PERTUSSIS, DIPHTHERIA, CHOLERA TOXINS--INHIBITOR DESIGN

Project: Research project

Project Details

Description

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.
StatusFinished
Effective start/end date7/1/936/30/94

Funding

  • National Institute of Allergy and Infectious Diseases

ASJC

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Toxicology
  • Physical and Theoretical Chemistry

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