[CANCER RESEARCH 38, 1916-1921, July 1978] 0008-5472/78/0038-OOOOS02.00 Deoxyadenosine Antagonism of the Antiviral Activity of 9-/3-D- Arabinofuranosyladenine and 9-/3-D-Arabinofuranosylhypoxanthine1

Sandra H. Smith, Charles Shipman, Jr., and John C. Drach

Dental Research Institute and Department of Oral Biology, School of Dentistry [S. H. S., C. S., J. C. D.], and Department of Microbiology, School of Medicine [C. S.¡,The University of Michigan, Ann Arbor, Michigan 48709

ABSTRACT or humans (3, 8, 44, 45) when used in its antiviral dose range. Oeoxyadenosine but not adenosine reversed the anti We have been studying the biochemical basis for the viral activity of 9-ß-D-arabinofuranosyladenine (ara-A) and selective toxicity of ara-A toward HSV-1 and have found that 9-ß-D-arabinofuranosylhypoxanthine (ara-H) when used in the drug inhibits viral DNA synthesis to a greater extent the presence of coformycin, an inhibitor of adenosine than it does host cell DNA synthesis (34, 36). On the basis deaminase. of this in vitro work, we have postulated that the high In suspension cultures of KB cells, 10 /tM ara-A inhibited therapeutic index noted in vivo is related to the preferential the replication of herpes simplex virus type 1 by 80%. inhibition of viral DNA synthesis (11). Concomitant addition of 50 /¿Mdeoxyadenosine reduced Another in vitro approach to elucidate the mechanism of the antiviral activity of 10 /MMara-A to only 40% inhibition. action of ara-A has involved the attempt to antagonize its Adenosine failed to antagonize the antiviral activity. In antiviral activity through the concurrent use of naturally monolayer cultures of KB cells, the 50% inhibitory concen occurring nucleosides. Based on the ability of 2'-deoxycy- tration of ara-A was increased from 1.5 to 2.9 /IM by 2 /purine nucleosides would plot method indicated that the antagonism was competi reverse the antiviral activity of ara-A. However, studies by tive. de Rudder and Privat de Garilhe (30) and by Miller ef al. (22) The antiviral activity of ara-H also was antagonized by failed to demonstrate any antagonism of the antiviral activ deoxyadenosine. The 50% inhibitory concentration of ara ity of ara-A by adenosine or by dAdo. Sidwell et al. (37) also bi was increased from 42 //M to 70, 91, or 121 //M by the found that the inhibition of pseudorabies virus-induced concurrent addition of 5, 10, or 20 /nM deoxyadenosine. cytopathic effect by ara-A was not reversed by 5 times the Competitive antagonism could not be demonstrated. concentration of adenosine, dAdo, or other nucleosides. In the absence of the adenosine deaminase inhibitor, Similar results were obtained by Scheffler ef al. (31), who neither ara-A nor ara-H was antagonized by deoxyadeno found that the inhibition of adenovirus multiplication by sine. Since such inhibitors were not available until re ara-A was not antagonized by a 2 mw concentration of cently, previous investigators were unable to observe the adenosine or other ribosyl nucleosides. antagonistic capacity of deoxyadenosine. In the current study we have reexamined the question of whether adenosine or dAdo can antagonize the antiviral INTRODUCTION activity of ara-A. A different experimental design was used wherein the activity of adenosine deaminase (EC 3.5.4.4.) The activity of ara-A3 against several DNA virus infections was blocked by the use of coformycin (25) and drug effects has been documented by extensive studies in cell culture were measured on the replication of HSV-1, not on plaque (35) and in experimental animals (38). Clinical studies now production. Under these conditions we found that the have proven the usefulness of the drug against herpetic antiviral activity of both ara-A and its deaminated metabolite keratitis (26), disseminated herpes zoster (44), and herpes ara-H was antagonized by dAdo. simplex encephalitis (45). The current interest in treating herpesvirus infections with ara-A has arisen largely because the drug does not produce serious toxicity in animals (18) MATERIALS AND METHODS

