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Magnetic nanoparticle hyperthermia induced cytosine deaminase expression in microencapsulated E. coli for enzyme-prodrug therapy.

ABSTRACT: Engineered bacterial cells that are designed to express therapeutic enzymes under the transcriptional control of remotely inducible promoters can mediate the de novo conversion of non-toxic prodrugs to their cytotoxic forms. In situ cellular expression of enzymes provides increased stability and control of enzyme activity as compared to isolated enzymes. We have engineered Escherichia coli (E. coli), designed to express cytosine deaminase at elevated temperatures, under the transcriptional control of thermo-regulatory ?pL-cI857 promoter cassette which provides a thermal switch to trigger enzyme synthesis. Enhanced cytosine deaminase expression was observed in cultures incubated at 42°C as compared to 30°C, and enzyme expression was further substantiated by spectrophotometric assays indicating enhanced conversion of 5-fluorocytosine to 5-fluorouracil. The engineered cells were subsequently co-encapsulated with magnetic iron oxide nanoparticles in immunoprotective alginate microcapsules, and cytosine deaminase expression was triggered remotely by alternating magnetic field-induced hyperthermia. The combination of 5-fluorocytosine with AMF-activated microcapsules demonstrated tumor cell cytotoxicity comparable to direct treatment with 5-fluorouracil chemotherapy. Such enzyme-prodrug therapy, based on engineered and immunoisolated E. coli, may ultimately yield an improved therapeutic index relative to monotherapy, as AMF mediated hyperthermia might be expected to pre-sensitize tumors to chemotherapy under appropriate conditions.

SUBMITTER: Nemani KV

PROVIDER: S-EPMC4417421 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Magnetic nanoparticle hyperthermia induced cytosine deaminase expression in microencapsulated E. coli for enzyme-prodrug therapy.

Nemani Krishnamurthy V KV Ennis Riley C RC Griswold Karl E KE Gimi Barjor B

Journal of biotechnology 20150325

Engineered bacterial cells that are designed to express therapeutic enzymes under the transcriptional control of remotely inducible promoters can mediate the de novo conversion of non-toxic prodrugs to their cytotoxic forms. In situ cellular expression of enzymes provides increased stability and control of enzyme activity as compared to isolated enzymes. We have engineered Escherichia coli (E. coli), designed to express cytosine deaminase at elevated temperatures, under the transcriptional contr ...[more]

PMID: 25820125

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Project description:BackgroundEnzyme prodrug therapy shows promise for the treatment of solid tumors, but current approaches lack effective/safe delivery strategies. To address this, we previously developed three enzyme-containing fusion proteins targeted via annexin V to phosphatidylserine exposed on the tumor vasculature and tumor cells, using the enzymes L-methioninase, purine nucleoside phosphorylase, or cytosine deaminase. In enzyme prodrug therapy, the fusion protein is allowed to bind to the tumor before a nontoxic drug precursor, a prodrug, is introduced. Upon interaction of the prodrug with the bound enzyme, an anticancer compound is formed, but only in the direct vicinity of the tumor, thereby mitigating the risk of side effects while creating high intratumoral drug concentrations. The applicability of these enzyme prodrug systems to treating prostate cancer has remained unexplored. Additionally, target availability may increase with the addition of low dose docetaxel treatment to the enzyme prodrug treatment, but this effect has not been previously investigated. To this end, we examined the binding strength and the cytotoxic efficacy (with and without docetaxel treatment) of these enzyme prodrug systems on the human prostate cancer cell line PC-3.ResultsAll three fusion proteins exhibited strong binding; dissociation constants were 0.572 nM for L-methioninase-annexin V (MT-AV), 0.406 nM for purine nucleoside phosphorylase-annexin V (PNP-AV), and 0.061 nM for cytosine deaminase-annexin V (CD-AV). MT-AV produced up to 99% cell death (p?<?0.001) with limited cytotoxicity of the prodrug alone. PNP-AV with docetaxel created up to 78% cell death (p?<?0.001) with no cytotoxicity of the prodrug alone. CD-AV with docetaxel displayed up to 60% cell death (p?<?0.001) with no cytotoxicity of the prodrug alone. Docetaxel treatment created significant increases in cytotoxicity for PNP-AV and CD-AV.ConclusionsStrong binding of fusion proteins to the prostate cancer cells and effective cell killing suggest that the enzyme prodrug systems with MT-AV and PNP-AV may be effective treatment options. Additionally, low-dose docetaxel treatment was found to increase the cytotoxic effect of the annexin V-targeted therapeutics for the PNP-AV and CD-AV systems.

Project description:Yeast cytosine deaminase (yCD) catalyzes the hydrolytic deamination of cytosine to uracil as well as the deamination of the prodrug 5-fluorocytosine (5FC) to the anticancer drug 5-fluorouracil. In this study, the role of Glu64 in the activation of the prodrug 5FC was investigated by site-directed mutagenesis, biochemical, nuclear magnetic resonance (NMR), and computational studies. Steady-state kinetics studies showed that the mutation of Glu64 causes a dramatic decrease in k(cat) and a dramatic increase in K(m), indicating Glu64 is important for both binding and catalysis in the activation of 5FC. (19)F NMR experiments showed that binding of the inhibitor 5-fluoro-1H-pyrimidin-2-one (5FPy) to the wild-type yCD causes an upfield shift, indicating that the bound inhibitor is in the hydrated form, mimicking the transition state or the tetrahedral intermediate in the activation of 5FC. However, binding of 5FPy to the E64A mutant enzyme causes a downfield shift, indicating that the bound 5FPy remains in an unhydrated form in the complex with the mutant enzyme. (1)H and (15)N NMR analysis revealed trans-hydrogen bond D/H isotope effects on the hydrogen of the amide of Glu64, indicating that the carboxylate of Glu64 forms two hydrogen bonds with the hydrated 5FPy. ONIOM calculations showed that the wild-type yCD complex with the hydrated form of the inhibitor 1H-pyrimidin-2-one is more stable than the initial binding complex, and in contrast, with the E64A mutant enzyme, the hydrated inhibitor is no longer favored and the conversion has a higher activation energy, as well. The hydrated inhibitor is stabilized in the wild-type yCD by two hydrogen bonds between it and the carboxylate of Glu64 as revealed by (1)H and (15)N NMR analysis. To explore the functional role of Glu64 in catalysis, we investigated the deamination of cytosine catalyzed by the E64A mutant by ONIOM calculations. The results showed that without the assistance of Glu64, both proton transfers before and after the formation of the tetrahedral reaction intermediate become partially rate-limiting steps. The results of the experimental and computational studies together indicate that Glu64 plays a critical role in both the binding and the chemical transformation in the conversion of the prodrug 5FC to the anticancer drug 5-fluorouracil.

Dataset Information

Magnetic nanoparticle hyperthermia induced cytosine deaminase expression in microencapsulated E. coli for enzyme-prodrug therapy.

Publications

Magnetic nanoparticle hyperthermia induced cytosine deaminase expression in microencapsulated E. coli for enzyme-prodrug therapy.

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