Metabolism in human cells of the D and L enantiomers of the carbocyclic analog of 2'-deoxyguanosine: substrate activity with deoxycytidine kinase, mitochondrial deoxyguanosine kinase, and 5'-nucleotidase

Abstract

The carbocyclic analog of 2'-deoxyguanosine (CdG) has broad-spectrum antiviral activity. Because of recent observations with other nucleoside analogs that biological activity may be associated the L enantiomer rather than, as expected, with the D enantiomer, we have studied the metabolism of both enantiomers of CdG to identify the enzymes responsible for the phosphorylation of CdG in noninfected and virally infected human and duck cells. We have examined the enantiomers as substrates for each of the cellular enzymes known to catalyze phosphorylation of deoxyguanosine. Both enantiomers of CdG were substrates for deoxycytidine kinase (EC 2.7.1.74) from MOLT-4 cells, 5'-nucleotidase (EC 3.1.3.5) from HEp-2 cells, and mitochondrial deoxyguanosine kinase (EC 2.7.1.113) from human platelets and CEM cells. For both deoxycytidine kinase and mitochondrial deoxyguanosine kinase, the L enantiomer was the better substrate. Even though the D enantiomer was the preferred substrate with 5'-nucleotidase, the rate of phosphorylation of the L enantiomer was substantial. The phosphorylation of D-CdG in MRC-5 cells was greatly stimulated by infection with human cytomegalovirus. The fact that the phosphorylation of D-CdG was stimulated by mycophenolic acid and was not affected by deoxycytidine suggested that 5'-nucleotidase was the enzyme primarily responsible for its metabolism in virally infected cells. D-CdG was extensively phosphorylated in duck hepatocytes, and its phosphorylation was not affected by infection with duck hepatitis B virus. These results are of importance in understanding the mode of action of D-CdG and related analogs and in the design of new biologically active analogs.

FIG. 1

Effects of dCyd and MPA on the phosphorylation of d-CdG in intact human cells. CEM cells (A) or HEp-2 cells (B) were incubated with 8 μM [³H]d-CdG alone or in combination with 5 μM MPA or 200 μM dCyd. After 8 h the acid-soluble metabolites of d-CdG were separated by SAX HPLC, and the radioactivity of each fraction was determined. TP, triphosphate; DP, diphosphate; MP, monophosphate.

FIG. 2

Effect of HCMV infection on the metabolism of d-CdG. HCMV-infected and noninfected MRC-5 cells (confluent monolayer of MRC-5 cells in 75-cm² flasks) were treated with 5 μM [³H]d-CdG for 8 h 0, 1, 2, 3, 4, and 5 days after virus infection. After incubation with [³H]d-CdG, the cells were collected and the incorporation of d-CdG into DNA (A) and the incorporation of d-CdG triphosphate (CdG-TP) (B) into all of the cells receiving each treatment were determined.

FIG. 3

Activities of dCyd kinase and 5′-nucleotidase in noninfected and HCMV-infected cells. Cell extracts were prepared from noninfected and HCMV-infected MRC-5 cells 5 days after infection. The phosphorylating activities of Ino (A) and d-CdG (B) were determined in these extracts by using conditions optimal for the measurement of 5′-nucleotidase activity (100 mM imidazole [pH 6.5], 500 mM NaCl, 50 mM MgCl₂, 5 mM ATP, and 10 mM IMP). The phosphorylating activities of dCyd (C) and d-CdG (D) were determined in these extracts by using conditions optimal for the measurement of dCyd kinase activity (50 mM imidazole [pH 7.4], 25 mM dithiothreitol, 2 mM ATP, 2.5 mM MgCl₂, 1 mg of bovine serum albumin per ml, and 5% glycerol).

FIG. 4

Effects of dCyd, Ino, and MPA on the phosphorylation of d-CdG. HCMV-infected MRC-5 cells were incubated with 8 μM [³H]d-CdG alone or in combination with 5 μM MPA, 200 μM dCyd, or 100 μM Ino plus a potent inhibitor of purine nucleoside phosphorylase (BDG). After 8 h the acid-soluble metabolites of d-CdG were separated by SAX HPLC, and the radioactivity of each fraction was determined. TP, triphosphate; DP, diphosphate; MP, monophosphate.