Ehrlich II –2nd World Conference on Magic Bullets

Selective Stimulation Of Human Natural Killer Cells Proliferation By Novel Fucosylated Acidic Glycan Drugs: Possible Therapeutic Use For Treating Cancer And Viral/Retroviral Infections MISEVIC GN, MISEVIC NJ, SUMANOVSKI LT Gimmune GmbH, Zug, Switzerland Background: Definition of natural killer (NK) cells function is to kill cancer and viral/retroviral infected cells. Selective stimulation of different subsets of NK cells proliferation is the prerequisite for specific and effective target killing. The nature of the molecules responsible for such stimulation was not completely established and consequently therapy using NK route could not be valuably achieved. Our aims were: 1) To find a new class of drug molecules which can induce selective proliferation of different subsets of NK cell populations in humans. 2) To widen and complement therapeutic methods for treating cancer and viral/retroviral infections using NK cells as the “cellular magic bullet”. Methods: 1) Fucosylated acidic glycans isolated by chromatography and electrophoresis from sponges were sequenced using NMR and MS. 2) Ex vivo stimulation of human NK cells proliferation by these compounds was tested using peripheral blood mononuclear cells (PBMC) cultured in supplemented homologues serum for 1-3 weeks and measured by FACS analyses using antibodies markers for cell identification: CD3, TCR αβ, TCR γδ, CD4, CD8 - T cells; CD 16, CD56 - NK cells; CD20 - B cell; CD 14 monocytes. 3) Ex vivo killing of human tumor cells and viral infected cells with human NK cells were examined microscopically. Results: 1) 33 novel fucosylated acidic glycan compounds were obtained. 2) Treatment of PBMC cultures with these compounds resulted in selective stimulation of proliferation of different NK cell subsets from 1-5% to 30-80%. Untreated controls remained at level of 1-5 %. No significant stimulation of B or T cells is observed. NK cells of all humans tested, from a variety of ethnic and racial groups could be significantly stimulated. 3) The obtained human NK cells showed massive and continuous killing of target human tumor or viral infected cells during five weeks of co-cultured period under condition of 1000 fold target cells excess. Conclusions: 1) Novel class of 33 fucosylated acidic glycans compounds was isolated. 2) These drug compounds selectively stimulated proliferation of different subsets of human NK cells in vitro. 3) NK “cellular magic bullet” therapeutic effect for treatment of cancer and viral/retroviral infections is suggested to widen and complement existing treatments. Authors’ disclosure statement: This abstract contains novel and unpublished data.

Chemistry Of Nitrogen And Sulphur Based Compounds: An Approach Towards The Discovery Of Magic Bullets MISHRA AK Department of Chemical Technology, University of Johannesburg, Doornfontein Campus, Johannesburg, South Africa Background: The synthesis, physico-chemical investigation and biological studies of metal complexes of thiodimaine ligands are described which act as magic bullet for drug discovery in the area of bio-inorganic chemistry. Aims: 1) To synthesized platinum (IV) and palladium (II) complexes of the thiodiamines and characterized them by elemental analysis, IR, mass, electronic and ^1H NMR spectroscopic studies. 2) To screen the complexes for cytotoxicity, in vitro antifungal and in vitro antibacterial activities. In vitro antifungal and in vitro antibacterial studies were performed against fungal and bacterial strains, Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger and Escherichia coli respectively. Methods: The following scheme involves for the synthesis of thiodiamine ligand and their metal complexes Results: A) The electronic spectra of the thiodiamines show spectral bands because of ^* and n^* transitions. Strong bands 340 nm is assignable to a combination of metal ligand charge transfer and d-d band. Strong bands at 2920-3250 cm^-1 in thiodiamines are assigned due to –NH stretching vibrations of NH2 and NH groups. The NH stretching of ligand has been found to shift to higher frequencies, the change being associated with coordination of terminal NH2 nitrogen to the metal ion. Conclusions: 1) The spectral studies indicate that the complexation takes place to the metal ion is through nitrogen and sulphur. 2) The in vitro antifungal activity of complexes as compared with standard drug Amphotericin B shows the minimum inhibitory concentrations (MICs) by microbroth dilution assays (MDA) and percent spore germination inhibition assays (PSGIA) are found to be 125-500 mg/mL. The in vitro antibacterial study of the complexes as compared with standard drug gentamycin shows the bacterial strains with the zone of inhibition were observed, 8-10 mm. The cytotoxic activity on primary adenocarcinoma show good activity at 100 mM solution as compared to standard drug cisplatin.

