Ehrlich II –2nd World Conference on Magic Bullets

Vitamin K3 Is A Potent Inhibitor Of Angiogenesis And DNA Polymerase MATSUBARA K^1,2, KAYASHIMA T^1, MIZUSHINA Y^2,3 ^1Hiroshima Univ., Higashihiroshima, Japan; ^2Cooperative Research Cent. Life Sci., Kobe-Gakuin Univ., Kobe, Japan; ^3Kobe-Gakuin Univ., Kobe, Japan Background: Recent studies demonstrate that vitamins not only play essental roles in cellular reactions but also suppress angiogenesis. Angiogenesis is involved in various diseases including cancer, atheroscrelosis and diabetic rethinopathy. Thus, anti-angiogenic activity of vitamins may contribute to human health. We have also shown that vitamin B6 and D3 and vitamin E derivative, which have anti-angiogenic activity, inhibit DNA polymerase. Althgouh anti-angiogenic activity of vitami K2 was demonstrated, it remained unknown whether vitamin K3 had the activities. In this study, we show anti-angiogenic activity of vitami K3 and the mechanisms and its inhibitory effect on DNA polymerase activity. Methods: Anti-angiogenic activity of vitamin K3 was ascertained in an ex vivo angiogenesis model using a rat aortic ring. To elucidate the mechanisms, its effect on in vitro angiogenesis models using human umbilical vein endothelial cells (HUVECs) with regard to HUVEC growth, tube formation and chemotaxis were evaluated. Effect of vitamin K3 on DNA polymerase activities was also examined. Results: Vitamin K3 suppressed angiogenesis in a rat aortic ring assay. HUVEC tube formation and proliferation were suppressed at 25 M and 5 M, respectively. These inhibitory effects were dose-dependent maners. HUVEC migration induced by vascular endothelial growth factor (VEGF) was also suppressed by vitamin K3 at 5 M. Vitamin K3 selectively inhibited DNA polymerase , however vitamin K1 and K2 had no effect on DNA polymerase activities. Conclusions: 1) Vitamin K3 exerted anti-angiogenic activity through inhibiting important angiogenesis processes. 2) Vitamin K3 selectively inhibited DNA polymerase , however vitamin K2, which has anti-angiogenic activity, had no effect. 3) Vitamin K3 could be a potent anti-cancer agent.

Global And Local Structure Of Cisplatin And DNA Base Pair Complex: A Theoretical Study MATSUI T^1, SHIGETA Y^2, HIRAO K^1 ^1Univ. of Tokyo, Tokyo, Japan; ^2Univ. of Hyogo, Aioi, Japan Background: Since cisplatin was discovered by Rosenberg in 1969, platinum complexes have received much attention for their effect as antitumor drugs. Although other anticancer drugs now replace cisplatin, it is used as a benchmark comparator for new drugs. It causes a cell disorder that leads to apoptosis of the living cell. The distorted DNAs are observed in X-ray analysis at 1.65–2.50 Å resolutions and in NMR experiments. Experimentally, it is known that the bridged structure consists of 65% 1,2-d (GpG), 25% 1,2-d (ApG), and the rest is other bridged structures. There are two possible influences of Pt-DNA formation: 1) global structural changes, such as the distortion of the DNA structure, or 2) local structural changes, such as a DNA mutation. Quantum chemistry enables us to understand the chemical reaction in small molecule systems. The aim of this study is to understand the tendencies of bridged structure qualitatively and to focus on the DNA mutation reactions from the view of computational chemistry. In this abstract, we discuss the base-preference of cisplatin. Methods: Because of the computational cost, we confined the system extremely small as two base pairs and cisplatin. We used density functional theory (DFT) and ONIOM method. We also assumed that the Pt atom binds to N7 of G and A. We assumed the binding energy as the energy difference between [Pt(NH3)4]^2++ B1+B2 and Cis-(B1-Pt-B2)+ 2NH3. Results: Following table shows the binding energy of d(B1pB2) (in kcal/mol). Sequence B1=G, B2=G B1=A, B2=G B1=A, B2=A Energy 73.8 56.1 21.3 The bases are likely to bind to the Pt complex in the order d(GpG), d(ApG) and d(ApA). In particular, the binding energy of the d(ApA) is remarkably low in this calculation. Conclusions: From the energetics of two base pairs with the cisplatin, it is theoretically confirmed that the Pt complex is likely to bind in the order d(GpG), d(ApG) and d(ApA), and the Pt atom prefers the N7 site of guanine to that of adenine. This result supports the experimental evidence, where the structure d(ApA) is seldom observed at room temperature. In the presentation, we will discuss the DNA mutation reaction caused by cisplatin binding.