Materials. ara-A and ara-H were provided through the courtesy of Dr. Harold E. Machamer and Dr. Robert Hodges, 1Presented in part to the Sixteenth Interscience Conference on Antimi Parke, Davis and Co., Detroit and Ann Arbor, Mich. The crobial Agents and Chemotherapy, Chicago. III., October 27, 1976 (39). Supported by USPHS Grant DE 02731 from the National Institute of Dental purity of ara-H was determined by high-pressure liquid chro- Research. matography and was found to be >99%; the preparation 2To whom requests for reprints should be addressed, at the School of contained <0.2% ara-A. Adenosine and dAdo were pur Dentistry, The University of Michigan, Ann Arbor, Mich. 48109. 3The abbreviations used are: ara-A, 9-0-D-arabinofuranosyladenine; HSV- chased from Sigma Chemical Co., St. Louis, Mo. Coformy 1, herpes simplex virus type 1; dAdo, 2'-deoxyadenosine; ara-H, 9-/3-D- cin, a known adenosine deaminase inhibitor, was the gift of arabinofuranosylhypoxanthine; PFU, plaque-forming units, U,,, 50% inhibi tory; ara-ATP, the 5'-triphosphate of 9-p-D-arabinofuranosyladenine. Professor H. Umezawa, Institute of Microbial Chemistry, Received December 5, 1977; accepted March 28, 1978. Tokyo, Japan. u-Arginine hydrochloride and 4-(2-hydroxy-