Ruthenium Complexes In Cancer Therapy: In Vitro And In Vivo Studies MISHRA L^1, KOIRI R^2, TRIGUN SK^2 ^1Department of Chemistry, Faculty of Science, Banaras Hindu University, Varanasi, India; ^2Department of Zoology, Faculty of Science, Banaras Hindu University, Varanasi, India Background: Ruthenium-protein interaction leading to modulate the enzymes of cancer associated metabolic events is an evolving concept for anticancer ruthenium complexes. Lactate dehydrogenase (LDH; EC: 1.1.1.27) is found as the critical enzyme implicated in maintaining the tumor growth via executing ‘Warburg effect’ in cancerous cells. Although implication of M4-LDH in tumor development is quite evident (Koukourakis et al., 2003; Fantin et al., 2006), baring some exception (Niakan, 2001), reports are scanty on LDH inhibition dependent regression of tumors in vivo. Methods: Several Ru(ll) and Ru(lIl) polypyridyl complexes containing azolo/aryl-diazo pentane 2,4-dione, derivatives, 2,6-(2'-benzimidazolyl)-pyridine, 1,2,4-triazole derivative ,chalcones ,and flavones as co-Iigands developed in our laboratory are tested against various tumor cell lines at Univ. of North Carolina USA for their cytotoxic and anti H IV activities [Bioorg. Med. Chem., 9, 1667,2001. Indian J. Experimental Biology, 42,660-666, 2004.J. Inorganic Biochemistry,99,1113-1118,2005]. To understand their mode of action on a preliminery level, their interaction with DNA (calf-Thymus) are monitored by UV.visible luminescence and NMR spectroscopic techniques. (New J. chem. (Royal Society Journal) 24, 505, 2000; Indian J. chem. 39A, 1295,2000). Results: Ru(ll) and Ru(lIl) polypyridyl complexes containing azolo/aryl-diazo pentane 2,4-dione, derivatives, 2,6-(2'-benzimidazolyl)-pyridine, 1,2,4-triazole derivative ,chalcones ,and flavones as co-Iigands showed cytotoxic activity as Ic50 0.8 and1.14I-μg/ml respectively against ovarian carcinoma and lung adeno carcinoma. This type of preliminary results suggested them as future drugs. We could recently describe LDH as a potential target of some metal complexes in vitro and in vivo including Ru (II) –Flavone and Ru (II) -CNEB .(Mishra et al., Indian J. Experimental Biology, 42,660-666, 2004. and Current Enzyme Inhibition 3, 243-353, 2007). Conclusions: Currently we, have been exploring the mechanism of the action of such classes of drugs The results shows that Ru(II)-CNEB is able to decline M4-LDH significantly and to induce mitochondrial dysfunction – cytochrome c release- apoptotic pathway in DL cells in vivo. A concomitant reduction of DL cell viability with a significant improvement in the general living parameters of the tumor bearing mice, without producing any physiological and metabolic toxicity. The current findings will be presented which provides a biochemical basis to evaluate/screen anti tumor activity of related metal complexes on a number of tumor models.

