title: Development of a cancer-marker activated enzymatic switch from the herpes simplex virus thymidine kinase.
abstract: Discovery of new cancer biomarkers and advances in targeted gene delivery mechanisms have made gene-directed enzyme prodrug therapy (GDEPT) an attractive method for treating cancer. Recent focus has been placed on increasing target specificity of gene delivery systems and reducing toxicity in non-cancer cells in order to make GDEPT viable. To help address this challenge, we have developed an enzymatic switch that confers higher prodrug toxicity in the presence of a cancer marker. The enzymatic switch was derived from the herpes simplex virus thymidine kinase (HSV-TK) fused to the CH1 domain of the p300 protein. The CH1 domain binds to the C-terminal transactivation domain (C-TAD) of the cancer marker hypoxia inducible factor 1α. The switch was developed using a directed evolution approach that evaluated a large library of HSV-TK/CH1 fusions using a negative selection for azidothymidine (AZT) toxicity and a positive selection for dT phosphorylation. The identified switch, dubbed TICKLE (Trigger-Induced Cell-Killing Lethal-Enzyme), confers a 4-fold increase in AZT toxicity in the presence of C-TAD. The broad substrate specificity exhibited by HSV-TK makes TICKLE an appealing prospect for testing in medical imaging and cancer therapy, while establishing a foundation for further engineering of nucleoside kinase protein switches.
authors.affiliation: Chemical Biology Interface Graduate Program, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA.
authors.forename: Nirav Y
authors.initials: NY
authors.lastname: Shelat
authors.affiliation: Department of Chemical & Biomolecular Engineering, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA.
authors.forename: Sidhartha
authors.initials: S
authors.lastname: Parhi
authors.affiliation: Department of Chemical & Biomolecular Engineering, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA oster@jhu.edu.
authors.forename: Marc
authors.initials: M
authors.lastname: Ostermeier
mesh_terms: D014408:Biomarkers, Tumor / Q000378:metabolism*; D004789:Enzyme Activation; D015723:Gene Library; D010766:Phosphorylation / Q000187:drug effects; D011355:Prodrugs / Q000378:metabolism; D000072417:Protein Domains; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018139:Simplexvirus / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D013936:Thymidine / Q000378:metabolism; D013937:Thymidine Kinase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015215:Zidovudine / Q000494:pharmacology
chemical_list: D014408:Biomarkers, Tumor; D011355:Prodrugs; D011993:Recombinant Fusion Proteins; D015215:Zidovudine; D013937:Thymidine Kinase; D013936:Thymidine
keywords: HSV; directed evolution; herpes simplex virus thymidine kinase; protein switch; thymidine kinase