Developments in engineering the herpes simplex virus thymidine kinase for enzyme prodrug therapy using bacterial systems
Shelat, Nirav Y.
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The continuous search for better cancer therapies has fueled investigations into a diverse array of approaches aimed at treating the disease. An ongoing challenge in this field of research is the development of highly effective treatments that are also highly discriminating towards cancer. Early treatments used small molecule drugs targeting metabolic systems to exploit the higher metabolic rates of cancer cells for specific toxicity. In efforts to improve specificity, some of these small molecule drugs were later converted to prodrugs that could be activated by specific enzymes and utilized in gene directed enzyme-prodrug therapy (GDEPT). The herpes simplex virus thymidine kinase (HSV-TK) is one such enzyme that is used in GDEPT. Its promiscuity towards phosphorylating nucleoside analog prodrugs has led a strong drive to implement it in GDEPT-based cancer therapies. Efforts to engineer HSV-TK towards selective activity against prodrugs have proven to be moderately successful. Here, we describe progress that has been made in advancing HSV-TK’s utility in GDEPT. In order to produce selective prodrug activation in cancer cells, we used directed evolution to engineer HSV-TK into a protein switch that can be activated in the presence of the C-terminal transactivation domain (C-TAD) of the cancer-specific marker, hypoxia inducible factor 1α (HIF1-α). The CH1 domain of the p300 protein is capable of binding to the C-TAD and was inserted into the amino acid backbone of HSV-TK to sensitize the enzyme to the presence of the cancer marker. The protein switch, dubbed TICKLE (Trigger-Induced Cell-Killing Lethal Enzyme), conferred a 4-fold increase in toxicity towards bacterial cells from azidothymidine (AZT) prodrug in the presence of C-TAD compared to the toxicity in the absence of C-TAD. The studies suggested that the switch was capable of phosphorylating AZT as well as thymidine (dT); thus, other nucleoside analogs could also act as substrates and increase TICKLE’s prospects for utilization in various applications such as cancer therapy and as a molecular reporter gene. In addition, our attempts to address challenges in selecting for kinase activity in protein switch variants led to the development of a new bacterial positive selection for nucleoside kinases. The new selection overcomes toxicity issues present in previous selections and removes limitations that could enable the discovery previously undiscovered high activity variants of HSV-TK. The advances made in HSV-TK engineering in this work expand our knowledge of protein switch development and manipulation of bacteria for kinase selection. The progress can be directly applied to creating better enzymatic switches and HSV-TK variants.