Perfluorocarbons(PFC) As Universal Remedial Of Free Radical Homeostasis At The Wide Spectrum Of Various Metabolic Disorders Miskevich D^1, Petushok N^1, Borodinsky A^1, Gerasimchyk P^2 ^1Regulation of metabolism department, Institute of Pharmacology and Biochemistry, Grodno, Belarus.; ^2Grodno state Hospital, Grodno, Belarus Background: Routine biomedical applications for PFC and their emulsions include their use as a gas carriers, due to their ability transport and upload O2 and CO2. It’s ability to dissolve great volumes of non polar gases (O2 and CO2) really dramatic. Oxygen solubility in PFC liquids is about 20–25 times greater than in either water or blood plasma under the same conditions. Also, it has been shown both in vitro and in vivo ability of PFC to modulate of physiologically significant pool of NO and other gases inorganic mediators. So, from our point of view, experience of use PFC may be extended for the account of wide spectrum metabolic disorders which caused or accompanied by free radical(FR) imbalance. Methods: A. Experiments were performed on male Wistar rats (180g). Animals fed ethanol (EtOH) in drug dose (3.5 g/kg, intragastrically, 25% solution) twofold per day during 42 days. I group was sacrificed after 1 day, II-III groups were sacrificed after 7days following the last EtOH injection. During the last 7 days of experiment, animals of II group were two fold PF injected (1ml/100 gr. i.v.), III group were two fold NaCl 0.09% solution (injected 1ml/100 gr.i.v.). B. Experiments were performed on male Wistar rats (160g). Experimental animals fed EtOH in drug dose (4.0 g/kg, intragastrically, 25% solution) twofold per day during 60 days, but control animals were treated by NaCl 0.09% solution the same period. All animals were sacrificed after 12 hours after last injection. Group I was injection by EtOH only, group II was treated ethanol and last 3 weeks simultaneously EtOH and PF(1ml/100 gr. i.v., one fold per week ). The activities of FR utilising enzymes, markers of alcohol injury of cells aminotransferases, gamma-glutamyltranspeptidase (GGTP), level of thiobarbituric acid-reactive substances (TBARS) and nitrite (NOx) were measured using Greiss reagent assay. C. The accessible literature about experience PFC application in clinic and experiments has been analysed and systematized. Results: In studies A. and B. animals which fed EtOH, have strongly pronounced oxidative stress (FR processes activated, increased level of TBARS, greatly activated activity of GGTP, damaged hepatocyte membranes). PF application normalized level of lipid peroxidation and activity of GGTP, but strong reduced both liver and plasma NOx content. In other words PF can effective correct metabolic changes in the liver caused by EtOH administration. C. Experience PFC application were shown similar immunomodulating, antioxidant, membrane-stabilizing and disintoxicating properties at the wide spectrum disorders. Conclusions: Results suggest that the PF application at various metabolic disorders (cardiology, oncology, ophthalmology etc.) had one general basis, founded on modulation level of FR, restoration cell membranes and may be a conceptually new pharmacological tool for various metabolic complications associated with free radical imbalance. In the work were discussed main mechanisms of the obtained findings.

