and its congener 9-{[2-hydroxy-1-(hydroxymethyl)ethoxy]-methyl}guanine (BW B759U) exhibit comparable antiviral activity against . However, BW B759U is a more potent inhibitor of both human CMV and Epstein-Barr virus (EBV) in vitro than is ACV (1-3, 5). As is the case with ACV, BW B759U is activated to the triphosphate form in HSV-and VZV-infected cells and the initial step of this phosphorylation is associated with the activity of the virus-specified thymidine kinase (TK) (refs. 4, 6-8; unpublished data Table 1). In the present study, we report that BW B759U, in contrast to ACV, is preferentially activated in human diploid fibroblasts infected with HCMV. MATERIALS AND METHODSCells and Virus. Human foreskin fibroblast (HFF) cells were derived in this laboratory from tissues obtained from Duke University Hospital Pediatrics Department (Durham, NC) and were used up to passage 11. Human diploid embryonic lung fibroblast (MRC-5) cells were obtained from American Type Culture Collection and were used between passage 22 and passage 26. Monolayer cultures were grown in Eagle's minimal essential medium (ME medium) containing 50 units of penicillin per ml and 50 ,g of streptomycin per ml supplemented with 10% fetal bovine serum (Sterile Systems, Logan, Utah) and 1% L-glutamine. HCMV strain AD169 was also obtained from American Type Culture Collection. Kerr strain of HCMV was obtained from E. S. Huang (Cancer Research Center, University of North Carolina, Chapel Hill). The clinical strain Wade was isolated. from a congenitally infected infant by J. Zeller (Duke University Hospital Infectious Diseases Laboratory).Virus stocks of AD169 were stored in liquid nitrogen and were amplified before use by low multiplicity of infection (moi) passage in MRC-5 cells [<0.05 plaque-forming units (pfu) per cell]. Supernatant virus was used at time of peak titer (days 8-11) and was back titrated at the time of use to determine approximate moi.Cells and virus stocks were routinely checked for mycoplasma contamination by electron microscopy and [125i]iododeoxycytidine autoradiography methods.Cell Cytotoxicity Assay. The cytotoxicity of the nucleoside analogs was determined by Naomi Cohn (The Wellcome Research Laboratories) by using a cell growth assay that allows Abbreviations: EBV, Epstein-Barr virus; HCMV, human cytomegalovirus; HSV, herpes simplex virus; VZV, varicella zoster virus; ACV, acyclovir or 9-[(2-hydroxyethoxy)methyl]guanine; FIaC, 2'-fluoro-5-iodoarabinosylcytosine; BW B759U, 9-{[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl}guanine, also abbreviated 2' NDG, BIOLF-62, and DHPG by other investigators; HFF, human foreskin fibroblast; TK, thymidine kinase; pfu, plaque-forming unit(s); moi, multiplicity of infection. 2473The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
We have isolated a human cytomegalovirus mutant that is resistant to the antiviral drug 9-{[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl}guanine (BW B759U), yet exhibits wild-type sensitivity to inhibitors of herpesvirus DNA polymerases such as phosphonoformic acid and aphidicolin. Cells infected with the mutant accumulate -1/1Oth the amount of drug triphosphate as do those infected with the wild-type parent. This reduction in drug triphosphate could not be attributed to altered drug uptake or to reduced stability of the triphosphate, once formed. The induction of normal nucleoside and deoxynucleoside triphosphates and certain cellular nucleoside kinases was comparable in mutant and wild-type virus infections. These results provide strong evidence for the importance of phosphorylation in the selectivity of this antiviral compound and raise the possibility that human cytomegalovirus encodes a nucleoside kinase. The mutant may identify the existence of a cytomegalovirus function whose properties could facilitate genetic analysis of this important pathogen.Human cytomegalovirus (HCMV) infections can produce serious consequences, including birth defects in newborns and life-threatening and sight-threatening disease in immunosuppressed individuals, such as transplant and cancer chemotherapy recipients as well as patients with acquired immunodeficiency syndrome (1).Currently, there are no licensed treatments for HCMV infections in the U.S. One promising anti-HCMV agent is the deoxyguanosine analog, 9-{[2-hydroxy-1-(hydroxymethyl)-ethoxy]methyllguanine (BW B759U; also known as 2'NDG, DHPG, and BIOLF-62) (2-4). BW B759U is a congener ofthe antiherpetic drug acyclovir (ACV), which is licensed for the treatment of herpes simplex virus (HSV). However, BW B759U is much more potent against HCMV than is ACV (2-6). Recent clinical studies suggest that BW B759U has consistent antiviral activity against HCMV in vivo and may be useful in treatment of HCMV disease (7-9).BW B759U is converted to its triphosphate form in HCMVinfected cells 10-100 times more efficiently than in uninfected cells, and much more efficiently than is ACV (5, 6). BW B759U triphosphate can then inhibit HCMV-induced DNA polymerase with Ki values below 1.5 x 10-6 M (4, 6, 10). As yet, an enzyme(s) specifically induced by HCMV infection that phosphorylates BW B759U has not been identified. Furthermore, it has not been possible to detect a HCMVencoded enzyme with properties similar to the HSV-encoded thymidine kinase (TK) (refs. 11 and 12; J.A.F. and K.K.B., unpublished data), an enzyme that is principally responsible for the monophosphorylation of BW B759U and ACV in HSV-infected cells (13-15). On the other hand, it has been shown that HCMV induces increases in host cell nucleoside kinases (5,6,11,12,16,17), but none of these has been identified as the enzyme responsible for BW B759U phosphorylation. Thus, it is still an open question whether BW B759U anabolism in the HCMV-infected cell results from the activity of an unidentified virus-encoded enzyme or ...
