AB5 bacterial toxins are the major virulence factors in diseases that affect over a billion people in the world each year. The mode of action of these toxins is a multi-step process which involves toxin assembly, translocation and catalytic activity. None of the steps are fully understood, as cellular biology studies are limited by the lack of potent, stage-specific inhibitors. Therefore, the goal of this work is to design and discover such compounds. The known three-dimensional crystal structures of E.coli heat-labile enterotoxin, cholera toxin and of their complexes with carbohydrates are well-suited for a protein structure-based design cycle supported by complementary thermodynamical characterization to arrive at tightly binding ligands. Targets are the GM1 oligo-saccharide receptor binding site of the B subunits and the structural integrity of the AB5 holotoxins. Receptor antagonists and assembly inhibitors will be arrived at by: (1) screening of potential ligands generated by combinatorial oligosaccharide synthesis; (2) in computro docking experiments; and (3) de novo ligand design. All three strategies will be embedded in an integrated ligand discovery and design cycle. Experimental feedback at both the energetic and structural level, by calorimetry and X-ray crystallography, will continuously validate the in computro approach and give insight in the binding process. The biological effect of potent inhibitors will be tested in cell culture systems. Whereas the immediate goal is to arrive at molecular tools for the study of the multi-step intoxication by AB5 toxins, the long-term goal is to provide leads and lay the foundation for the development of prophylactic drugs against the intoxication by all AB5 toxins. It is also anticipated that ligands developed for the oligo-saccharide receptor binding B subunits may have therapeutic properties for the treatment of Guillaume-Barre syndrome, and may become molecular research tools for studying cell interactions.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29GM054618-02
Application #
2701771
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1997-05-01
Project End
2002-04-30
Budget Start
1998-05-01
Budget End
1999-04-30
Support Year
2
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Washington
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
135646524
City
Seattle
State
WA
Country
United States
Zip Code
98195