Aptamers As Magic Bullets And Delivery Vehicles In Disease Imaging And Therapy MISSAILIDIS S Department of Chemistry and Analytical Sciences, The Open University, Milton Keynes, UK Background: The term of ‘magic bullet’, coined so long ago, would be most appropriate for these novel targeting modalities termed aptamers. Aptamers are nucleic acid in nature, and although they are larger than traditional chemotherapy agents (~10kDa), they are smaller than antibodies and other protein-based biologics. Furthermore, they have high affinity and specificity for their target, significantly higher than that of small molecules or peptide and even antibody therapeutics. Method: Aptamers are usually selected by the SELEX methodology and we have used anything between 1 and 10 round of selection and amplification to isolate high affinity and specificity ligands. These are subsequently modified to ensure resistance to nuclease degradation and their target affinity is assessed by SPR, spectroscopic and immunological and immunohistochemical techniques, whilst the pharmacokinetic and tumour penetration properties have been assessed in experimental model systems. Results: Aptamers are ideal magic bullets that can travel throughout the body with minimal immunogenic or toxic effects, without entering cells that are not carrying the aptamers’ cell-surface receptor target, bind specifically and inhibit the action of their target. This has led to the success of aptamers in the market as antiangiogenic agents with aptamers against VEGF for the treatment of macular degeneration, and as inhibitors of nucleolin in clinical trials against cancer and our own aptamers in preclinical studies with excellent tumour penetration properties. Furthermore, where the cell-surface receptor is not part of a vital pathway and its blockade does not lead to cell kill, aptamers have sufficient mass and relevant pharmacokinetic properties to act as carriers of toxins, chemotherapy agents or radionuclides to exert their therapeutic effects. Finally, this therapeutic action of aptamers is complemented with their potential as diagnostic and imaging agents, when their carrier allows distant recognition, such as gamma emitters, contrast agents, NIR emitters or fluorescent agents. An overview of the aptamers’ characteristics and applications in disease diagnosis and therapy, examplified with novel results from own research and that of other groups will be presented.

Toll-Like Receptor (TLR) Agonists And The Induction Of The Innate Immune Response MITCHELL WM^1, STRAYER DS^2, CARTER WA^2 ^1Vanderbilt University, Nashville, USA; ^2Hemispherx Biopharma, Philadelphia, USA Background: TLRs are pattern recognition sensors that induce cellular immune responses to the systemic presence of a variety of microbes. TLR agonists are in active development as pharmaceuticals for a variety of indications. The most advanced in clinical development are –CpG- (TLR9), LPS (TLR4), and dsRNA (TLR3) agonists. Nine of the ten human TLRs utilize a common intracellular signaling pathway. TLR3 employs a unique pathway that may be responsible in part for its relative lack of toxic inflammatory responses. TLR3 ligands have been demonstrated to have broad anti-viral, immunomodulatory, and anti-proliferative responses in a wide spectrum of pre-clinical studies. The parent TLR3 agonist, Poly I:Poly C, is not limited to TLR3 activation and is associated with a variety of toxic responses in humans. The analog, Poly I:Poly C12U, is active only as a ligand for TLR3 and is not associated with dose limiting toxicities which facilitates clinical utilization for a variety of indications. Methods: A variety of studies have demonstrated the utility of Poly I:Poly C12U as an inducer of innate immune responses in the prophylaxis and treatment of human diseases. These include:1) Phase 2 and 3 clinical trials for chronic fatigue syndrome (CFS), 2) utilization as an immune enhancer for avian HPIV vaccines, and 3) treatment of human renal cell carcinoma. Results: 1) Double-blind, placebo-controlled, Phase 2 and 3 clinical trials with Poly I:Poly C12U have demonstrated efficacy in primary end-points and a variety of secondary end-points in well defined CFS. These include exercise tolerance, decrease in drug usage for symptoms of CFS, and a decrease in physician and patient assessments of disease symptoms. Poly I:Poly C12U was well tolerated; 2) The intranasal administration of Poly I:Poly C12U with a Japanese seasonal influenza vaccine in mice provided protection against challenge from the 3 viruses represented in the vaccine as well as challenge with avian H5N1 HPIVs; 3) An open-label study in human renal cell carcinoma (Elson Risk Group =3) was compared to historical controls stratified to Risk Groups 1-5 over a 2 year observation period. Poly I:Poly C12U provided significantly improved survival compared to Risk Groups 2- 5. Conclusions: Poly I:Poly C12U is a well-tolerated and active TLR3 agonist under current clinical development for a variety of indications. Authors’ disclosure statement: Studies on Poly I:Poly C12U have been funded by Hemispherx Biopharma, by contract through NIH (NIAID/ NCI), or by the NIID (Japan). WMM is an independent member of the BOD of Hemispherx Biopharma.

