Potentiation of antiherpetic activity of acyclovir by ribonucleotide reductase inhibition.

Abstract

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 (...

2-Acetylpyridine 5-[(dimethylamino)thiocarbonyl]-thiocarbonohydrazone (A1110U), a potent inactivator of ribonucleotide reductases of herpes simplex and varicella-zoster viruses and a potentiator of acyclovir.

“…Effects of Al11OU on Cellular Deoxynucleoside Triphosphate and ACVTP Levels. In confirmation of earlier reports (17,26), Table 1 shows that ACV caused the dGTP pool to increase in HSV-1 infected cells. The addition of A111OU, as expected, decreased the dGTP pool by 90%o.…”

“…Further evidence that the time-dependent decrease in the rate of product formation was due to inactivation of the ribonucleotide reductases and not to the modification of another reagent in the reaction mixture was obtained when additions of fresh enzyme to totally inhibited reactions produced a second cycle of decelerating activities. The apparent inactivation ofthese enzymes is similar to that observed for A723U with HSV-1 and VZV ribonucleotide reductases (9,17). However, A111OU is about 30-fold more potent than A723U and is among the most potent inactivators of HSV ribonucleotide reductase reported to date (6,20,30).…”

“…The mechanism by which A111OU potentiates ACV is similar to that of A723U (17). Both ribonucleotide reductase inactivators prevent the build-up of dGTP, which in their absence would be concomitant to the inhibition of viral DNA synthesis by ACVTP.…”

“…Sci. USA 86 (1989) fi-/ / --anism for elevating the concentrations of ACVTP is not yet understood, as described before (17), it is most likely related to the inhibition of ribonucleotide reductase. Recently, Karlsson and Harmenberg (31) reported that inhibition of HSV ribonucleotide reductase by A723U decreases the excretion of thymidine by infected cells.…”

“…To prevent this competition, inhibitors of the viral ribonucleotide reductases were sought. 2-Acetylpyridine 4-(2-morpholinoethyl)thiosemicarbazone (A723U) was found to inactivate HSV-1 ribonucleotide reductase (17) as well as VZV ribonucleotide reductase (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).…”