Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.5.4.1 (cytosine deaminase)
 747 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Use of highly potent small interfering RNAs (siRNAs) can substantially reduce dose-dependent cytotoxic and off-target effects. We developed a genetic forward approach by fusing the cytosine deaminase gene with targets for the robust identification of highly potent siRNAs from RNA interference (RNAi) libraries that were directly delivered into cells via bacterial invasion. We demonstrated that two simple drug selection cycles performed conveniently in a single container predominately enriched two siRNAs targets the MVP gene (siMVP) and one siRNA targets the egfp gene (siEGFP) in surviving cells and these proved to be the most effective siRNAs reported. Furthermore, the potent siRNAs isolated from the surviving cells possessed noncellular toxic characteristics. Interestingly, the length of highly potent siMVPs identified could be as short as 16-mer, and increasing the length of their native sequences dramatically reduced RNAi potency. These results suggest that the current approach can robustly discover the most potent and nontoxic siRNAs in the surviving cells, and thus has great potential in facilitating RNAi applications by minimizing the dose-dependent and sequence nonspecific side effects of siRNAs.
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PMID:Forward and robust selection of the most potent and noncellular toxic siRNAs from RNAi libraries. 1904 72

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.
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PMID:Antitumor therapy mediated by 5-fluorocytosine and a recombinant fusion protein containing TSG-6 hyaluronan binding domain and yeast cytosine deaminase. 1926 97