Ehrlich II –2nd World Conference on Magic Bullets

Results: The key to the novel one-pot procedure for producing single tubulysin is our discovery that an N,O-acetal can be converted in situ in stabilized N-acyliminium ion and subsequent reacted with a nucleophile. The FA-spacer was synthesized using solid phase protocol. The conjugates were prepared applying an original one-pot synthetic approach. Finally, the release of the parent drug from the conjugate was demonstrated.

Conclusions: These studies demonstrate a universal, simple, and easily scalable synthetic approach to natural tubulysins and their FA conjugates. All compounds exhibit potent cytotoxicity towards FR-positive KB cells. The conjugates have demonstrated their selective targeting of FR-expressing cell lines.

Adenosine-Based Therapies for Hearing Loss VLAJKOVIC SM^1, GUO CX^1, WONG A^1, LEE KH^1, GUPTA R^1, HOUSLEY GD^1,2, THORNE PR^1 ^1The University of Auckland, Auckland, New Zealand; ^2University of New South Wales, Sydney, Australia Background: Oxidative stress is the key element in the pathogenesis of many forms of cochlear injury, for example from noise exposure, cytotoxic drugs and aging. Hearing loss from noise exposure (NIHL) is a leading occupational disease, with more than 10% of the population at risk world-wide. This study investigates adenosine-based experimental strategies to stem NIHL. Here we present the first viable pharmacological intervention that can ameliorate noise-induced cochlear injury in the post-exposure period. Methods: Wistar rats were exposed to narrow-band noise (8-12 kHz, 110 dB SPL, 24 hours) sufficient to induce permanent hearing loss. Gene expression levels of adenosine receptors in the noise-exposed cochlea were studied using quantitiative RT-PCR. Selective adenosine A1 receptor agonist Adenosine Amine Congener (ADAC) lacking cardiovascular side effects was administered intraperitoneally (100 µg/kg/day) at time intervals after noise exposure. Hearing thresholds were assessed using auditory brainstem responses (ABRs) and the hair cell loss was evaluated by quantitative histology. Free radical damage induced by reactive oxygen and reactive nitrogen species was assessed using 4-hydroxynonenal and nitrotyrosine markers respectively. Results: Adenosine A1 receptors were up-regulated during sustained noise exposure suggesting their role in cochlear response to noise stress. The treatment with ADAC after noise exposure led to a substantial recovery of hearing thresholds at all frequencies tested (4-24kHz). Earlier treatment starting at 6 hours after noise exposure provided greater recovery than late treatment starting at 24 hours post-noise. These results were upheld by increased survival of sensory hair cells and reduced nytrotyrosine immunoreactivity in ADAC-treated cochlea. The activation of A1 adenosine receptors thus ameliorated damage to the sensorineural tissues in the cochlea, leading to the functional recovery of auditory thresholds. Conclusions: This study strongly suggests that ADAC could be a valuable treatment for noise-induced cochlear injury in instances of both acute exposure to impulse noise and to chronic or intermittent exposures of longer duration. ADAC may also become a drug of choice for other inner ear pathologies based on oxidative stress. Authors’ disclosure statement: All studies were approved by the University of Auckland Animal Ethics Committee. Supported by the RNID (UK), Deafness Research Foundation (NZ), Auckland Medical Research Foundation, and Health Research Council (NZ).	Then - Macrocirculation and Antibacterial Treatment – and Now: Microcirculation and Antiviral Treatment VOGEL GE, BACK TH, BLASINI R, BRANDL R, HECKLER R, VON ITZSTEIN M, KOMM CH, MANYCH M, MÖLLER R, SCHÖTTLER M, SCHREIBER M, SCHWAIGER M In this era of antiviral treatment inflammation is increasingly being suspected again of also playing a role in cancer, arteriosclerosis, multiple sclerosis, diabetes and arthritis (www.griffith.edu.au). As early as 1860 Robert Koch, and later Paul Ehrlich (syphillis and heart disease), saw a connection between inflammation and arteriosclerosis. The discovery of antiviral treatment against influenza (Mark v. Itzstein, 1993) drew attention to the endothelium (endotheliotropism of the virus). There is no infection that does not develop via vessels, endothelium and coagulation (Norbert Heimburger). Endothelial dysfunction and inflammatory processes are important risk factors. Chronic inflammatory metabolic condition affects almost all cells and their microcirculation. The important thing for us in this regard is that the first contact for the acutely ill patient is a family doctor with a new quality (ESWI III, Portugal, 9/2008, Abstract #1325371, #1324914). Thus we were able to treat as outpatients 233 patients of all ages and both sexes with an influenza infection (confirmed by PCR), none of whom had to be admitted to hospital. We also applied this knowledge in the treatment of patients with acute respiratory tract infections caused by other viruses (RSV et al. reported on 1408 patients) and found that 37% of these patients had developed Community Acquired Pneumonia (CAP). The approach in future will be an exact identification of the virus followed by the development of a specific antiviral treatment. Our guiding principle, which we have followed consistently in our approach, namely: „If you understand influenza you understand the whole of virology“ (England 1950), summarizes all the knowledge and experience gained with regard to microcirculation and microinflammation that should be applied forcefully. The costs for the health care system in Germany alone are predicted to reach 100 billion Euros – a worthwhile endeavour.
