub-populations. We have developed a novel method for the efficient encapsulation of cisplatin in a lipid formulation that may reduce the side effects and increase the therapeutic efficacy of cisplatin in the clinic (Burger et al., Nat. Med. 8, 2002). The method is unique in that it generates nanocapsules, nanoprecipitates of cisplatin covered by a single lipid bilayer. The nanocapsules exhibit an in vitro cytotoxicity up to 100-fold higher than the free drug toward some but not all human ovarian carcinoma cells. Here we report on the mechanism underlying the cell line dependence of the increased cytotoxicity.
Methods: Cellular platinum accumulation and platinum-DNA adduct formation were analyzed by non-flame atomic absorption spectroscopy. The interaction of fluorescently labeled cisplatin nanocapsules with ovarian tumor cells was investigated by confocal fluorescence microscopy. Endocytic pathways were down-regulated using siRNAs.
Results: The increased cytotoxicity of cisplatin nanocapsules was shown to result from enhanced cellular uptake of encapsulated cisplatin as compared to the free drug, leading to increased formation of platinum-DNA adducts. The origin of the increased accumulation of cisplatin from nanocapsules in the cells was investigated by confocal fluorescence microscopy using nanocapsules containing a fluorescent cisplatin derivative. The results showed that intact nanocapsules are taken up by an energy-dependent mechanism. Co-localization of the fluorescently labeled nanocapsules with markers of early and late endosomes indicated uptake via endocytosis. Transfection of siRNAs against clathrin heavy chain and caveolin-1 in cell lines that differ in sensitivity to cisplatin nanocapsules, revealed that the increased cytotoxicity only occurs after caveolin-1 mediated endocytosis.
Conclusion: The high cytotoxicity of cisplatin nanocapsules in human ovarian carcinoma cells strictly depends on uptake by caveolin-1 mediated endocytosis.
Pneumocystisjirovecii Dihydropteroate Synthase Gene Mutations and Sulfa Resistance de la HORRA C 1,2,MORILLA R1, RIVERO L2, FRIAZA V1, GUTIERREZ S2, RESPALDIZA N1, MARTIN-JUAN J3, MONTES-CANO MA1,4, MEDRANO FJ1,2, VARELA JM1,2, CALDERÓN EJ1,2.
1CIBER of Epidemiology and Public Health, 2Internal Medicine, 3Pneumology and 4Immunology Services, Virgen del Rocío University Hospital, Seville, Spain.
Background: Sulfa drugs, trimethoprim-sulfamethoxazole and dapsone, are mainstays for prophylaxis and treatment of Pneumocystis pneumonia (PcP), a life-threatening disease in immunosuppressed patients. The inability to culture Pneumocystis has led to develop molecular techniques based on identification of punctual mutations on the Dihidropteroate Synthase gene (DHPS) that cause sulfa resistance in other microorganisms. A key issue is whether the emergence of DHPS mutations is result of transmission between patients or arises from selection by the pressure of sulfa drugs, two possibilities are not mutually exclusive. The role of Pneumocystis colonized subjects in transmission of DHPS mutations still unknown. The aim is to provide epidemiological data of P. jirovecii DHPS mutations among PcP patients and immunocompetent colonized subjects.
Methods: The study included 47 PcP patients and 75 Pneumocystis colonized subjects during 2001-2007 identified by nested PCR at mtLSUrRNA gene. DHPS mutations were studied by Restriction Fragment Length Polymorphism using Acc I and Hae III at nucleotide positions 165 and 171 respectively.
Results: The analysis showed a 19.7% prevalence of DHPS gene mutations in the overall population. , All possible polymorphisms described were identified. There were not difference between the frequency of DHPS mutations in PcP patients and colonized subjects (23.4% vs 17.3%; p = 0,75). A trend towards decreased frequency was observed during this period (31.3% of DHPS-mutations during 2001 to 11.6% at 2007).
Conclusions: Similar DHPS pattern was observed in PcP patients and immunocompetent colonized subjects suggests that both group could share a common transmission cycles of mutated strains, and arise question about the role that colonized subjects could represent as reservoir for DHPS mutations with ability to transmit them to immunocompromised hosts susceptible to PCP.
A Simple Microwave-assisted Synthesis of Sulfonamides directly from Sulfonic Acids DE LUCA L1, GIACOMELLI G1 1Univ. of Sassari, Sassari, Italy;
Background: Sulfonamides are an important class of pharmaceutical compounds with a wide spectrum of biological activities. Sulfonamides drugs have broad applications in many areas of clinical medicine, as excellent, diuretics, anticonvulsants, hypoglycemics HIV protease inhibitors, carbonic anhydrase, and caspase inhibitors and in particular as antibacterials.
Methods: We wish to report here an easy and convenient technique for the preparation of sulfonamides directly from sulfonic acid or its sodium salt, improved by microwave irradiation. The method consists of the addition of 1 equiv. of TCT to a mixture of 1 equiv. of sulfonic acid and 1 equiv. of triethylamine in acetone. The reaction was carried out under microwave irradiation in a sealed tube (10-mL pressure-rated reaction vial) in a self-tuning single mode irradiating synthetizer, operating at 80 °C for 20 min. After cooling, the precipitate formed is filtered off on Celite and the solution is added with 1.2 equiv. of NaOHaq., THF and an amine. The reaction mixture is newly exposed to microwave irradiation for 10 min at 50 °C in a sealed tube and then is filtered on Celite to eliminate the formed salts, diluted with DCM and washed with water, aqueous Na2CO3, diluted HCl, and brine. The target product is obtained in pure form and in practically quantitative yield, just by concentration of the DCM extracts at reduced pressure.
Results: A selection of sulfamides were synthesized from an array of sulfonic acids and the yields were satisfactory in all cases. The methodology is proficient and successful with aromatic and aliphatic sulfonic acids. The reaction is not limited to primary and secondary amines, but works well with hydrazines (entry 3) and amino acid derivatives (entry 4). Also anilines are applicable in the reaction (entry 1).
entry sulfonic acid amine product yield%
1 90
2 95
3 “ 92
4 “ 80
Conclusions: The methodology described represents a convenient,handly and high yielding synthesis of sulfonamides even in large scale, as it uses mild reaction conditions and cheap and commercially available reagents.
90
95
92
80