PERTUSSIS, DIPHTHERIA, CHOLERA TOXINS-INHIBITOR DESIGN

This research program addresses two major and current problems in infectious disease using novel technology. Toxin-directed transition state inhibitors offer a new approach to prevent the damage of bacterial exotoxins to human tissues. Antibiotic resistance is a global problem in infectious disease. Tissue- protective toxin inhibitors could act as antibiotics which are not expected to elicit resistance in the causitive organisms. The experimental approach is to use the frontier method of enzymatic transition state analysis and to apply it to the action of bacterial exotoxins. ADP-ribosylating bacterial exotoxins catalyze the covalent modification of GTP-binding proteins. Cholera, diphtheria and pertussis toxins ADP-ribosylate Gsalpha, eukaryotic elongation factor 2, and Gialpha proteins, respectively. Transition state analysis of bacterial ADP-ribosylating exotoxins will be used to design transition state inhibitors against cholera, diphtheria, pertussis and related exotoxins. Transition-state inhibitors against bacterial exotoxins are expected to protect against the exotoxins and thus ameliorate the damage caused in these childhood and endemic diseases. Transition state structure is determined by measuring kinetic isotope effects with NAD+ substrate labeled in all of the atomic positions expected to undergo bonding changes as bonds are broken and made at the enzyme-stabilized transition state. The ADP- ribosylated G-protein is analyzed for the isotopic discrimination of the incorporated ADP-ribose. The isotope effects are then corrected to reveal the full chemical expression of intrinsic isotope effects. An atomic model of all atoms at least two bonds from the reaction center is constructed which is constrained by the values of the kinetic isotope effects. Semiemperical and ab initio methods are used to complete the structure of the transition state molecules, with constraints at every step to comply with the experimental kinetic isotope effects. Transition state structures are mapped using the molecular electrostatic potential surface at the van der Waals radius and compared to that of the substrate. The relationship provides predictive value for transition state inhibitor design. Molecules with electronic similarity close to that of the transition state are synthesized and tested as transition state inhibitors. These procedures have resulted in the discovery of novel transition state inhibitors for several simple enzymatic reactions. The goal of this work is to extend transition state inhibitor design to the complex reactions catalyzed by bacterial ADP-ribosylating toxins.