Inhibition Of Tumor Metastasis And Angiogenesis By NK4, Bifunctional Inhibitor Of HGF-Met And Angiogenesis MATSUMOTO K Cancer Research Institute, Kanazawa University, Kanazawa, Japan Background: Hepatocyte growth factor (HGF) and the Met/HGF receptor tyrosine kinase play a crucial role in invasion and metastasis of a variety of cancer cells. HGF induces dissociation of cell-cell contact, breakdown of extracellular matrix, and concomitant migration of cells, thereby potently enhancing cancer invasion and metastasis. Thus, HGF-Met system is a notable molecular target in cancer therapeutics to inhibit tumor invasion and metastasis. Results: We prepared NK4, a competitive antagonist for HGF-Met. NK4 is an internal fragment of HGF that encompassing the N-terminal and subsequent four kringle domains. We thereafter found that NK4 acts as an angiogenesis inhibitor as well as HGF-antagonist. NK4 inhibits angiogenesis driven by vascular endothelial cell growth factor and basic fibroblast growth factor, as well as HGF. Through its binding to a protein differrent from Met receptor, NK4 inhibits cell surface assembly of fibronectin and integrin-mediated signaling, thereby inhibiting angiogenesis. Based on unique bifunctional characteristics of NK4, therapeutic approaches with NK4 have been examined. In a model of colon cancer metastasis, NK4 suppressed liver metastasis. In situ Met tyrosine phosphorylation was inhibited by NK4 and this was associated with inhibition of invasion and spreading of metastases in the liver. NK4 suppressed intrahepatic growth of metastases, mainly by inhibiting tumor angiogenesis. In an orthotopic implantation model of pancreatic cancer, NK4 suppressed peritoneal metastasis, tumor growth, and accumulation of ascites, and this was associated with prolonged survival. Conclusions: The invasive and metastatic behavior of cancer leads to difficulty in attaining a long-term survival. We propose that simultaneous targeting of tumor angiogenesis and the HGF-Met-mediated metastasis by NK4 may prove to be a new approach to treating patients with aggressive tumor. Reviews: Matsumoto K, Nakamura T. Mechanisms and significance of bifunctional NK4 in cancer treatment. Biochem Biophys Res Commun, 333: 316-327, 2005. Matsumoto K, Nakamura T. Hepatocyte growth factor and the Met system as a mediator of tumor-stromal interactions. Int J Cancer, 119: 477-483, 2006. Matsumoto K, et al. Hepatocyte growth aactor and Met in tumor biology and therapeutic approach with NK4. Proteomics, 8: 3360-3370, 2008.

The Effects Of Single-Dose Ethanol Administration To Aldehyde Dehydrogenase 2 Knock-Out Mice: Down-Regulation Of Expression Of Cytochrome P450 2E1 Mrna And Amelioration Of Oxidative Stress In Liver Tissue MATSUMOTO A^1, KAWAMOTO T^2, ISSE T^2, OYAMA T^2, OISHI H^1, ICHIBA M^1 ^1Saga University, Saga, Japan; ^2University of Occupational and Environmental Health, Kitakyushu, Japan Background: The polymorphism of aldehyde dehydrogenase 2 (ALDH2), denoted ALDH2*2, is very common in East Asian people. Acetaldehyde, an intermediate metabolite of ethanol, is metabolized very slowly in people with ALDH2*2 because the mutant ALDH2 protein lacks the activity of acetaldehyde metabolism. On the other hand, one of the cytochrome P450 enzymes, CYP2E1, is well known as an activator of carcinogens and a generator of oxidative stress, and CYP2E1 is induced by ethanol via gene transcriptional regulation. Oxidative stress is also generated when ethanol is metabolized with enzymes other than CYP2E1. Methods: To examine the consequences of the ALDH2*2polymorphism, Aldh2+/+ and -/- mice were orally administered ethanol at a dose of 5 g/kg body weight and the levels in liver tissue of CYP2E1 mRNA, malondialdehyde (MDA, an indicator of oxidative stress), and glutathione (GSH, a key antioxidant), were then analyzed 0 - 72 hours after administration. Results: The level of CYP2E1 mRNA 12 hours after ethanol administration in Aldh2-/- mice was significantly lower than that in the 0 hour Aldh2-/- group and that in the 12 hour Aldh2+/+ group (*, ^¶: p < 0.05). The levels of MDA were significantly lower in Aldh2-/- mice than in Aldh2+/+ mice at 12 hours after ethanol administration, while levels of GSH were significantly higher in Aldh2-/- mice than in Aldh2+/+ mice at 6 and 12 hours after administration. Conclusions: 1) Single-dose ethanol administration down-regulates the expression of cytochrome p450 2E1 mRNA in the presence of inactive ALDH2. 2) A lack of ALDH2 ameliorates ethanol-induced oxidative stress in liver tissue.