Helicobacter Cysteine-Rich Proteins A And -C, Two Novel Signalling Molecules Form The Family Of Sel1-Like Repeat Proteins Involved In The Modulation Of Pathogen/Host Interactions MITTL P Biochemisches Institut, Universität Zürich, Schweiz The spiral-shaped gram-negative -proteobacterium H. pylori settles in the gastric lumen of primates and higher vertebrates and shows an exceptional adaptation to its ecological niche, resulting in long-lasting infections of the gastric mucosa. The persistence of H. pylori infections is most likely a consequence of its ability to modulate the innate immune system of the host. To identify novel virulence factors we initiated an inverse genetics approach, focusing on a family of secreted proteins that are specific for å-proteobacteria. Due to the high content of disulfide bridges this family was designated Helicobacter cysteine-rich proteins (Hcp). The crystal structures of HcpB and -C served as the prototype structures for the large family of Sel1-like repeat proteins that participate as protein/protein-interaction modules in signaling pathways of bacteria and eukaryotes but not in archea. Expression of Hcps under in vivo conditions was confirmed by the detection of elevated anti-Hcp IgG titres in H. pylori positive patients. Since Hcp knock-out experiments suggested that Hcps might be important for the interaction between the bacterium and its host, we investigated the effects of recombinant Hcps on mammalian leukocytes. HcpA elicits the release of cytokines from naive mouse splenocytes and also affects the morphology and adherence of monocytes in vitro. The sequence analysis of Hcp homologues from a large collection of H. pylori strains uncovered amino acid positions that seem to be important for the functionality of Hcps and for the adaptation to different host populations. Thus, Hcp serve as bacterial signaling molecule that modulate important properties of mammalian immune cells. To gain deeper insights into the adaptation of H. pylori to different host populations we are currently working towards the analysis of the Hcp signal transduction mechanisms. Recent progress in the identification and validation of eukaryotic Hcp receptor molecules will be discussed.

Cadmium Stress Associated Protein Mediated Resistance to Fusarium Infection in Wheat MITTRA B^1 , GHOSH P^2, HENRY SL^3, PATTNAIK SS^4 , DAS TK^5 , GHOSH S^6, BABU CR^7 AND MOHANTY P^8 ^1,4 Fakir Mohan University,Balasore,School of Biotechnology,India; ^2,6Department of Biophysics, University of Delhi, South Campus, New Delhi ,India;^3 Department of Plant Sciences, Institute of Bio-medical and Life Sciences, Universityof Glasgow, UK;^5 Electron Microscopic Facility, All India Institute of Medical Sciences, New Delhi,India ^7 CEMDE,School of Environmental Studies,Delhi University,India52001, India;^8Regional Plant Resource Center,Bhubaneswar,India. Background: Plants do not have a defined immune system directly comparable to that of animals. However, plants respond to pathogen attack by a variety of biochemical means known as defense responses. Particularly, during fungal infection, plants synthesize low molecular mass inhibitory compounds such as, phytoalexins and accumulate pathogen-related (PR) proteins. In many plants, enhanced disease resistance is accompanied by the activation of genes encoding pathogen-related (PR) proteins [30]. Recently, the defense-role of sugarcane glycoproteins against the smut disease in sugarcane caused by Ustilago scitaninea was demonstrated. Similarly, there has been increasing evidence for direct influences of strobilurins, a fungicide, on plant defense physiology, which suggest that in addition to their fungicidal activity, strobilurin also enhances the capability of plants to ward off viral and bacterial pathogens. The anti fungal activity of a strobilurin compound, F-500 (Pyraclostrobin) has been tested in tobacco plants which, enhanced plant resistance against tobacco mosaic virus (TMV) and bacterial pathogen, Pseudomonas syringae pv tabaci . Some chemicals and metal ions are anti fungal in that the treatment of seeds prevents or arrests fungal infection to plants. Cadmium salts