Budesonide - Breath-giving innovation for asthma patients VOLOVITZ B Schneider Children's Medical Center of Israel, Petach Tikva, Israel Background: Asthma affects an estimated 300 million people worldwide, causing absenteeism from school and work, disability, and even death. Asthma was initially considered a disease of the smooth airway muscles, and treatment consisted of bronchodilators targeted at reducing the muscle constriction in order to relieve symptoms of wheezing and shortness of breath. It was not until the 1980s that researchers recognized asthma as an inflammatory disease wherein symptoms occur only when the underlying inflammation remains untreated or insufficiently controlled. Methods: Review of the literature indicates that experts and physicians agreed that maintenance therapy in all patients with persistent asthma should be based on an anti-inflammatory drug, preferably an inhaled corticosteroid. Budesonide is an inhaled corticosteroid introduced in 1981 for the treatment of asthmatic inflammation. It has since been investigated in more than 600 published studies and become the first-line therapeutic agent for asthma. Budesonide is currently approved in 112 countries, and has been used to date on more than 13 billion treatment days. Results: Budesonide has been shown to be both effective and safe for the treatment of asthma, with improved ratio of topical to systemic potency. Budesonide significantly improved patient health-related quality of life and airway function, and has dramatically changed the life for patients with asthma. Clinical studies found it to be more effective than cromoglycate, theophylline, bronchodilators, and epidemiological studies reported a significant decrease in mortality, hospitalizations, sick-listings, and disability. Budesonide has also been central to many recent evidence-based innovations such as: once-daily administration and protection against both early and late asthmatic reaction and exercise-induced bronchoconstriction. Furthermore, researchers found that with early dose adjustments of budesonide, asthma exacerbations were avoided. Data on the safety of budesonide are extensive and comprehensive. Children treated with long-term budesonide achieved normal final height, and its use during pregnancy and lactation was not associated with adverse effects in either mother or fetus. Conclusion: Budesonide is effective and safe for the long-term treatment of asthma. Its use has revolutionized asthma therapy and provided patients with a better quality of life.	The Development of Influenza Virus Sialidase Inhibitors as Anti-Influenza Drugs VON ITZSTEIN M Institute for Glycomics, Griffith University, Gold Coast Campus, Queensland, Australia [e-mail] The important, and sometimes essential, roles of carbohydrates and the proteins that recognise them in biological processes are diverse [1]. When these roles are associated with a disease a potential drug discovery opportunity is presented. For example, a range of clinically significant pathogens, including viruses, parasites and bacteria utilise carbohydrates and their associated proteins to invade their host, facilitate their lifecycle and as a consequence produce disease [2]. Viruses such as influenza virus, rotavirus, and dengue virus all have essential carbohydrate recognition processes in their replicative cycles that present possible drug discovery targets [2,3]. We have had a long-term interest in influenza virus and the discovery of novel influenza virus sialidase inhibitors that has now provided a novel class of anti-influenza drugs [4-6]. Some of our most recent work and advances towards the development of drugs to treat or prevent influenza, including pandemic influenza, will be presented. [1] Carbohydrate-based Drug Discovery, edited by Wong, C-H, Wiley-VCH, Weinheim (2003).[2] Carbohydrates in Chemistry and Biology, edited by Ernst, B, Hart, GW, Sinaÿ, P, Wiley-VCH, Weinheim (2000). [3] Rich JR, Gehle D, von Itzstein M: Design and synthesis of sialidase inhibitors for influenza virus infections. In Comprehensive Glycoscience, edited by Kamerling JP, Boons G-J, Lee YC, Suzuki A, Taniguchi N, Voragen AGJ Elsevier (2007) pp 885-922. [4] von Itzstein et al (1993). Nature, 363:418-423. [5] Mann, MC, Islam, T, Dyason, JC, Florio, P, Trower, CJ, Thomson, RJ and von Itzstein, M (2006). Glycoconj. J., 23:127-133. [6] von Itzstein, M (2007). Nature Drug Discov Rev, 6:967-974. [7] Haselhorst T, Garcia J-M, Islam T, Lai JCC, Rose FJ, Nicholls JM, Peiris JSM, von Itzstein M (2008). Angew Chem Int Ed, 47:1910-1912.