Compound A723U, a 2-acetylpyridine thiosemicarbazone, produced apparent inactivation of herpes simplex virus type 1 (HSV-1) ribonucleotide reductase. Inactivation occurred after A7M3U formed a reversible complex with the enzyme and only while the enzyme was catalyzing the formation of deoxynucleotides. A723U inhibited HSV-1 replication at concentrations that were not toxic to the confluent host cells. Most importantly, A723U and acyclovir (ACV) were found to exhibit mutual potentiation of their antiviral activities. Subinhibitory concentrations of either compound greatly reduced the ED50 (median effective dose) of the other. Studies of the deoxynucleotide pool sizes and the levels of ACV triphosphate, (ACV-P3) revealed that A723U not only significantly reduced the pool of dGTP but also increased the level of ACV-P3 in infected cells. The net result was an 80-fold increase in the ratio of ACV-P3 to dGTP. This should greatly facilitate the initial binding of ACV-P3 to HSV-1 DNA polymerase and probably accounts for the mechanism of potentiation.Acyclovir (ACV), a clinically useful antiherpetic agent, is selectively phosphorylated by herpes simplex virus (HSV)-encoded thymidine kinase (1, 2). Cellular enzymes (3, 4) then catalyze the conversion of ACV monophosphate to the triphosphate form (ACV-P3), which is the fully activated metabolite that is toxic to the virus. Its mechanism of action appears to be the inhibition ofviral DNA synthesis (5), which is probably the result of the selective inactivation by ACV-P3 of the HSV type 1 (HSV-1)-encoded DNA polymerase (6). Prior to inactivation, an obligate reversible complex (Michaelis type) is formed between ACV-P3 and the susceptible enzyme (6). Since dGTP can competitively prevent this initial binding of ACV-P3 (6-8), and since the level of dGTP characteristically increases after treatment of HSV-1-infected cells with ACV (9), dGTP has the potential to protect the polymerase from inactivation. Furthermore, an agent that blocks the synthesis of dGDP would decrease the intracellular dGTP pool. It follows that such an agent should promote the ACV-P3 inactivation ofthe HSV DNA polymerase. In the present study, we have investigated an inhibitor of HSV-1 ribonucleotide reductase, the enzyme that catalyzes the synthesis of dGDP in HSV-1 § and appears to be essential to HSV replication (11,12). This inhibitor was found to be tolerated by the host cells and to significantly potentiate the antiviral activity of ACV. In addition to its predicted suppression of the dGTP pool, this ribonucleotide reductase inhibitor also unexpectedly produced a marked increase in the level of ACV-P3. MATERIALS AND METHODSChemical Synthesis. Burroughs Wellcome compound A723U [2-acetylpyridine 4-(2-morpholinoethyl)thiosemicarbazone] was prepared by refluxing 2-acetylpyridine and 4-(2-morpholino)thiosemicarbazide (molar ratio, 1.1-1.0) for 75 min in 95% ethanol containing 2% (vol/vol) glacial acetic acid. The product was obtained as yellow-tinted crystals by recrystallization from 95% ethanol (...
The ability of LM cells, thymidine kinase-deficient LM cells (LMTK-), and LMTK-cells transformed to the LMTK+ phenotype by herpes simplex virus type 1 genetic information (LH7 cells) to anabolize the acyclovir congener ganciclovir was examined. About 50-fold more ganciclovir triphosphate was produced by LH7 cells than by either LM or LMTK-cells. Growth inhibition studies indicated that 180 and 120 ,uM ganciclovir were required to achieve 50% growth inhibition of LM and LMTK-cells, respectively; only 0.07 ,uM ganciclovir was necessary to achieve 50% inhibition of LH7 cells. DNA synthesis in the transformed cells was significantly reduced by ganciclovir treatment, whereas ganciclovir had little effect on DNA synthesis in the nontransformed cells. Alkaline sucrose gradient sedimentation analysis of transformed cellular DNA indicated that LH7 DNA synthesized in the presence of ganciclovir chased into mature DNA. Both LM and LH7 DNA synthesized in the presence of ganciclovir exhibited a concentration-dependent reduction in the rate of elongation into mature DNA. Finally, [14C]ganciclovir was incorporated internally into the growing DNA chains of L117 cells.
(9) and to potentiate the antiherpetic activities of ACV. In addition to producing the expected decrease in the dGTP pool, A723U also caused the pool of ACVTP to increase by 10-fold (17).In the present study, the properties of 2-acetylpyridine 5-[(dimethylamino)thiocarbonyl]thiocarbonohydrazone (A111OU) were investigated. This compound was found to be considerably more potent than A723U. At submicromolar concentrations, A111OU inactivated the ribonucleotide reductases of these three herpes viruses. It also markedly potentiated the activity of ACV against the viruses in vitro. Furthermore, as reported elsewhere ( §, ¶), A111OU and ACV produced significantly synergistic therapy as topical treatment of HSV-infected animals. MATERIALS AND METHODSReagents. All reagents not described below were obtained as previously described (11).Synthesis of A111OU. A mixture of 24.2 g (0.203 mol) of 4,4-dimethylthiosemicarbazide, 26.6 g (0.220 mol) of 2-acetylpyridine, 2.5 ml of glacial acetic acid, and 100 ml of 95% (vol/vol) ethanol was refluxed for 1.5 hr and then incubated overnight at room temperature. Thick orange needles contaminated with a small amount of a light-colored solid were collected, washed with 20 ml of ethanol, and, while still damp, added to 450 ml of boiling methanol. After 10 min, the mixture was filtered. The undissolved solid was re-incubated with 50 ml of boiling methanol for 3 min and then filtered. After 15 min at room temperature, the yellow product recrystallized from the combined filtrates and was collected by filtration.
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