Purification of human cytidine deaminase: Molecular and enzymatic characterization and inhibition by synthetic pyrimidine analogs

Cacciamani, Tiziana; Vita, Alberto; Cristalli, Gloria; Vincenzetti, Silvia; Natalini, Paolo; Ruggieri, Silverio; Amici, Adolfo; Magni, Giulio

doi:10.1016/0003-9861(91)90543-r

Cited by 48 publications

(45 citation statements)

References 28 publications

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“…The presence of sulphydryl groups in the catalytic site of human CDA has been previously evidenced by the dependence of enzyme activity on reducing agents, by the substrate protection on p-chloromercuribenzoate inhibition and by the strong inhibition exerted by 5-chloromercuricytidine (12). By amino acid composition 9 cysteine residues per enzyme subunit were detected: these data are in agreement with those deduced from the sequence (13).…”

Section: Discussionsupporting

confidence: 85%

“…CDA incubated with 10mM cytidine was the control. The catalytic activity was assayed spectrophotometrically as described previously (12), monitoring at 290nm because of the interference of the chelating agents at 282nm. Determination of sulphydryl groups.…”

Section: Methodsmentioning

confidence: 99%

“…Other authors demonstrated that the presence of zinc is characteristic of several deaminases (3)(4)(5) and that zinc plays an important role on the substrate deamination. The human form of CDA was studied in several tissues including liver (6)(7)(8), spleen (9), granulocytes (10), lymphocytes (11) and placenta (12). Although the human CDA gene has been identified (13) …”

Section: Introductionmentioning

confidence: 99%

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Human placenta cytidine deaminase: a zinc metalloprotein

et al. 1997

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Summary: Cytidine deaminase, a tetrameric enzyme purified from human placenta, was shown to contain a single atom of tightly bound zinc per subunit by Inductively Coupled Plasma Optical Emission Spectrometry analysis. The metal appears to be involved in catalysis, as suggested by the inhibition exerted by 1,10-phenanthroline and dipicolinic acid. This hypothesis is further supported by the finding that the presence of substrate protects the enzymatic activity from dipicolirtic acid inhibition. Furthermore the total cysteine residues per subunit were investigated by sulphydryl groups titrating agents.

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Section: Discussionsupporting

confidence: 85%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Human placenta cytidine deaminase: a zinc metalloprotein

et al. 1997

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“…CD catalyzes the deamination of Cyd, dCyd, and several cytidine analogs used as antiviral or anticancer drugs and thus often reduces their pharmacological activity. In vivo studies have shown marked tissue and species differences in the extent of deamination of (d)Cyd and (d)Cyd analogs and higher levels of deaminase activity in human malignant tumors (28). In vitro, kinetic differences have been observed in CD purified from human or canine liver (29).…”

Section: Resultsmentioning

confidence: 99%

“…Human CD activity was determined with a direct spectrophotometric assay based on the decrease in absorbance at 282 nm when the dCyd is deaminated (28). The standard reaction mixture (1 ml) contained 100 mM Tris⅐HCl, pH 7.5, without or with a 0.13 mM concentration of the compound to test.…”

Section: Chemicalsmentioning

confidence: 99%

Lack of Enantiospecificity of Human 2′-Deoxycytidine Kinase: Relevance for the Activation of β-l-Deoxycytidine Analogs as Antineoplastic and Antiviral Agents

et al. 1997

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SUMMARYWe demonstrate that human 2Ј-deoxycytidine kinase (dCK) is a nonenantioselective enzyme because it phosphorylates ␤-D-2Ј-deoxycytidine (D-dCyd), the natural substrate, and ␤-L-2Ј-deoxycytidine (L-dCyd), its enantiomer, with the same efficiency. Kinetic studies showed that L-dCyd is a competitive inhibitor of the phosphorylation of D-dCyd with a K i value of 0.12 M, which is lower than the K m value for D-dCyd (1.2 M). Chemical modifications of either the base or the pentose ring strongly decrease the inhibitory potency of L-dCyd. L-dCyd is resistant to cytidine deaminase and competes in cell cultures with the natural D-dCyd as substrate for dCK, thus reducing the incorporation of exogenous [ 3 H]dCyd into DNA. L-dCyd had no effect on the pool of dTTP deriving from the salvage or from the de novo synthesis, does not inhibit short term RNA and protein syntheses, and shows little or no cytotoxicity. Our results indicate a catalytic similarity between human dCK and herpetic thymidine kinases, enzymes that also lack stereospecificity. This functional analogy underlines the potential role of dCK as activator of L-deoxycytidine analogs as antiviral and antineoplastic agents and lends support to the hypothesis that herpesvirus thymidine kinase might have evolved from a captured cellular dCK gene, developing the ability to phosphorylate thymidine and retaining that to phosphorylate deoxycytidine.

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Delineation of the Molecular Mechanisms of Nucleoside Recognition by Cytidine Deaminase through Virtual Screening

Costanzi

Vilar

Micozzi³

et al. 2011

ChemMedChem

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Cytidine deaminase (3.5.4.5, CDA), an enzyme of the pyrimidine salvage pathways, is responsible for the degradation and inactivation of several cytidine-based antitumoral drugs, such as cytarabine, gemcitabine, decitabine, and azacytidine. Thus, CDA inhibitors are highly sought after as compounds to be co-administered with said drugs in order to improve their effectiveness. Alternatively, the design of antitumoral drugs not susceptible to the action of CDA is also regarded as an attractive solution. Here, we describe a virtual screening for CDA ligands based on chemical similarity and molecular docking. The campaign led to identification of three novel inhibitors and one novel substrate, with a 19% hit rate, and allowed a significant extension of the structure-activity relationships, also in light of the compounds that resulted inactive. The most active compound identified through the screening was the inhibitor pseudoisocytidine, which has the potential to serve as a lead for highly stable compounds. The study also delineated the detrimental effect of 5-aza and 6-aza substitutions, the incompatibility of the presence of an amino group at the 3′-position, as well as the presence of very strict steric requirements around the 2′-arabino position and, even more, the N4-position. Importantly, these features can be exploited for the design of novel antineoplastic agents resistant to the action of CDA.

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Purification of human cytidine deaminase: Molecular and enzymatic characterization and inhibition by synthetic pyrimidine analogs

Cited by 48 publications

References 28 publications

Human placenta cytidine deaminase: a zinc metalloprotein

Human placenta cytidine deaminase: a zinc metalloprotein

Lack of Enantiospecificity of Human 2′-Deoxycytidine Kinase: Relevance for the Activation of β-l-Deoxycytidine Analogs as Antineoplastic and Antiviral Agents

Delineation of the Molecular Mechanisms of Nucleoside Recognition by Cytidine Deaminase through Virtual Screening

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