Chemical Postevolution of Antibacterial Natural Products VON NUSSBAUM F Bayer Schering Pharma AG, Medicinal Chemistry Europe, D-42096 Wuppertal, Germany Multi-resistant and hyper-virulent microbes have become a physicians’ nightmare in hospitals and in the community (USA300).[1] These aggressive “superbugs” demand “superdrugs” addressing novel therapeutic approaches without cross-resistance to antibiotics in clinical use. Natural products have been the most important source for new antibacterial drug classes.[2] Even today, natural antibiotic lead structures have not lost there value as guideposts for novel targets and future therapy (Reversed Genomics). Most natural products cannot be used in the clinics. However, medicinal chemistry provides the tools to transform natural products into drugs with improved pharmacokinetic and toxicological properties (Chemical Postevolution).[2b] We have investigated the medicinal chemistry of natural depsipeptide antibiotics such as the katanosines[3] or enopeptins as valuable lead structures on our search for new antibacterial therapies.[4] By their modular structure, cyclic peptide antibiotics[5] are particularly well suited for systematic chemical modifications by means of semisynthesis and de novo synthesis. Synthetic strategies, structure-activity relationships, in vitro potency and in vivo efficacy will be discussed. [1] S. J. Projan, P. A. Bradford, Curr. Opin. Microbiol. 2007, 10, 441–446. [2] a) M. S. Butler, A. D. Buss, Biochem. Pharmacol. 2006, 71, 919–929; b) F. von Nussbaum, M. Brands, B. Hinzen, S. Weigand, D. Häbich, Angew. Chem. 2006, 118, 5194–5254; Angew. Chem. Int. Ed. 2006, 45, 5072–5129. [3] F. von Nussbaum, S. Anlauf, J. Benet-Buchholz, D. Häbich, J. Köbberling, L. Musza, J. Telser, H. Rübsamen-Waigmann, N. A. Brunner, Angew. Chem. 2007, 119, 2085–2088; Angew. Chem. Int. Ed. 2007, 46, 2039–2042. [4] a) K. H. Michel, R. E. Kastner (Eli Lilly), US 4492650, 1985; b) H. Brötz-Oesterhelt, D. Beyer, H.-P. Kroll, R. Endermann, C. Ladel, W. Schroeder, B. Hinzen, S. Raddatz, H. Paulsen, K. Henninger, J. E. Bandow, H. G. Sahl, H. Labischinski, Nat. Med. 2005, 11, 1082–1087; c) B. Hinzen, S. Raddatz, H. Paulsen, T. Lampe, A. Schumacher, D. Häbich, J. Benet- Buchholz, R. Endermann, H. Labischinski, H. Brötz-Oesterhelt, ChemMedChem 2006, 1, 689–693. [5] F. von Nussbaum, S. Anlauf, C. Freiberg, J. Benet-Buchholz, J. Schamberger, T. Henkel, G. Schiffer, D. Häbich, ChemMedChem, 2008, 3, 619–626.	Wheat and barley for celiac patients by molecular silencing of the immunogenic endosperm proteins VON WETTSTEIN D^A,B,C,D, BRUEGGEMANA R, GAMINI C. KANNANGARAA, NII ANKRAHA, RUSTGIA S, LANGENC G, KOGELC KH, PANGD J, LIUD B ^aDepartment of Crop and Soil Sciences and ^bSchool of Molecular Biosciences, Washington State University, Pullman WA 99164-6420, USA, ^cInstitute for Phytopathology and Applied Zoology, Justus-Liebig-University, Giessen, D-35392, ^dKey Laboratory of Molecular Epigenetics, Institute of Genetics and Cytology, Northeast Normal University, Changchun 130024, P.R. China Celiac disease is the most common food-sensitive enteropathological condition in humans. It is caused by an autoimmune reaction against wheat, barley and rye grain storage proteins. In human leukocyte antigen (HLA) DQ2- (or DQ8-) positive individuals exposure to epitopes of these prolamin proteins leads in celiacs to a painful chronic erasure of the microvilli of the epithelium in the intestine and a permanent intolerance of dietary prolamins. Despite its prevalence in most populations comprising 24.4 million registered celiac individuals world-wide, the only effective therapy is strict dietary abstinence from these food grains. Estimates suggest that for every registered celiac there are 50 unrecognized individuals. Cereal prolamins are of two types: high molecular weight glutenins (HMWG) with