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DHS Summer Student Project Report [electronic resource].

Tetanus and botulinum neurotoxins are among the most potent toxins known to man (Montecucco et al. al., 1995). Produced by the Clostridium tetani and Clostridium botulinum bacteria, respectively, these toxins concentrate in presynaptic axons and inhibit the release of neurotransmitters leading to paralysis and possibly death. Due to the potency of this lethal class of neurotoxins, we have undertaken a project to develop high affinity ligands that specifically bind to these toxins. Such compounds can have significant implications in both the design of detection systems to monitor for the possible release of these neurotoxins into the public and also the design of possible therapeutics to treat individuals exposed to tetanus or botulinum neurotoxins. The Clostridial neurotoxins are synthesized as 150 kDa proteins that are post-translationally cleaved into N- and C-terminal fragments held together by a single disulfide bond. The tetanus C-terminal fragment (TetC) has been shown to bind specifically to gangliosides present on the neuronal membrane surface and facilitate endocytosis of the toxin (Morris et al., 1980). Once the toxin is internalized in a membrane-bound vesicle, the light chain (N-terminal fragment) translocates to the cytosol where it interferes with neurotransmitter release. Previous work has demonstrated that various small molecule and peptide-based compounds bind to TetC, albeit in different locations. Among these molecules are the anticancer agent doxorubicin (Dox) and the tripeptides WEY and YEW (Figure 1; Cosman et al. al., 2002). The crystal structure of botulinum toxin and Dox (PDB code: 1I1E) demonstrates that Dox binds in a surface groove of in C-terminal fragment that is conserved in both botulinum and tetanus toxins. Similarly, YEW has been shown to bind to a second binding site that is highly conserved and also relatively close to the binding site of Dox. Thus, in our quest to design and synthesize high affinity ligands, we proposed to link Dox and YEW (or WEY) in hopes of creating a bidentate ligand. In theory, such a ligand could have a binding affinity approaching the product of the two binding affinities of the individual ligands. For my internship project, I was charged with the task of creating libraries of compounds linking Dox and YEW (or WEY) with linkers of varying lengths (Figure 2a). In addition, I was to attach a fluorescein dye to the molecules (Figure 2b) so that they could be used to develop a fluorescence polarization (FP) binding assay. The FP assay will greatly increase the ease with which future ligands can be rapidly screened and binding affinities can be accurately determined. As a side project, I worked on optimizing the conditions necessary to employ the Huisgen 1,3-dipolar cycloaddition reaction to be able to optimize linker lengths and possibly compound solubility (Huisgen, 1984). This reaction, often termed ''click chemistry'', utilizes molecules terminally functionalized with either an acetylene moiety or an azide. In the presence of a copper(I) catalyst, the alkyne and azide undergo a step-wise cycloaddition reaction to link the two molecules together via the formation of a 1,4-disubstituted triazole ring (Figure 3; Rostovtsev et al., 2002). By varying the length of the tethers between the terminal acetylene or azide and their respective molecules, the overall length of the linker between the two molecules can be ''fine tuned'' by one carbon unit at a time. At the completion of my internship I had synthesized conjugates of Doxorubicin and N-acyl-WEY linked together by linkers having 0-2 polyethylene glycol (PEG) linkers. These compounds are currently being used in experiments that employ electrospray ionization mass spectrometry (ESI-MS) to determine whether they bind to TetC with higher affinity than either Dox or WEY alone. I also synthesized the fluorescein tagged versions of the same three molecules. It is expected that these molecules will be used in the near future ...

Online OSTI

Community indicators for your city : student independent project, Lyndon B. Johnson School of Public Affairs, the University of Texas at Austin

Reconfigurable suspension technology [electronic resource].

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Chinese Studies

Chinese Studies