| The goals of this research project fall into two major categories, both relating to structure-based drug design for CT (cholera toxin) and LT (heat-labile enterotoxin), which are homologous ABS toxins produced as primary virulence factors by V. cholerae and E. coli, respectively. The first major goal was to set in motion a new area of investigation, fondly called by our group structure-based “plug” design. The idea was to find small molecules that may be able to interrupt protein-protein interactions during toxin assembly and prevent incorporation of the A-subunit by occupying or plugging its space in the growing B-pentamer. We describe our discovery of the first potential inhibitor of ABS assembly, and a high-resolution crystal structure of MDT (3-methylthio-diphenyl-triazolium) bound in the B-pentamer pore. Subsequently, the structure-based “plug” design cycle was pursued using MDT as a lead molecule. Additional leads having five-fold symmetry were pursued by de novo design. Further structural knowledge about the “tail” region, the portion of the A-subunit that directly contacts the B-pentamer pore, may assist future work in the realm of finding assembly inhibitors. This region is the most structurally divergent in the currently solved structures of wild-type CT and LT and has been implicated in the different toxicity levels of CT and LT. Two hybrid ABS toxins, differing in the tail region only, were studied by x-ray crystallography. The second major goal of this thesis work was in the area of structure-based drug design for the A1-subunit. A crystal structure of activated A1 has yet to be solved, and would aid in the development of high-affinity inhibitors for the enzymatic portion of LT or CT. Initial expression, purification, and characterization of several recombinant A1 constructs, three of CT and three of LT, are described. Special attention is given to the issues of refolding and auto-modification, which we believe are crucial in understanding how to arrive at pure protein, and which may assist future scientists interested in studying LT-A1 and CT-A1. |