a molecular structure of elastic fibrils that form dityrosine cross-links during dough formation and baking, and gliadins. The gene promoters of the gliadin-type proteins, but not those of HMWG, are silenced by DNA methylation in vegetative tissues. This methylation is removed during grain development by a 5-methylcytosine deglycosylase to permit transcription and protein synthesis. Silencing of the enzyme by mutation or by RNAi technology in the endosperm prevents the synthesis of gliadins, which contain the vast majority of the different celiac causing epitopes and only the HMWG prolamins will be produced in fully viable plants. Gliadin-type prolamins are of no importance for baking since wheat HMWG has been demonstrated to be alone sufficient to produce high quality breads. This is probably the first project using interference with DNA de-methylation to obtain a remedy for an autoimmune disease.
Fentanyl Analogues: Structure-Activity-Relationship (SAR) Study VUČKOVIĆ S, TODOROVIĆ Z, NEŠIĆ Z, IVANOVIĆ M, STOJANOVIĆ R, DIVAC N, PROSTRAN M University of Belgrade, Belgrade, Serbia Background: Fentanyl is the prototype of the 4-anilidopiperidine class of synthetic opioid analgesics. This study aimed to examine antinociceptive activity of newly synthesized fentanyl analogues supstituted at the position 3, or at the position 4 of the piperidine ring, and to establish structure-activity-relationship (SAR). Methods: The antinociceptive activity was determined by tail-immersion test in rats of both sexes (200-250 g). All drugs were given intraperitoneally. The antinociceptive activity was reversed by subcutaneous administration of naloxone (non-selective opioid antagonist). Results: It was revealed that the analgesic activity in both series of 3-supstituted and 4-supstituted fentanyl analogues is influenced by the steric factor (eg. voluminosity of the group and the cis/trans isomerism), and not by the chemical nature of the substituent. The evaluation of an open chain fentanyl analogues, also suggests the influence of the steric factor upon the analgesic activity and in particular, the importance of the piperidine ring as a key pharmacophore. Conclusions: The potency and the duration of action of these novel fentanyl analogues are interesting from the aspect of SAR studies, and some of them have potential promise for clinical application. This work is supported by Ministry of Science of Serbia (grant no. 145001).	Synthesis of a Novel Subunit Vaccine against HIV-1: Native Envelope Proteins in Lipid Bilayer of Inactivated Virions Devoid of p24, RT and Viral Nucleic Acids VYAS GN Univ. of California, San Francisco, CA, U.S.A. Background: HIV vaccine development remains a continuing challenge. It is our objective to prepare inactivated HIV-1 isolates devoid of p24, RT, and viral RNA, but retaining the quaternary structure of their native envelope proteins. A pool of such isolates derived from human blood can safely elicit protective antibodies against HIV-1. Methods: Plasma from five blood donors with acute HIV infection (PHIV: NAT+ but antibody-) was used for virus isolation. PHIV isolates co-cultured with PHA-stimulated PBMC from transfusable blood were pooled and reacted with Benzonase (BZ) to hydrolyze free cellular/viral DNA/RNA and filtered through a 300 kD membrane. Beta-cyclodextrin (BCD) was used to extract membrane cholesterol and permeabilize the purified virions, allowing BZ to hydrolyze virion-associated DNA/RNA. The quaternary structure of HIV-envelope proteins (gp41 and gp120) was restored by reloading the virion shells with Cholesterol. Results: The PHIV isolates co-cultured with single donors’ PBMC yielded p24 widely ranging between 2.6-175 ng, whereas PBMC pooled from 3-4 unselected donors uniformly yielded 174-177 ng/million cells. Magnetic beads coated with anti-CD45 removed cellular