Thymidine, phosphorylation

Deoxyuridine is also phosphorylated by extracts of E. coli, but at only one-third the rate of thymidine phosphorylation (83). Purification of E. coli extracts has revealed the presence of a kinase for each of the deoxyribo-nucleotides found in DNA (108). The conversion of uridine to uridine 5 -phosphate with ATP as phosphorylating agent has been demonstrated in yeast (83) and fiver (109). 

Expressed as ppmoles of thymidine phosphorylated per 10 cells per minute of incubation. iBased on setting the average of the mutants equal to 1.00. 

With the aid of cytosine permease, flucytosine reaches the fungal cell where it is converted by cytosine deaminase into 5-fluorouracil [51-21-8]. Cytosine deaminase is not present in the host, which explains the low toxicity of 5-FC. 5-Fluorouracil is then phosphorylated and incorporated into RNA and may also be converted into 5-fluorodeoxyuridine monophosphate, which is a potent and specific inhibitor of thymidylate synthetase. As a result, no more thymidine nucleotides are formed, which in turn leads to a disturbance of the DNA-synthesis. These effects produce an inhibition of the protein synthesis and cell repHcation (1,23,24). 5-Fluorouracil caimot be used as an antimycotic. It is poorly absorbed by the fungus to begin with and is also toxic for mammalian cells. 

Amino-5-iodo-2, 5 -dideoxyuridine [56045-73-9] (13) C2H22IN2O4, was synthesized ia 1975 (27) and was found effective against herpes keratitis ia rabbits (28). This compound is markedly less cytotoxic than IdU, iadicating that it may have a safer and more specific mode of antiviral activity. A potential limitation of this group of nucleosides is their specificity, for they fail to inhibit all strains of herpes vimses. The specific antiviral activity of (13) is considered to be a result of the incorporation of the 5 -Ai-phosphate into both viral and host DNA in infected cells, but not into the DNA of normal cells. Phosphorylation of (13) occurs only in herpes vims-infected cells, brought about by a vims-induced thymidine kinase (29). 

BVdU differs from IdU and F TdU by being specifically phosphorylated in the 5 -position by herpes simplex vims type-1 (HSV-1) induced thymidine kinase. This restricts its action to cells infected by HSV-1. It is less active against genital herpes (HSV-2). HSV-l-induced thymidine kinase converts BVdU to the corresponding 5 -mono- and diphosphate, but HSV-2-induced thymidine kinase stops at the stage of the 5 -phosphate of BVdU. Apparendy, cellular kinases phosphorylate BVdU-5 -diphosphate to the corresponding 5 -triphosphate, which inhibits HSV-1 DNA polymerase to a greater extent than similar cellular DNA polymerases. 

The antiviral mechanism of action of acyclovir has been reviewed (72). Acyclovir is converted to the monophosphate in herpes vims-infected cells (but only to a limited extent in uninfected cells) by viral-induced thymidine kinase. It is then further phosphorylated by host cell guanosine monophosphate (GMP) kinase to acyclovir diphosphate [66341 -17-1], which in turn is phosphorylated to the triphosphate by unidentified cellular en2ymes. Acyclovir triphosphate [66341 -18-2] inhibits HSV-1 viral DNA polymerase but not cellular DNA polymerase. As a result, acyclovir is 300 to 3000 times more toxic to herpes vimses in an HSV-infected cell than to the cell itself. Studies have shown that a once-daily dose of acyclovir is effective in prevention of recurrent HSV-2 genital herpes (1). HCMV, on the other hand, is relatively uninhibited by acyclovir. 

Moderate in vivo antiherpes vims activity was demonstrated by 9-P-Dxylofuranosylguanine [27462-39-1] (xylo-G, 38), C qH N O, and the 5 -mono-and 3, 5 -cycHc phosphates of (38), although none was as active as ara-A (89). Generally, guanine base-modified analogues of acyclovir are less active than acyclovir because they are not readily phosphorylated by herpes thymidine kinase. 

All NRTIs, as exemplified for AZT (Fig. 7), act in a similar fashion following their uptake by the cells, they are phosphorylated successively to their 5 -monophosphate, 5 -diphosphate, and 5 -triphosphate form (De Clercq 2002). Unlike the first phosphorylation step in the metabolic pathway of the acyclic guanosine analogues (see above), which is carried out by a virus-encoded enzyme (thymidine kinase), the first as well as the subsequent phosphorylations of the 2, 3 -dideoxynucleosides are carried out by cellular enzymes, that is, a 2 -deoxynucleoside (e.g., dThd) kinase, a 2 -deoxynucleotide (e.g., dTMP) kinase, and a (2 -deoxy)nucleoside 5 -diphosphate (NDP) kinase. 

FIAC- (also FMAU-, FAU-, and FAC-)resistant variants of HSV-1 and -2 are unable - " to phosphorylate these nucleosides because of decreased activity of the thymidine kinase (TK) and, therefore, are 6,000-fold less pathogenic " (for the HSV-1 variant) than the parent virus. This decreased TK activity (production) seems to be one of the characteristic features of resistant viruses. 

Deoxy-5 -fluorothymidine (838) was prepared by Langen and Kowol-jj. 796,797 fpQjyj 5 -0-tosyl precursor by treatment with fluoride. Compound 838 cannot be phosphorylated enzymically owing to the lack of OH-5, but it inhibits the growth of carcinoma cells. This was explained as follows the thymidine 5 -monophosphate (thymidylate) kinase in carcinoma cells, catalyzing the transformation of thymidine 5 -monophosphate into the diphosphate, is inhibited by 838, thus preventing the synthesis of... 

While mammahan cells reutilize few free pyrimidines, salvage reactions convert the ribonucleosides uridine and cytidine and the deoxyribonucleosides thymidine and deoxycytidine to their respective nucleotides. ATP-dependent phosphoryltransferases (kinases) catalyze the phosphorylation of the nucleoside diphosphates 2 "-de-oxycytidine, 2 -deoxyguanosine, and 2 -deoxyadenosine to their corresponding nucleoside triphosphates. In addition, orotate phosphoribosyltransferase (reaction 5, Figure 34-7), an enzyme of pyrimidine nucleotide synthesis, salvages orotic acid by converting it to orotidine monophosphate (OMP). 

The phosphorylation of the 3 -OH of a 5 -substituted thymidine was achieved with 2,4,6-triisopropylbenzenesulfonyl-3-nitro-1,2,4-triazole [49a]... 

When administered as valaciclovir, aciclovir is released during absorption, and 60% of the drug reaches the bloodstream, as described above. Site activation also occurs in herpesvirus-infected cells where aciclovir is biochemically transformed to the phosphorylated active drug by virus-specific thymidine kinase [74]. 

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