Developing an Enzymatic Switch from HSV-Thymidine Kinase As a Potential Cancer Therapeutic

Developing an enzymatic switch from HSV-­-thymidine kinase as a potential cancer therapeutic.

Nirav Y. Shelat and Marc Ostermeier

The development of ligand-­-activated protein switches holds tremendous potential for protein-­-based therapeutics.A specific application of engineered switches is in gene directed enzyme-­-prodrug therapy (GDEPT).GDEPT discriminately kills cancer cells through targeted DNAdelivery of a suicide gene, but is limited by the specificity of gene delivery methods.An engineered protein switch would be able to add another level of specificity towards cancer cells beyond that of the DNA delivery method and make GDEPT a more attractive therapeutic strategy. To this end, we have developed a directed evolution approach for creating a protein switch from the herpes simplex virus thymidine kinase (HSV-­-TK), an enzyme often used in GDEPT with the prodrug ganciclovir. The desired switch would couple the intrinsic HIF1a-­- binding affinity of the CH1 domain from the p300 protein to the activity of the HSV-­- TK enzyme, creating a HSV-­-TK protein switch that is active only when HIF1-­-a is available. Since HIF1-­-a is a cancer marker, such a switch would cause the activation of the prodrug, ganciclovir, into ganciclovir monophosphate, specifically in cancer cells. Our strategy to make such switches involved the generation of a DNA library that had variants with a CH1 domain inserted in various locations in the HSV-­-TK gene. A two tier selection scheme was implemented to then select for fusions which possessed the desired ?OFF? and ?ON? states. Upon screening, we identified CH1-­- HSV-­-TK gene fusions that conferred a switch phenotype to E.coli cells that is dependent upon the induced expression of HIF1-­-a. These constructs will serve as a basis for future rounds of directed evolution that will further increase the activity window between the ?ON? and ?OFF? states of the HSV-­-TK protein switch