microvesicles from culture supernatants, yielding purified virions (0.6 ug/ml p24), which contained HIV-RNA ~2X10e9 copies/ml (CpmL). After reaction with 300 or 500 mM BCD, virions were consistently negative in PBMC co-cultures. Results tabulated below show residual p24 and HIV-RNA CpmL after each successive step of viral inactivation. HIV-1 reacted with p24 (%) HIV-RNA 0.3 mM BCD 10 1.1X10e4 0.5 mM BCD 4 6.2X10e3 0.3 mM BCD + 1XBZ 2 2.8X10e3 0.3 mM BCD + 2XBZ 0.8 1.2X10e2 0.5 mM BCD + 2XBZ 0.8 0.8X10e2 Thus, viral inactivation with BCD led to loss of >10e5 HIV-RNA CpmL amplifiable by RT-PCR; this was further reduced to 0.8X10e2 by additional reaction with BZ to hydrolyze virion-associated RNA. Since HIV-1 SF2 vaccine stock contained ~10e5 TCID50 and 6.4X10e9 RNA CpmL, the minimal chimpanzee infectious dose (CID-50) of 1X10e2.3 is equivalent to ~5.4X10e 6.7 CpmL. Thus, combined treatment with 0.3 mM BCD and BZ would provide a product with safety margin that is ~2.6X10e3.7 below the minimal in vitro infectivity in chimpanzees. The forgoing results indicate the safety and efficacy of PHIV inactivation, which eliminated p24 and HIV-RNA but retained ~85-90% of the gp120. Conclusions: Our preliminary results provide an impetus for pragmatic refinements and a rational basis for seeking FDA-IND and IRB approvals for a pilot clinical trial of the candidate vaccine for immunotherapy in individuals whose HIV infection is controlled with HAART. Ultimately, broadly neutralizing antibody response in uninfected/at-risk individuals would permit serological distinction between infection and protective immunization.
Proteomic analysis of human breast cancer cells derived from metastatic versus non-metastatic tumors Vydra J^1, Selicharova I^2, Smutna K^2, Sanda M^2, Matouskov E^3, Bursíkova E^3, Prchalova M^3, Velenska Z^4, Coufal D^5, Jiracek J^2 ^1Department of Oncology, 1^st Faculty of Medicine, Charles University Prague, Czech Republic ^2Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic ^3Institute of Biochemistry and Experimental Oncology, 1^st Faculty of Medicine, Charles University Prague, Czech Republic ^4Institute of Pathology 1^st Faculty of Medicine, Charles University Prague and General Teaching Hospital, Prague, Czech Republic ^5Institute of Computer Science, Academy of Sciences of the Czech Republic, Prague, Czech Republic Background: Breast carcinomas are a heterogeneous group of tumors diverse in behavior, outcome and response to therapy. Identifications of proteins that reflect the tumor biology can improve the diagnosis, prediction, treatment selection and targeting of therapy. Methods: Primary cultures of epithelial cells from malignant breast tumors were studied by two-dimensional electrophoresis (2DE). Differentially expressed protein spots were identified by mass spectrometry. The samples were obtained from women who underwent partial breast resection or radical mastectomy for breast cancer at the General Faculty Hospital in Prague. The patients were treated according to stage-adjusted therapeutic standards. We estimated the clinical outcome of the patients. The 23 patients with follow-up at least three years were chosen for further analysis. The patients were divided in two groups: distant metastases-free after three years and patients with proven distant metastases. 2-DE gels in pH range 4-7 were prepared. Spot densities in 2-DE protein maps were subjected to statistical analyses and data-mining analysis. Proteins in selected spots were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Results: Three protein spots were significantly altered between the metastatic and nonmetastatic groups. The correlations were proven at the 0.05 significance level. Nucleophosmin was increased in the group with metastases. The levels of 2,3-trans-enoyl-CoA isomerase and glutathione peroxidase 1 were decreased.