Consideration Of Metachromatic Spectral Changes Of Toluidine Blue Staining Of DNA Depending On Its Helical Structure MATSUMOTO S^1, SATO S^1, FREIVALDS T^2, ERENPREISA J^3 ^1Yokohama Natl. Univ., Yokohama, Japan; ^2Univ. of Latvia and ^3Biomed. Centre, Riga, Latvia Background: Metachromasy (metachromasia) is a well-known observation in biological and medical experiments. This phenomenon often allows to discriminate a certain substrate or tissue from others and thus metachromatic staining has been used as a cytological and diagnostic tool. Metachromatic colour changes have been considered as being caused by dye aggregation. However, the structure of the dye aggregates is still unknown. In this study, as a first step for our goal to understand structure-function relationships of biomolecules by means of metachromatic staining, the metachromatic spectral changes of Toluidine blue(TB) with DNAs were considered by computational approach. Methods: TB-DNA complex models were constructed by the optimized molecular structure of TB calculated by quantum calculations (Software: CAChe ver 5.2, Hamiltonian: AM1 for structure optimization and ZINDO/S for electronic properties) and crystal structures of DNAs obtained from Protein Data Bank (B-DNA: 1BNA and A-DNA: 440D). The dimethyl amino group of TB was assumed to bind to a phosphate group of DNAs by electrostatic interaction. Displacement energies for the TB aggregates were estimated by the extended-dipole model using the calculated transition dipole moment of TB corresponding to its visible absorption. Results: In TB-B-DNA complexation, the two adjacent dye molecules gave a hypsochromic spectral shift in the range from 117 to 330 cm^-1. The resulting spectral shifts are strongly related with the geometrical parameters of DNA. The twist angle and the alpha-angle were found to play an important role in the determination of the dimer geometry. A small difference between the alpha-angles of adjacent phosphate groups resulted in a parallel arrangement of TB molecules producing a large spectral shift. In TB-A-DNA complexation, on the other hand, the geometrical limitation for TB-dimer formation was revealed due to a short distance between adjacent phosphate sites. These results suggested that the dimer formation is considerably affected by the helical structure of DNA. Conclusions: 1) In the complexation of TB with the B-DNA, hypsochromic TB dimers were found to form with the DNA. Their spectral shifts are strongly influenced by the twist angle and the alpha-angle. 2) The dimer formation is sterically restricted in the tightly rolled A-DNA.

Innovations In Organoboron Chemistry Essential To The Discovery Of Bortezomib MATTESON DS Department of Chemistry, Washington State University, Pullman, WA, USA Bortezomib, currently approved for treating relapsed multiple myeloma and undergoing testing for other kinds of anticancer activity, is the first successful proteasome inhibitor and the first useful pharmaceutical containing boron. Before bortezomib could be synthesized and tested, a series of fundamental discoveries in the chemistry of boronic esters was required. These discoveries began in my laboratory when it was found that halogen atoms on a carbon attached to boron undergo unusually facile boron assisted displacement by nucleophiles (1963). We reported a dialkylamino boronic acid in 1968. Biochemist Gustav E. Lienhard suggested that the boronic acid analogue of N-acetylphenylalanine, PhCH2CH(NHAc)B(OH)2, ought to be a good chymotrypsin inhibitor (1971). After a variety of failed attempted syntheses, we found that amino boronic esters having an NH group decomposed readily (1979). Finally, it was found that the facilitation of nucleophilic halide substitution by adjacent boron is so great that lithiohexamethyldisilazane, LiN(SiMe3)2, could be used to introduce the amino nitrogen, and prompt acylation after protonolysis of the silyl groups led to the stable targeted N-acetylphenylalanine analogue, which was indeed a good inhibitor of chymotrypsin (with K. M. Sadhu and G. E. Lienhard, 1981). In the meantime, other discoveries in my laboratory had led to an efficient general asymmetric synthesis of the requisite chloro boronic ester precursors (1980). Soon after our initial report, du Pont chemists extended and refined our chemistry to produce several interesting serine protease inhibitors, including highly active elastase and thrombin inhibitors, and studied their mechanisms of action in great detail, but did not find clinically useful drugs. Julian Adams and coworkers reported the activity of bortezomib in 1998 after screening numerous potential inhibitors of proteasome 26S. There are still unsolved problems regarding the synthesis of highly functionalized organoboron compounds. Among the more interesting of these are boron analogs of nucleosides. Some of the author’s current interests and recent progress on these problems will be described.

