DEVELOPMENT OF A VEROTOXIN INHIBITOR WlTH ORIENTED CARBOHYDRATES BY USING CARBOSILANE DENDRIMERS

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1) Preparation of carbosilane dendrimers with different numbers of terminal functional unit and different core structures. We developed the first and the second generation of carbosilane dendrimers which have 9, 18 and 36 trisaccharides, sugar moiety of globotriaosyl ceramide, as functional terminal units in their structures (referred to as SUPER TWIG). Inhibitory effects of these compounds on the binding activity of radio-labeled Stx to target cells and on the cytotoxic activity of Stx were investigated. We found that at least 6 trisaccharides present in one molecule are enough for the inhibitory effects of these SUPER TWIGs on these biological activities of Stx. Furthermore, we found that relative distances of the trisaccharides present in SUPER TWIGs rather than the number of trisaccharides play an important role in the high affinity binding to Stx.2) Inhibitory effects of SUPER TWIGs in vivo. Inhibitory effects of SUPER TWIGs on the lethality of Stx2 were investigated in mice model … More , in which lethal dose of Stx2 was intravenously administered with or without each SUPER TWIG. Among all the SUPER TWIGs developed in this study, i. e. SUPRER TWIG (0)3, (0)4, (1)4, (1)6, (1)9, (2)18, and (2)36 (the numbers in parentheses indicate the generation numbers; the zero, the first, and the second generation of the SUPER TWIGs have one, three, and five linear silicon atoms, respectively, in the core structures), which have 3, 4, 6, 9, 18, and 36 trisaccharides, respectively, in their structures, only SUPER TWIGs (1)6 and (2)18 could effectively inhibited the lethality of Stx2. One of the major common characteristics of these two SUPER TWIGs is their dumbbell shape, in which they have 3 and 9 trisaccharides, respectively, at the both end of their structures in a symmetrical manner. On the other hand, dissociation constants (KD value) of SUPER TWIGs (1)4, (1)6, (1)9, (2)18, and (2)36 for the binding to the B-subunits of Stx1 and Stx2 ,which were determined by using Biacore system, were very low in a similar range, suggesting that these SUPER TWIGs bind to the B-subunits with high affinities. In contrast, KD values of SUPER TWIGs (0)3 and (0)4, the zero generation SUPER TWIG, was very high. These results indicate that not only the high affinity binding to Stx but also another factor, such as the dumbbell shape of the molecule, are required for the efficient inhibitory effects of the SUPER TWIGs in vivo. At present, the precise mechanisms by which SUPER TWIGs (1)6 and (2)18 but not others effectively inhibit the lethality of Stx2 in vivo remains to be elucidated, our findings indicate that SUPER TWIGs (1)6 and (2)18 can be therapeutic agents against infections of Stx-producing E. coli O 157:H7. Less