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1978 American Association for Cancer Research. dAdo Antagonism of ara-A and ara-H ethyl)-1-piperazineethanesulfonic acid were purchased from method of least squares (12). The U»concentrations were Calbiochem, La Jolla, Calif. Donor calf serum (Flow Labo interpolated directly from the regression lines. Identity of ratories, Inc., Rockville, Md.) was extensively heated at 56° any 2 dose-response regression lines was tested with 1-way for 12 hr to inhibit the serum-associated deamination of covariance analysis (12) by calculating p values for the nucleosides and nucleoside analogs (33). Fetal calf serum equality of the regressions and slopes. and horse serum were purchased from K C Biological, Inc., Double Reciprocal Plot Analysis. Double reciprocal plot Lenexa, Kans. analysis was used to establish the mechanism of the antag Cell Culture Techniques. The source of cells and HSV-1 ; onistic effect (13). Values for these plots were obtained in the routine growth and passage of BHK 21/4 cells and KB the following manner: (a ) the mean virus titer (control virus cells; the propagation and titration of HSV-1; and the titer) was calculated from the virus titers of all cultures techniques used for the enumeration of cells, the determi containing no agonist in a particular series of experiments; nation of cell viability, and the detection of Mycoplasma (b) values for the inhibition of virus replication, expressed contamination have been described previously (36). as percentage of control virus titer, were interpolated from Protocol for Studies in Suspension Culture. Suspension the dose-response regression line at the corresponding cultures of KB cells containing 2 to 3 x 10s cells/ml were drug concentrations; (c ) the percentage of the control virus pelleted and resuspended in 2 to 4 ml of HSV-1 stock titer at each drug level was converted back to a virus titer suspension at a multiplicity of 3 to 4 PFU/cell. Following a with the use of the mean titer described in a; (d) this value 1-hr period of viral adsorption at 37°,the cells were washed was subtracted from the mean control virus titer to deter 2 times in minimal essential medium with spinner salts, mine the change in virus titer (A); (e) the reciprocal of the divided into replicate cultures, and resuspended in the same change in virus titer (1/A) was regressed on the reciprocal medium containing 1.2 mM arginine, 3.5 /MM coformycin, of the agonist concentration by the method of least squares and 10% extensively heated calf serum (33) with or without (12). Values of Kx (the dissociation constant of the agonist ara-A, dAdo, or adenosine. The cultures were incubated at drug-receptor complex, analogous to Km in enzyme kinet 37°for 24 hr; aliquots were removed aseptically at 4, 12, ics) and K, (the dissociation constant of the antagonist and 24 hr and stored at -76° for subsequent virus assay. drug-receptor complex, analogous to K¡inenzyme kinetics) HSV-1 titers were determined with the use of monolayer were calculated from the slopes and x-intercepts of the cultures of BHK 21/4 cells grown in 60- x 15-mm Contur double reciprocal regression lines. plastic tissue culture dishes (LUX Scientific Corp., Thou sand Oaks, Calif.). The cells were planted at 1 x 106 cells/ dish in Ham's Nutrient Mixture F-12 supplemented with 5% RESULTS unheated fetal calf serum or 5% unheated horse serum. Preliminary Studies. Initial experiments were performed After 22 to 24 hr, the cells were 90% confluent and were with suspension cultures of KB cells infected with HSV-1 so inoculated with 0.2 ml of the virus suspension to be assayed that virus titers could be measured conveniently at selected and incubated in a humidified 3% CO2-97% air atmosphere times. HSV-1-infected cultures were exposed to ara-A alone at 37°for 1 hr to permit viral adsorption. After adsorption or ara-A plus 5 times the concentration of adenosine or the cells were overlaid with 5 ml of F-12 medium containing dAdo, and the resulting titers were compared to cultures 0.5% methocel (4000 centipoises/sec; Dow Chemical Co., with no drug. Coformycin was used in all cultures to inhibit Midland, Mich.), and incubated for an additional 3 days. adenosine deaminase activity totally. Chart 1 illustrates that After fixation with neutral formalin, the cells were stained 10 fjLM ara-A, a concentration previously found to reduce with Giemsa tissue stain, Wolbach modification (Paragon HSV-1 titers by approximately 1 log (34), inhibited viral C. and C. Co., Inc., Bronx, N. Y.), and virus titers were replication by 80% in these experiments. The presence of calculated from the number of PFU. 50 /*M dAdo antagonized the effect of ara-A, reducing Protocol for Studies in Monolayer Culture. KB cells were antiviral activity to 40% inhibition. In contrast 50 /¿Maden planted in 25-sq cm plastic tissue culture flasks (Falcon osine was without antagonistic effects. In the absence of Plastics, Oxnard, Calif.) at 2 to 2.5 x 106 cells/flask. After a ara-A, neither 50 /¿Madenosine nor 50 ¡J.MdAdo affected 24-hr incubation, each culture was infected with HSV-1 at a HSV-1 replication (data not shown). multiplicity of 5 to 8 PFU/cell. Following virus adsorption dAdo Antagonism. The antagonistic effect of dAdo on for 1 hr at 37°,the virus inoculum was aspirated and the cell the antiviral activity of ara-A was investigated in more detail sheet was washed twice with warm medium. Five ml of with monolayer cultures of HSV-1-infected KB cells. Its minimal essential medium with Hanks' salts containing 10% effect on the antiviral activity of ara-H also was examined. extensively heated calf serum, 1.2 mivi arginine, and selected Virus titers were measured following incubation of infected combinations of coformycin, ara-A, ara-H, and dAdo were cultures with selected concentrations of ara-A and dAdo. added to each flask. Drugs and dAdo were added to the Coformycin was present in all cultures to prevent the medium immediately before use. The cultures were incu deamination of adenine nucleosides. Chart 2 illustrates bated for an additional 12 or 21 hr and frozen at -76°. that, in the absence of dAdo, 10 /MMara-A reduced HSV-1 Following 3 cycles of alternate freezing and thawing, the titers to 8% of the control titer. Increasing concentrations virus titers of the cell lysates were determined according to of dAdo progressively antagonized the activity of ara-A. the plaque assay described above. Maximum antagonism was produced by concentrations of Statistical Analyses. Dose-response relationships were dAdo >20 /MM at which 10 /XM ara-A produced only 30% constructed by linearly regressing values of the percentage inhibition of HSV-1 replication. In separate experiments of the control virus titer on log drug concentrations by the (data not presented), the antiviral activity of lower concen-

