Thymidine kinase substrate specificity

Thymidine kinase Substrate specificity (gene therapy) 43-fold increase in sensitivity to gancydovir in hamster cells Cassette mutagenesis + selection + screening E. coli [85]... 

The antiviral mechanism of action of triflnridine involves inhibition of viral DNA synthesis. Triflnridine monophosphate irreversibly inhibits thymidylate synthetase, and trifluridine triphosphate is a competitive inhibitor of thymidine triphosphate incorporation into DNA by DNA polymerases. Trifluridine is incorporated into viral and ceUnlar DNA. Trifluridine-resistant HSV with altered thymidine kinase substrate specificity can be selected in vitro, and resistance in clinical isolates has been described. 

The 2 -chloro and 2 -bromo congeners of either 748 (FIAC) or 758 (FMAU) are more cytotoxic than FIAC and FMAU, suggesting that these chloro and bromo nucleosides, in contrast to the 2 -fluoro compounds, are comparatively better substrates for deoxycytidine kinase of human lymphocytes than the substrates for viral-specific thymidine kinase. The disposition of the 2 -fluoro group may also be important from the biological viewpoint. It should be noted that the structural difference between RNA and DNA is at the 2 -position. The ribo type of analog (738) of FIAC is 10 times less effective in suppression of HSV replication than is FIAC. Thus Fox, and Watanabe and coworkers concluded that the 2 - up fluorine disposition and the species of the substituent at C-5 are the two important factors influencing antiviral activity. Nevertheless, the mechanism of action of 2 -deoxy-2 -fluorocytidine (737) on certain herpes viruses, including HSV-1... 

Answer C. To inhibit DNA polymerases in HSV, acyclovir must undergo initial monophos-phoryktion by a viral specific thymidine kinase (TK). Most HSV strains resistant to acyclovir lack this enzyme and are thus TK- strains. A few strains of HSV are resistant to acyclovir by structural changes in TK that lower substrate affinity or by mutations in the gene that encode viral DNA polymerases. 

Recently, it has been shown that some virus-infected cells induce virus-specific enzymes such as thymidine kinase or DNA polymerase. These virus-induced enzymes have been proven to differ from their cellular enzymes in terms of such properties as substrate specificity. 

In addition to thymidine, the enzyme will accept as substrates deoxyiui-dine and various 5-position derivatives including those with fluoro, bromo, and iodo substituents. The kinase is specific for deoxyribonucleosides and will not phosphorylate deoxycytidine. 

Fractionation of extracts from calf thymus for kinase activity toward deoxyribonucleoside monophosphates has revealed the presence of at least four separate enzymes the kinase activities for dAMP, dGMP, dCMP, and dTMP are separate entities in this tissue 31). Each has been partly purified and examination of substrate specificities showed that the kinases for deoxyadenylate and deoxyguanylate also phosphorylate the ribosyl homologues, adenylate and guanylate, respectively. The deoxycytidylate kinase accepts as substrates both cytidylate and uridylate, but will not phosphor> late deoxyuridylate. The calf thymus thymidine monophosphate... 

On the other hand, the most potent among these in the cytosine series are DMDC (3a) and FDMDC (3b), the cytotoxicities of which are comparable to that of SMDC (li). It is important to note that FDMDC would act as a 5-fluorocytosine derivative but not as a 5-fluorouracil derivative via the deamination by cytidine deaminase since DMDC was reported not to be a substrate of cytidine deaminase from mouse kidney.33 The 5-methylcytosine derivative 3f was about 170 times less active than DMDC and FDMDC. The other 5-halogeno- and 5-ethyl derivatives, 3c-e and h, were found to be devoid of the activity. Therefore, it is conceivable that the order of cytotoxicity might be related to the substrate specificity of the first activation enzymes, such as thymidine kinase or deoxycytidine kinase. 

Comparison of the substrate specificities of human thymidine kinase 1 and 2 and deoxycytidine kinase toward antiviral and cytostatic nucleoside analogues,... 

The kinase which converts deoxycytidine to its 5 -monophosphate has been studied most extensively in preparations from calf thymus 35, 36). The preferred substrate is deoxycytidine, for which the Michaelis constant (5 X 10 M) is much lower than that of two other substrates, deoxyadenosine and deoxyguanosine. Cytidine, uridine, and thymidine are not phosphorylated by this enzyme. Deoxycytidine kinase is subject to a complex pattern of allosteric regulation by nucleotides. The end product of deoxycytidine phosphorylation, dCTP, is a potent inhibitor this inhibition is reversed by dTTP. The enzyme has a rather broad specificity for the phosphate donor, with the triphosphates of the natural ribo- and deoxyribonucleosides being substrates the inactivity of dCTP is a notable exception. 

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