IM28 Inhibiting HIV1 Replication, Glucose, Lipids, Hemoglobin Levels And Nitric Oxide (NO) In HIV1 Patients: Study From Gabon MAKA G, MAVOUNGOU VP, AKOUME NDONG MY, ONGALI B, MAVOUNGOU E, MAVOUNGOU D Centre de Recherche sur les Pathologies Hormonales (CRPH), Université des Sciences de la Santé, Libreville, Gabon Background: IM28, an analog of dehydroepiandrosterone (DHEA) demonstrates deep in-vitro HIV-1 antiviral activity including, the inhibition of the reverse transcriptase activity, the restriction of envelop proteins mediating cell-cell fusion (gp120/gp41) between infected and healthy cells and the suppression of 3TC/AZT resistant clinical isolate HIV-1. Aim: To study the effects of IM28 or DHEA on a short term pre-clinical trial of six months on 201 HIV-1 patients among which 90 carried opportunistic pathologies such as diabetes, hypertension, tuberculosis, malaria, skin rash, digestive rash and urinal rash, facial paralysis, language troubles, memory deficit, anorexia and anxiety. Methods: Patients received 50 mg/day/70Kg of IM28 or DHEA and were weekly monitored by our physicians according to the research guidelines of our institution. Results: No side effects attributable to IM28 were noticed regarding hepatic, cardiac and renal functions evaluation as no significant difference was seen with baseline of urea, creatinine, GOT and TGP. By contrast to patients treated with DHEA, normalization of glycaemia, increased body weight, CD4 (p<0.01), lymphocytes and haemoglobin levels (p<0.001) paralleled by significant reduction of platelets, antigenemia p24 (p<0.001) and viral load (p<0.01) were observed in patients under IM28 treatment. In addition, IM28 normalised body weight, lipids, glucose levels and blood pressure in obese, hypertensive and diabetes patients more to reduce significantly the percentage of opportunistic affections such as tuberculosis, malaria, skin rash, digestive rash, urinal rash, stroke, facial paralysis, language and memory troubles, dementia and anxiety. Moreover, the body temperature which is always higher in HIV1 patients and persisting under HAART treatment was reduced and normalised in patients under IM28. Conclusion: Data suggest that, the use or the substitution of DHEA which is already used as supplement of HIV-1 treatment by IM28 should represent a better therapeutic avenue for HIV-1 and opportunistic related diseases as well as for cardiovascular diseases. Magic bullet effects of IM28 may be partly due by nitric oxide (NO) through the normalisation of his reservoir haemoglobin levels.

A Concerted Approach Using Physical Chemical Methodologies, Computational Chemistry And 3D QSAR Studies Aiming To Develop Novel Analgesic Cannabinoid Analogs MAVROMOUSTAKOS T^1,2, DURDAGI S^2,3, REIS H^2, KOUKOULITSA C^2, PAPADOPOULOS^2 ^1Department of Chemistry, University of Athens, Zographou, Athens, Greece, ^2Institute of Organic and Pharmaceutical Chemistry, The National Hellenic Research Foundation, Greece, ^3Department of Biology Chemistry and Pharmacy, Freie Universitat Berlin, Germany The C-1'-dithiolane ^8-tetrahydrocannabinol (^8-THC) amphiphilic analogue (–)-2-(6a,7,10,10atetrahydro-6,6,9-trimethylhydroxy-6H-dibenzo[b,d]pyranyl)-2-hexyl-1,3dithiolane (AMG3) is considered as one of the most potent synthetic analgesic cannabinoid (CB) ligands. Its structure is characterized by a rigid tricycle and flexible alkyl chain segments. Its conformational and ADME properties have been explored using a combination of physical chemical methodologies, computational chemistry and 3D QSAR studies. More particularly, a strategy is developed in which its conformational properties are studied in an ascending complexity. Thus, the conformational properties of AMG 3 are explored in vacuum, in amphoteric solvents, in the receptor site and in a bilayer environment that closely simulates the biological one. Two sites are discovered in the binding pocket (see Figure below ) which show distinct conformational preferences and structural requirements (S1 specific and S2 specific). These sites can be explored for a future design and may constitute a magic bullet target for potential analgesic drugs. Two examples of potential magic bullet targets are given below.