Pertussis, diphtheria and cholera infections affect an estimated 100 million people each year with an estimated 1 million fatalities. Recently, local epidemics have occurred. In 1991 alone, a cholera epidemic in the Americas caused 391,000 reported cases and nearly 4,000 reported deaths. The cellular pathologies are caused by bacterial toxins which attach to and penetrate the cellular membrane to release a catalytically active peptide toxin. The catalytic peptides of these three toxins use NAD+ as a substrate and catalyze the adenosine diphosphate ribosylation of cellular GTP-binding proteins which alter normal G-protein functions. the catalytic subunit of pertussis toxin ADP-ribosylates the inhibitory G-protein, Gialpha, preventing GDP-GTP exchange, thereby inactivating it and preventing it from inhibiting adenylate cyclases. diphtheria toxin ADP-ribosylates eukaryotic elongation factor-2 at a dipthamide residue, a unique modified histidine, thus inactivating the factor. In cholera, ADP ribosylation of Gsalpha causes large increases in cAMP levels in intestinal epithelial cells, resulting in Na+ efflux, diarrhea and often fatal dehydration. With increasing world population and the decline of preventive health care delivery, pertussis, diphtheria and cholera are expected to become endemic and increasingly epidemic. A powerful adjunct to immunization or rehydration therapy could be provided by inhibitors specific for the cellular actions of the toxins. These inhibitors could also provide a rescue paradigm for recombinant toxin therapy of cancer. Recent advances in the analysis of enzymatic transition state structure makes it possible to establish the geometric and electronic nature of enzymatic transition states. These structures provide blueprints for the logical design of specific tight-binding inhibitors. ADP-ribosylation reactions are attractive targets for transition state structure analysis. Experiments are proposed to synthesize heavy-atom labeled NAD+ molecules as substrates for pertussis, diphtheria and cholera toxin A chains (the catalytic peptides). These substrates will be used to determine the heavy-atom kinetic isotope effects for the toxins. Pre-steady state and steady state kinetic studies will determine the intrinsic kinetic isotope effects. Transition state structures will be determined from intrinsic kinetic isotope effects using bond-order bond-vibrational analysis and molecular orbital calculations. These transition state structures will form the information required for logical design of transition state inhibitors. Inhibitors will be designed and synthesized to inhibit the ADP-ribosylation reaction of pertussis toxin A chain.
|Effective start/end date||7/1/93 → 1/31/05|
- Infectious Diseases
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