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Antitumor therapy mediated by 5-fluorocytosine and a recombinant fusion protein containing TSG-6 hyaluronan binding domain and yeast cytosine deaminase.

ABSTRACT: Matrix attachment therapy (MAT) is an enzyme prodrug strategy that targets hyaluronan in the tumor extracellular matrix to deliver a prodrug converting enzyme near the tumor cells. A recombinant fusion protein containing the hyaluronan binding domain of TSG-6 (Link) and yeast cytosine deaminase (CD) with an N-terminal His(x6) tag was constructed to test MAT on the C26 colon adenocarcinoma in Balb/c mice that were given 5-fluorocytosine (5-FC) in the drinking water. LinkCD was expressed in Escherichia coli and purified by metal-chelation affinity chromatography. The purified LinkCD fusion protein exhibits a K(m) of 0.33 mM and V(max) of 15 microM/min/microg for the conversion of 5-FC to 5-fluorouracil (5-FU). The duration of the enzyme activity for LinkCD was longer than that of CD enzyme at 37 degrees C: the fusion protein retained 20% of its initial enzyme activity after 24 h, and 12% after 48 h. The LinkCD fusion protein can bind to a hyaluronan oligomer (12-mer) at a K(D) of 55 microM at pH 7.4 and a K(D) of 5.32 microM at pH 6.0 measured using surface plasmon resonance (SPR). To evaluate the antitumor effect of LinkCD/5-FC combination therapy in vivo, mice received intratumoral injections of LinkCD on days 11 and 14 after C26 tumor implantation and the drinking water containing 10 mg/mL of 5-FC starting on day 11. To examine if the Link domain by itself was able to reduce tumor growth, we included treatment groups that received LinkCD without 5-FC and Link-mtCD (a functional mutant that lacks cytosine deaminase activity) with 5-FC. Animals that received LinkCD/5-FC treatment showed significant tumor size reduction and increased survival compared to the CD/5-FC treatment group. Treatment groups that were unable to produce 5-FU had no effect on the tumor growth despite receiving the fusion protein that contained the Link domain. The results indicate that a treatment regime consisting of a fusion protein containing the Link domain, the active CD enzyme, and the prodrug 5-FC is sufficient to produce an antitumor effect. Thus, the LinkCD fusion protein is an alternative to antibody-directed prodrug enzyme therapy (ADEPT) approaches for cancer treatment.

SUBMITTER: Park JI

PROVIDER: S-EPMC2706098 | biostudies-literature | 2009 May-Jun

REPOSITORIES: biostudies-literature

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Antitumor therapy mediated by 5-fluorocytosine and a recombinant fusion protein containing TSG-6 hyaluronan binding domain and yeast cytosine deaminase.

Park Joshua I JI Cao Limin L Platt Virginia M VM Huang Zhaohua Z Stull Robert A RA Dy Edward E EE Sperinde Jeffrey J JJ Yokoyama Jennifer S JS Szoka Francis C FC

Molecular pharmaceutics 20090501 3

Matrix attachment therapy (MAT) is an enzyme prodrug strategy that targets hyaluronan in the tumor extracellular matrix to deliver a prodrug converting enzyme near the tumor cells. A recombinant fusion protein containing the hyaluronan binding domain of TSG-6 (Link) and yeast cytosine deaminase (CD) with an N-terminal His(x6) tag was constructed to test MAT on the C26 colon adenocarcinoma in Balb/c mice that were given 5-fluorocytosine (5-FC) in the drinking water. LinkCD was expressed in Escher ...[more]

PMID: 19265397

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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

Antitumor therapy mediated by 5-fluorocytosine and a recombinant fusion protein containing TSG-6 hyaluronan binding domain and yeast cytosine deaminase.

Publications

Antitumor therapy mediated by 5-fluorocytosine and a recombinant fusion protein containing TSG-6 hyaluronan binding domain and yeast cytosine deaminase.

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