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io 20 30 40 50 60 DEOXYADENOSINECONCENTRATION Chart 2. dAdo antagonism of the antiviral activity of ara-A and ara-H. Monolayer cultures of KB cells were infected with HSV-1 at a multiplicity of 5 to 8 PFU/cell. Following virus adsorption the cultures were incubated at 37° 1 «10- in fresh medium with 3.5 ¿iMcoformycin and either no drug or the indicated concentrations of ara-A, ara-H. and dAdo. After 12 or 21 hr, the cultures were harvested and assayed in triplicate for plaque-forming ability on BHK 21/4 cells. The data are expressed as the percentage of the mean virus titer J_ in cultures containing no agonist (approximately 2.5 ±0.7 x 107 PFU/ml of 8 12 16 20 24 cell lysate). INCUBATION TIME (hr) Chart 1. Effect of adenosine (ADO) and dAdo on the inhibition of HSV-1 replication by ara-A. A suspension culture of KB cells was infected with HSV- 1 at a multiplicity of 3 to 4. Following virus adsorption the culture was antiviral activity (Chart 36). The regression line for ara-H divided into 4 replicate cultures containing 1.8 to 2.5 x 105 cells/ml and incubated at 37°in fresh medium containing 3.5 ^M coformycin. Cultures alone was distinct from the regression lines for ara-H plus 5 contained either no drug. 10 ^M ara-A 10 ¿iMara-Aplus 50 I./Madenosine. /UMdAdo (p = 0.18), ara-H plus 10 ^M dAdo (p = 0.009) or 10 fiM ara-A plus 50 IJ.MdAdo. Aliquots were removed aseptically from the and ara-H plus 20 /H.MdAdo(p = 0.02). The \Mconcentration infected cultures at 4. 12. and 24 hr and assayed for plaque-forming ability for ara-H without dAdo was 42 /J.M,and this increased to 70, on BHK 21/4 cells. Each assay was performed in triplicate; titers are the average of 2 experiments. 91, and 121 fj.M by the addition of 5, 10, and 20 ^M dAdo, respectively. trations of ara-A (1 or 2 pM) was completely reversed by 20 Dose-response experiments such as those described to 60 fjiM dAdo. Surprisingly, dAdo also antagonized the above also were performed in the absence of coformycin. activity of ara-H but to a lesser extent (Chart 2). Under this condition dAdo failed to antagonize the antiviral Dose-Response Relationships. In order to characterize activity of either ara-A or ara-H. The dose-response regres the antagonistic effect of dAdo more fully, a series of dose- sion line for ara-A alone was coincident with the regression response experiments was performed using either ara-A or lines for ara-A plus dAdo (p = 0.91 and 0.999 comparing ara-H as the agonist and dAdo as the antagonist. Four ara-A alone with ara-A plus 25 or 50 ¡J.MdAdo,respectively). selected concentrations of ara-A (plus coformycin) were The Is»concentrations of ara-A derived from the regression utilized to evaluate the effects of 0, 2, and 10 /J.MdAdo. In lines were 21 /¿Mineach case, clearly illustrating a lack of the absence of the antagonist, the activity of ara-A was antagonistic effect. Similar results were observed in exper similar to that found previously (34). The addition of 2 or 10 iments with ara-H. The dose-response regression line for P.MdAdo reduced antiviral activity at all ara-A concentra ara-H without dAdo was equal to those for ara-H plus 2 or tions tested (Chart 3A). To establish firmly that the dose- 10 mw dAdo (p = 0.97 and 0.74, respectively). ara-H I5(, response curves obtained in the presence of antagonist concentrations derived from the 3 regression lines were 64, were distinct from the curve generated in its absence, the 62, and 48 /¿Mforara-H alone, ara-H plus 2 /uM dAdo, and equality of the dose-response regression lines was tested ara-H plus 10 /XMdAdo, respectively. The apparent differ with 1-way covariance analysis (12). The regression line for ences among the U»concentrations are not significant, ara-A without dAdo was separate from the regression lines however, owing to the near equality of the regression lines. for ara-A in the presence of 2 (J.MdAdo (p = 0.02) and 10 Nature of the Antagonism. The parallelism of the dose- UM dAdo (p = 0.001). I50concentrations of ara-A were response regression lines in Chart 3 suggested that the calculated from each regression line. The I5(lconcentration antagonism between ara-A or ara-H and dAdo was compet for ara-A without dAdo was 1.5 /J.M.This was increased to itive (13). For further exploration of the agonist-antagonist 2.9 fíMby2 /¿MdAdoand to 8.5 /¿Mby10 ¿IMdAdo. relationship, double reciprocal plots were constructed Similar dose-response experiments were performed with using values from the regression lines of Chart 3. The ara-H (plus coformycin) as the agonist. At the concentra plot for antagonism of ara-A by dAdo (Chart 4X\)shows the tions of ara-H tested (50, 100, and 200 /nM),all 3 concentra regression lines intersecting on or near the y-axis, thereby tions of dAdo (5, 10, and 20 /nM) caused a reduction of confirming the competitive nature of the interaction. The Kx

