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. 1998 Aug;42(8):2095-102.
doi: 10.1128/AAC.42.8.2095.

Mode of action of (1'S,2'R)-9-[[1',2'-bis(hydroxymethyl) cycloprop-1'-yl]methyl]guanine (A-5021) against herpes simplex virus type 1 and type 2 and varicella-zoster virus

N Ono  1 , S Iwayama, K Suzuki, T Sekiyama, H Nakazawa, T Tsuji, M Okunishi, T Daikoku, Y Nishiyama
Affiliations

Mode of action of (1'S,2'R)-9-[[1',2'-bis(hydroxymethyl) cycloprop-1'-yl]methyl]guanine (A-5021) against herpes simplex virus type 1 and type 2 and varicella-zoster virus

N Ono et al. Antimicrob Agents Chemother. 1998 Aug.

Abstract

The mode of action of (1'S,2'R)-9-([1', 2'-bis(hydroxymethyl)cycloprop-1'-yl]methyl)guanine (A-5021) against herpes simplex virus type 1 (HSV-1), HSV-2, and varicella-zoster virus (VZV) was studied. A-5021 was monophosphorylated at the 2' site by viral thymidine kinases (TKs). The 50% inhibitory values for thymidine phosphorylation of A-5021 by HSV-1 TK and HSV-2 TK were comparable to those for penciclovir (PCV) and lower than those for acyclovir (ACV). Of these three agents, A-5021 inhibited VZV TK most efficiently. A-5021 was phosphorylated to a mono-, di-, and triphosphate in MRC-5 cells infected with HSV-1, HSV-2, and VZV. A-5021 triphosphate accumulated more than ACV triphosphate but less than PCV triphosphate in MRC-5 cells infected with HSV-1 or VZV, whereas HSV-2-infected MRC-5 cells had comparable levels of A-5021 and ACV triphosphates. The intracellular half-life of A-5021 triphosphate was considerably longer than that of ACV triphosphate and shorter than that of PCV triphosphate. A-5021 triphosphate competitively inhibited HSV DNA polymerases with respect to dGTP. Inhibition was strongest with ACV triphosphate, followed by A-5021 triphosphate and then (R,S)-PCV triphosphate. A DNA chain elongation experiment revealed that A-5021 triphosphate was incorporated into DNA instead of dGTP and terminated elongation, although limited chain extension was observed. Thus, the strong antiviral activity of A-5021 appears to depend on a more rapid and stable accumulation of its triphosphate in infected cells than that of ACV and on stronger inhibition of viral DNA polymerase by its triphosphate than that of PCV.

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Figures

FIG. 1
FIG. 1
Phosphorylation of A-5021 by viral TKs and determination of the site of phosphorylation. A-5021 was incubated with HSV-1 (a) or VZV TK (b) at 37°C for 24 h. A-5021 monophosphate (A-5021MP) was detected by HPLC. The reaction solution of VZV TK was mixed with chemically synthesized A-5021–2′-monophosphate (MP) (C) or A-5021–1′-MP (d) and was analyzed by HPLC. Peak number 1, peak containing chemically synthesized A-5021–2′-MP and A-5021MP from viral TK; peak number 2, A-5021–1′-MP; peak number 3, A-5021MP from viral TK.
FIG. 2
FIG. 2
Structure of chemically synthesized A-5021 monophosphates. (a) A-5021–1′-monophosphate. (b) A-5021–2′-monophosphate.
FIG. 3
FIG. 3
HPLC analysis of A-5021 and its phosphate esters in HSV-1-infected MRC-5 cells. HSV-1-infected MRC-5 cells were treated with 1 mM [3H]A-5021 for 6 h, and the extract was analyzed by HPLC. MP, monophosphate; DP, diphosphate; TP, triphosphate.
FIG. 4
FIG. 4
Formation of phosphate esters of nucleoside analogs in virus-infected MRC-5 cells. MRC-5 cells infected with HSV-1 (1), HSV-2 (2), or VZV (3) were treated with [3H]A-5021 (a), [3H]ACV (b), or [3H]PCV (c) at the indicated concentrations. At the designated times, cells were extracted, and the extracts were analyzed by HPLC. Open circles, triphosphate; closed triangles, diphosphate; closed squares, monophosphate.
FIG. 5
FIG. 5
Concentration dependency of formation of triphosphate esters of nucleoside analogs in virus-infected MRC-5 cells. MRC-5 cells infected with HSV-1 (a) or HSV-2 (b) were treated with various concentrations of [3H]A-5021, 3H[ACV], or [3H]PCV for 6 h. Then cells were extracted, and the extracts were analyzed by HPLC. Open circles, A-5021 triphosphate; closed triangles, ACV triphosphate; closed squares, PCV triphosphate. Conc, concentration.
FIG. 6
FIG. 6
Intracellular stability of phosphate esters of nucleoside analogs in virus-infected MRC-5 cells. MRC-5 cells infected with HSV-1 (1), HSV-2 (2), or VZV (3) were treated with [3H]A-5021 (a) [3H]ACV (b), or [3H]PCV (c) at the concentrations shown for 5 h (HSV-1 and HSV-2) or 6 h (VZV). After extracellular compounds were removed, the levels of intracellular phosphate esters were analyzed by HPLC. Open circles, triphosphate; closed triangles, diphosphate; closed squares, monophosphate. T1/2, half-life.
FIG. 7
FIG. 7
Chain termination by A-5021 triphosphate (A-5021TP) or ACV triphosphate (ACVTP). HSV-2 DNA polymerase was reacted with M13mp18 single-stranded DNA and specific oligomer (17 mer) as a template and a primer, respectively, in the presence of fixed concentrations of natural dNTPs and various concentrations of A-5021TP or ACVTP (lane 1, 1 mM; lane 2, 300 μM; lane 3, 100 μM; lane 4, 30 μM; lane 5, 10 μM; lane 6, 3 μM; lane 7, 1 μM). Cont., no chain terminator. Lanes ddG, ddA, ddT, and ddC, dideoxy sequencing with ddGTP, ddATP, ddTTP, and ddCTP, respectively. Samples were analyzed on a 6% sequencing gel, followed by autoradiography.
FIG. 8
FIG. 8
DNA chain extension by HSV-2 DNA polymerase. The reaction was performed with M13mp18 single-stranded DNA and a 5′ 32P-end-labeled specific oligomer (17 mer) as the template and the primer, respectively, in the presence or absence of 50 μM dATP, dTTP, and/or inhibitor as shown. Lanes ddG, ddA, ddT, and ddC, dideoxy sequencing with ddGTP, ddATP, ddTTP, and ddCTP, respectively. Samples were analyzed on a 15% sequencing gel, followed by autoradiography.
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