are usually used as antifungal . However, cadmium is also phytotoxic, athigh concentration it retards plant growth and development . Exposure of plants to heavy metals like Cd2+ induces production of phytochelatin and some stress associated proteins,SAP. However, no report has been found on Cd2+ pre-exposure imparting resistance to fungal specification .In this communication, we report, for the first time, on a novel mode of antifungal activity of CdCl2 in that a pre-exposure of wheat seedlings to a mild dose of CdCl2 imparts immunity to plants against Fusarium infection.Aims:1) Structural characterization of cadmium associated stress protein(CSAP) induced in wheat with the treatment of a low dose CdCl2 .2)To assess the possible direct and/or indirect role of glutathione(GSH) against fungal infection in wheat apart from its heavy metal detoxification activity. Method: Surface sterilized wheat (Triticum aestivum L.) seeds(100), treated with 0.01 % HgCl2 (m/v) for 10 min and washed thrice in sterile distilled water, were treated with50 μM CdCl2 for 48 h at room temperature (28 ± 2 °C).The seeds were allowed to germinate on filter paper saturated with sterile distilled water. An equal number of seeds without Cd2+ treatment served as control. The Cd content of the control and the treated seedlings were estimated by atomic absorption spectrometry. Seven days old untreated (control) and CdCl2 pre-exposed seedlings with a well developed root system were grown in test tubes containing liquid MS (1/10 strength) medium supplemented with 4-day-old freshly cultured F. oxysporum (1x106) inoculums. The mild dose Cd2+ pre-exposed seedlings maintained in tubes containing F. oxysporum with MS basal medium were considered as test plants while the only Cd2+-exposed seedlings served as comparative control. Seedlings without Cd2+ pre-exposure (control) and seedlings grown in only F. oxysporum inoculums with MS basal medium were also maintained for comparison .A total 48 number of seedlings were used per each set of experiment and repeated for three times. All test and control plants were incubated at 26 °C ± 2 °C .Two seedlings per tubes were considered for better observation. The tolerance of the plant assessed was based on the survival and growth of the seedlings under stressed conditions. Growth and survival data were recorded for a period of another seven days. Fourteen days old germinated seedlings (500 mg) were removed from the seeds and crushed with 10mM sodium phosphate buffer (pH 7.55). Samples were then centrifuged at10000 rpm (2,500 g) using a centrifuge for 10 min. (Remi). The supernatant containing protein was collected and pellet was washed, re-centrifuged and then discarded. Protein was estimated according to Lowry et al. Protein samples were dialyzed against the same phosphate buffer with two changes for 24h each and finally concentrated by using polyethylene-glycol (PEG-60). Extracted protein was characterized on a 10% SDS-PAGE and the molecular weight of the particular protein band of control (without treatment), Fusarium treated, Cd2+ (50μM)-exposed and co-stressed (50μM Cd2+ pre-exposed and then infected with F.oxysporum) seedling was determined from their Rf values. The particular protein band of interest, which was expressed dominantly due to stress induction, was cut out from the particular gels and the protein was electro-eluted in a Bio-rad (Electro-eluter) apparatus, using the electro-elution buffer (Tris 25mM, glycine 192mM and SDS 0.1%, pH 8.3) at a constant current of 8-10mA/glass tube for 5h. The eluted protein was collected, and concentration was estimated and again ran on a 10% SDS-PAGE to establish its homogeneity. Further in-gel triptic digestion and liquid chromatography mass spectrometry (LC-MS/MS) analysis was done. The purified protein was dissolved in water at a concentration of 80 μg cm-3. The N-terminal sequence ofCSAP was obtained by automated Edman degradation,followed by HPLC and UV detection (Edman and Begg1967), using a PPSQ-21A protein sequencer (Shimadzu,Kyoto, Japan). Searches for sequence similarity were performed using Blast P databases. [Regrettably not enough space on this page.]