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120

IDO 200 ARA-H CONCENTRATION

Chart 3. Dose-response relationships between antiviral activity of ara-A 002 (A) or ara-H (B) and concentration of drug or of drug plus antagonist. [ARA- Experimental conditions were the same as described in the legend to Chart 2. The data are expressed as the percentage of the mean virus titer in Chart 4. Double reciprocal plot analysis of dAdo antagonism of the cultures containing no agonist (A, 2.2 ±0.5 x to7 PFU/ml cell lysate; fi, 2.7 antiviral activity of ara-A (A) and ara-H (B). Values of the reciprocal of the ±0.7 x 107 PFU/ml cell lysate). Regression lines were constructed by the change in virus titer in the presence of drug with or without antagonist were method of least squares. Each point represents the mean of 3 separate calculated from corresponding points on the lines in Chart 3 and regressed experiments. Bars. S.E. Values for ara-A plus 2 ¿iMdAdoand ara-H plus 5 on the reciprocal of drug concentration. fj.u dAdo are results from 1 experiment; other values are the average of duplicate experiments. not dAdo reversed the ability of ara-A to inhibit DMA synthe sis in TA3 cells, and Mülleref al. (23) found that dAdo value* for ara-A was 2.0 f¿M,andthe (Rvalue* for dAdo was partially negated ara-A-induced inhibition of L5178Y cell 2.7 fj.M. Regression lines in the double reciprocal plot for proliferation. In addition Hubert-Habart and Cohen (14) the antagonism of ara-H by dAdo (Chart 4B) did not have a reported that adenosine and adenine antagonized the lethal common intersection, indicating that a more complex rela effect of ara-A in Escherichia coli. In the present series of tionship may exist. experiments, antagonism of the antiviral effect of ara-A by dAdo was observed only when coformycin, an adenosine DISCUSSION deaminase inhibitor, was present. In the absence of cofor dAdo antagonized the antiviral activity of ara-A when mycin, both ara-A and dAdo were deaminated to the respec added to HSV-1-infected cultures of KB cells containing an tive hypoxanthine nucleosides. Consequently, the antago adenosine deaminase inhibitor. Such antagonism had not nistic effect of dAdo could not be detected, and the antiviral been observed by previous investigators (22, 30, 31, 37) activity of ara-A was reduced. The complete loss of the whose experiments were performed without an inhibitor. antagonistic activity of dAdo but only partial loss of the Brink and LePage (6) noted, however, that adenosine but antiviral activity of ara-A might be caused by a more rapid deamination of dAdo than of ara-A by adenosine deaminase ' Defined according to the method of Goldstein (13) (see "Materials and (1), as well as by the retention of some antiviral activity by Methods"). ara-H, the deaminated metabolite of ara-A.

