title: Advancements in the development of HIF-1α-activated protein switches for use in enzyme prodrug therapy.
abstract: While gene-directed enzyme prodrug therapy has shown potential as a cancer therapeutic in animal and clinical trials, concerns over the efficacy, selectivity, and safety of gene delivery vehicles have restricted its advance. In an attempt to relieve some of the demands on targeted gene delivery vehicles and achieve the full potential of enzyme prodrug therapy, cancer-targeted activity can be engineered into the enzyme itself. We previously engineered a switchable prodrug-activating enzyme that selectively kills human cancer cells accumulating the cancer marker hypoxia-inducible factor-1α (HIF-1α). This HIF-1α-activated protein switch (Haps59) is designed to increase its ability to convert the prodrug 5-fluorocytosine into the chemotherapeutic 5-fluorouracil in a HIF-1α-dependent manner. However, in cancer cell lines expressing Haps59 the 5FC sensitivity difference between the presence and absence of HIF-1α was not as large as desired. In this work, we aimed to improve the cancer specificity of this switch via a directed evolution approach utilizing random mutagenesis, linker mutagenesis, and random insertion and circular permutation. We identified improved HIF-1α-activated protein switches that confer E. coli with modest increases in HIF-1α-dependent 5FC toxicity. Additionally, the current bottleneck in the development of improved HIF-1α-activated protein switches is screening switch candidates in mammalian cells. To accommodate higher throughput and reduce experimental variability, we explored the use of Flp recombinase-mediated isogenic integration in 293 cells. These experiments raised the possibility that Haps59 can be activated by other interactors of the CH1 domain, and experiments in E. coli indicated that CITED2 can also activate Haps59. Although many CH1 binding partners are also oncogenes, CH1's promiscuous binding and subsequent off-target activation of Haps59 needs to be examined under normal physiological conditions to identify off-target activators. With aberrant activating molecules identified, further directed evolution can be performed to improve the cancer specificity of HIF-1α-activated protein switches.
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland, United States of America.
authors.forename: R Clay
authors.initials: RC
authors.lastname: Wright
authors.affiliation: Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland, United States of America.
authors.forename: Arjun
authors.initials: A
authors.lastname: Khakhar
authors.affiliation: Departments of Pathology and Oncology, Sol Goldman Pancreatic Cancer Center, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.
authors.forename: James R
authors.initials: JR
authors.lastname: Eshleman
authors.affiliation: Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, Maryland, United States of America.
authors.forename: Marc
authors.initials: M
authors.lastname: Ostermeier
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D055808:Drug Discovery / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005472:Fluorouracil / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D016296:Mutagenesis; D009369:Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D011355:Prodrugs / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D011355:Prodrugs; D005472:Fluorouracil