The new frontier in protein design is the creation of completely artificial enzymes. One attractive design target is the construction of improved catalytic agents for bioreductive cancer therapy. Currently, these therapies utilize naturally occurring enzymes, which in turn contain biological enzyme cofactors. A major drawback of using these enzymes is that the prodrugs which they activate are necessarily similar to naturally occuring metabolites. This results in significant activation of these prodrugs elsewhere in the body, causing chemotoxicity. I propose to design completely artificial enzymes that can activate new prodrugs which are orthogonal to human metabolism. This de novo design approach also lends itself well to the utilization of non-natural enzyme cofactors as the basis for tailored enzyme activities. This in turn widens the scope of chemical reactivity possible for the enzyme, permitting the design of the prodrug to focus on low nonspecific activity and consequently, low toxicity. In specific, I plan to computationally implant a flavin-dependent nitroreductase active site on a four alpha helix bundle scaffold. Then, using the NMR-directed iterative redesign process that I have had success with in the past, the enzyme will be redesigned to utilize the flavinlike safranine 0 molecule. This molecule has electron transfer energetics that are better suited and more robust than the biological flavin cofactors for activating nitraromatic prodrugs without catalyzing side reactions. The next step entails a simulataneous active site:prodrug redesign process wherein an optimally active non-toxic pairing can be created. This will be tested using human cell cultures, comparing prodrug toxicity with and without exposure to the enzyme. I am proposign to design improved enzymes for bioreductive cancer therapy. Designing and synthesizing proteins which are completely different from those that exist in the body allows us to also greatly redesign the drugs it activates. This should allow us to greatly reduce the toxicity normal encountered in chemotherapy.
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