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Although dAdo was used as an antagonist in the current known about how ara-H inhibits DNA synthesis, its pre series of experiments, the compound has agonist proper sumed mode of antiviral action (34, 36). However, the 5'- ties of its own. In 1965 Borman and Roizman (4) reported monophosphate of ara-H is active as an antiviral drug (2), that mM concentrations of dAdo inhibited the multiplication and this nucleotide is formed metabolically in erythrocytes of HSV-1. Other work performed before adenosine deami- (7). Furthermore, the bioconversion of 9-/3-D-arabinofura- nase inhibitors were available established that dAdo also nosylhypoxanthine 5'-monophosphate to 9-/3-D-arabinofur- lowered cell viability and inhibited DMA synthesis if high anosyladenine 5'-monophosphate is possible (5) and would concentrations were used (15, 16, 27). Recently, similar explain how an adenine nucleoside (dAdo) antagonized the cellular toxicities have been observed at much lower dAdo activity of a hypoxanthine nucleoside (ara-H). Recently, it concentrations (50 to 100 ¿¿M)inthe presence of adenosine has been demonstrated that 9-/3-D-arabinofuranosylhypo- deaminase inhibitors (19, 21). In the current studies, how xanthine 5'-monophosphate serves as a substrate for adeny- ever, dAdo showed no antiviral effects at concentrations up losuccinate synthetase (EC 6.3.4.4) (41) and that the result to 60 fjLM. ing arabinosyladenylosuccinate is cleaved to 9-ß-D-arabi- One of several mechanisms may be responsible for the nofuranosyladenine 5'-monophosphate by adenylosuccin- noted antagonism between ara-A and dAdo. Some of the ate lyase (EC 4.3.2.2). Significantly, dAMP is a competitive more obvious possibilities for the competitive interaction inhibitor of the lyase (40). Although the entire pathway has between ara-A and dAdo include the following, (a) compe not been observed in a single system, it appears that ara-H tition for a common transport protein. The transport of both can be converted to ara-A nucleotides, thereby providing at purine and pyrimidine nucleosides across the plasma mem least 2 points at which 2'-deoxyadenosine nucleotides brane is carried out in several mammalian cell lines by a could antagonize antiviral activity, i.e., at adenylosuccinate single, saturable carrier-mediated system (42). Many struc lyase and DNA polymerase. Consequently, our current data turally related nucleoside analogs competitively inhibit strengthen the possibility (20) that ara-H acts by conversion adenosine transport by this system. The most critical struc to nucleotides of ara-A. Proof of this assumption will require tural requirements for binding are the nature of the base additional experimental evidence. and the 3'-hydroxy configuration of the sugar, both of which are the same in dAdo and ara-A. Thus it is possible that these compounds interacted competitively at the level REFERENCES of transport into the cell, (b) Competition of ara-A and 1. Agarwal, R. P., Sagar, S. M.. and Parks, R. E., Jr. Adenosine Deaminase dAdo as substrates for the same kinase. The major intracel- from Human Erythrocytes: Purification and Effects of Adenosine Ana lular form of ara-A is the 5'-triphosphate (10, 23, 32), and logs. Biochem. Pharmacol.. 24: 693-701, 1975. 2. Allen, L. B., Hintz, C. J., Wolf, S. M., Huffman, J. H., Simon, L. N., there is good evidence that ara-A must be phosphorylated Robins, R. K., and Sidwell, R. W. Effect of 9-/3-D-Arabinofuranosylhypox- to ara-ATP in order to inhibit DNA synthesis (23, 32). anthine b Monophosphate on Genital Lesions and Encephalitis Induced by Herpesvirus hominis Type 2 in Female Mice. J. Infect. Diseases. '33 Because both ara-A and dAdo can serve as substrates for (Suppl.): A178-A183, 1976. 