Advancements in the development of HIF-1α-activated protein switches for use in enzyme prodrug therapy

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dc.contributor.authorWright, R. Clayen
dc.contributor.authorKhakhar, Arjunen
dc.contributor.authorEshleman, James R.en
dc.contributor.authorOstermeier, Marcen
dc.contributor.departmentBiological Systems Engineeringen
dc.contributor.editorLebedeva, Irina V.en
dc.date.accessioned2019-01-21T17:31:23Zen
dc.date.available2019-01-21T17:31:23Zen
dc.date.issued2014-01en
dc.date.updated2019-01-21T17:31:20Zen
dc.description.abstractWhile 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.en
dc.description.versionPublished versionen
dc.format.extentPages e114032en
dc.format.mimetypeapplication/pdfen
dc.identifier.doihttps://doi.org/10.1371/journal.pone.0114032en
dc.identifier.eissn1932-6203en
dc.identifier.issn1932-6203en
dc.identifier.issue11en
dc.identifier.orcidWright, Robert [0000-0001-7125-3943]en
dc.identifier.otherPMC4245239en
dc.identifier.otherPONE-D-14-30543 (PII)en
dc.identifier.pmid25426963en
dc.identifier.urihttp://hdl.handle.net/10919/86812en
dc.identifier.volume9en
dc.language.isoenen
dc.relation.urihttps://www.ncbi.nlm.nih.gov/pubmed/25426963en
dc.rightsCreative Commons Attribution 4.0 Internationalen
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/en
dc.subjectMD Multidisciplinaryen
dc.subjectGeneral Science & Technologyen
dc.subject.meshCell Line, Tumoren
dc.subject.meshHumansen
dc.subject.meshEscherichia colien
dc.subject.meshNeoplasmsen
dc.subject.meshFluorouracilen
dc.subject.meshAntineoplastic Agentsen
dc.subject.meshProdrugsen
dc.subject.meshMutagenesisen
dc.subject.meshHypoxia-Inducible Factor 1, alpha Subuniten
dc.subject.meshDrug Discoveryen
dc.titleAdvancements in the development of HIF-1α-activated protein switches for use in enzyme prodrug therapyen
dc.title.serialPLOS ONEen
dc.typeArticle - Refereeden
dc.type.dcmitypeTexten
dc.type.otherResearch Support, N.I.H., Extramuralen
dcterms.dateAccepted2014-11-03en
pubs.organisational-group/Virginia Tech/Agriculture & Life Sciencesen
pubs.organisational-group/Virginia Techen
pubs.organisational-group/Virginia Tech/All T&R Facultyen
pubs.organisational-group/Virginia Tech/Agriculture & Life Sciences/Biological Systems Engineeringen
pubs.organisational-group/Virginia Tech/Agriculture & Life Sciences/CALS T&R Facultyen

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