2'-deoxycytidine kinase from calf thymus (17), it is possible 3. Aronson, M. D., Phillips, C. F., Gump, D. W., Alberimi, R. J., and that dAdo antagonized the phosphorylation of ara-A. (c) Phillips, C. A. Vidarabine Therapy for Severe Herpesvirus Infections: An Unusual Syndrome of Chronic Varicella and Transient Immunologie Competitive inhibition of a DNA polymerase by ara-ATP. It Deficiency. J. Am. Med. Assoc.,235: 1339-1342, 1976. has been known for some time that ara-ATP inhibits at least 4 Borman, G. S.. and Roizman. B. The Inhibition of Herpes Simplex Virus Multiplication by Nucleosides. Biochim Biophys. Acta. 103: 50-59, 1 mammalian DNA polymerase by competition with dATP 1965. [see review by Cohen (10)]. Recent work by Mülleref al. 5. Brink. J. J., and LePage, G. A. Metabolism and Distribution of 9-/J-D- (24) and by us (28) indicates that ara-ATP inhibits HSV-1- Arabinofuranosyladenine in Mouse Tissues. Cancer Res.. 24. 1042-1049, 1964. induced DNA polymerase to a greater extent than either 6. Brink, J. J., and LePage. G. A. 9-/3-c-Arabinofuranosyladenine as an cellular DNA polymerases a or ß,and this inhibition is Inhibitor of Metabolism in Normal and Neoplastic Cells. Can. J. Bio competitive with respect to dATP. Such inhibition not only chem. ,43: 1-15, 1965. 7. Chang, T., and Glazko, A. J. Effect of an Adenosine Deaminase Inhibitor may explain the competitive antagonism reported in this on the Uptake and Metabolism of Arabinosyl Adenine (Vidarabine) by study but also may be related to the selective antiviral Intact Human Erythrocytes. Res. Commun. Chem. Pathol. Pharmacol.. activity of ara-A (11). Other work by Mülleref a/. (23) also 14: 127-140, 1976. 8. Chien. L T.. Whitley, R. J.. Nahmias, A. J.. Lewin, E. B., Linnemann, C. supports this idea and argues against the first 2 possibili C., Jr., Frenkel. L. D., Ballanti, J. A., Buchanan, R. A., and Alford, C. A., ties. These investigators found that increasing the extracel Jr. Antiviral Chemotherapy and Neonatal Herpes Simplex Virus Infection: A Pilot Study-Experience with Adenine Arabinoside (ARA-A). Pediat lular concentration of dAdo in the presence of a constant rics, 55: 678-685, 1975. amount of ara-A did not lower intracellular ara-ATP levels. 9. Chu, M. Y., and Fischer, G. A. A Proposed Mechanism of Action of 1-0- Intracellular dATP levels did increase, however, and the D-Arabinofuranosyl-cytosine as an Inhibitor of the Growth of Leukemic Cells. Biochem. Pharmacol., 11: 423-430, 1962. antiproliferative activity of ara-A diminished. 10. Cohen, S. S. The Lethality of Aranucleotides. Med. Biol., 54: 299-326, The nature and biochemical basis of dAdo antagonism of 1976. ara-H is more difficult to understand. dAdo was less effective 11. Orach, J. C., and Shipman, C.. Jr. The Selective Inhibition of Viral DNA Synthesis by Chemotherapeutic Agents: An Indicator of Clinical Useful as an antagonist of ara-H (Chart 2), and the nature of the ness? Ann. N. Y. Acad. Sci.. 284. 396-406, 1977. antagonism was not readily apparent (Chart 4B). Although 12. Fox, D. J., and Guire, K. E. Documentation for MIDAS: Michigan Interactive Data Analysis System, Ed. 3 Ann Arbor, Mich.: The Statistical the absence of competitive antagonism may be due to the Research Laboratory of the University of Michigan, 1976. variability of virus replication data, it is possible that ara-H 13. Goldstein. A.. Aronow, L., and Kaiman. S. M. Consequences of Drug- interacted with dAdo at more than 1 site. At the present Receptor Interactions: Analysis of the Graded Dose-Response Relation ship. In: Principles of Drug Action: The Basis of Pharmacology, Ed. 2, time the biochemical basis for the antagonism is particu pp. 82-111. New York, N. Y.: John Wiley and Sons. Inc.. 1974. larly difficult to conceptualize because virtually nothing is 14. Hubert-Habart, M., and Cohen, S. S. The Toxicity of 9-/3-o-Arabinofura-

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JULY 1978 1921

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1978 American Association for Cancer Research. Deoxyadenosine Antagonism of the Antiviral Activity of 9-β -d-Arabinofuranosyladenine and 9-β -d-Arabinofuranosylhypoxanthine

Sandra H. Smith, Charles Shipman, Jr. and John C. Drach

Cancer Res 1978;38:1916-1921.

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