HI I { “WNW!!!|H|llllIWIIHWHIHIIHHIIIHWW I—lN (BLOOD —1_‘_‘ THESIS This is to certify that the thesis entitled INHIBITION OF HERPESVIRUS REPLICATION, HERPESVIRUS- INDUCED DNA POLYMERASE, AND RETROVIRUS REVERSE TRANSCRIPTASE BY PHOSPHONOACETIC ACID AND PHOSPHONOFORMIC ACID presented by JOHN MARSHALL RENO has been accepted towards fulfillment of the requirements for PH.D. degreein BIOCHEMISTRY Mdfiw Major professor Date 5-16-80 0-7639 ! ,r.. .u. . ‘ A-ov fi-w')~.-‘_L__,.~‘. .m. W: 25¢ per ‘0 per 11:- RETURNING LIBRARY MATERIAL; Place In book nturn to rave charge fro- cIrculatton records INHIBITION OF HERPESVIRUS REPLICATION, HERPESVIRUS- INDUCED DNA POLYMERASE, AND RETROVIRUS REVERSE TRANSCRIPTASE BY PHOSPHONOACETIC ACID AND PHOSPHONOFORMIC ACID By John Marshal] Reno AN ABSTRACT OF A DISSERTATION Submitted to Michigan State University in partiaI fulfiIIment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1980 ABSTRACT INHIBITION OF HERPESVIRUS REPLICATION, HERPESVIRUS- INDUCED DNA POLYMERASE, AND RETROVIRUS REVERSE TRANSCRIPTASE BY PHOSPHONOACETIC ACID AND PHOSPHONOFORMIC ACID By John Marshall Reno Phosphonoacetic acid was an effective inhibitor of both the Marek's disease herpesvirus- and the herpesvirus of turkey-induced DNA polymerase. The apparent inhibition constants were I to 3 uM. Using the herpesvirus of turkey-induced DNA polymerase, the results of an enzyme kinetic inhibition analysis demonstrated that phosphono- acetic acid inhibits by interacting with the enzyme at its pyro- phosphate binding site and functioning as an alternate product inhibitor. Several analogs of pyrophosphate were also tested for inhibition of the herpesvirus-induced DNA polymerase. Only one, phosphonoformic acid, was found to be effective. The apparent inhibition constants were again I to 3 uM and the mechanism of inhibition was analogous to that of phosphonoacetic acid. Phosphono- formic acid was able to block the replication in cell culture of Marek's disease herpesvirus, the herpesvirus of turkeys, and herpes simplex virus. Phosphonoformic acid was not an effective inhibitor of a phosphonoacetate-resistant mutant of the herpesvirus of turkeys nor of its induced DNA polymerase. John Marshall Reno Phosphonoformic acid was effective in treating herpesvirus infections in animal model systems. When mice or guinea pigs were inoculated intravaginally with herpes simplex type 2 and treated intravaginally with l0% trisodium phosphonoformate beginning at times up to 24 hours post infection, a significant reduction in virus titer was obtained. Phosphonoformic acid was not effective in mice inocu- lated intraperitoneally or intracerebrally with herpes simplex virus or intraperitoneally with murine cytomegalovirus. The antiviral effects of phosphonoacetic acid and phosphono- formic acid are not entirely specific. Both compounds were shown to inhibit the DNA polymerase a from HeLa, Wi-38, and phytohemagglutin- stimulated lymphocytes, all human cells, and from Chinese hamster ovary cells and calf thymus. The apparent inhibition constants were about 30 uM. The mechanism of inhibition was analogous to that of the herpesvirus-induced DNA polymerase. The DNA polymerases B and y were not sensitive to either phosphonoacetic acid or phosphonoformic acid. Phosphonoformic acid, but not phosphonoacetic acid, was a potent inhibitor of DNA synthesis catalyzed by reverse transcriptase from avian myeloblastosis virus, Rous sarcoma virus, Moloney murine leukemia virus, and feline leukemia virus with poly(A)-oligo(dT) or 2+ activated DNA as substrate. With Mg as the cofactor, 50% inhibition in the rate of DNA polymerization was observed with about l0 uM 2+ as the cofactor, 50% inhibition was phosphonoformic acid. With Mn obtained with about 0.5 uM phosphonoformic acid. The endogenous reactions or reactions in which purified viral RNA was the substrate John Marshall Reno were much less sensitive to phosphonoformate. The RNase H activity of AMV reverse transcriptase wasrxm inhibited by either phosphonate 2+. Inhibition studies of both in the presence of either Mg2+ or Mn the DNA polymerization and the deoxyribonucleoside triphosphate- pyrophosphate exchange reaction were carried out. The results were different from those obtained with the herpesvirus-induced DNA polymerase and DNA polymerase a and are consistent with phosphono- formic acid interacting with the reverse transcriptase at the pyrophosphate binding site but functioning as a dead-end inhibitor. INHIBITION OF HERPESVIRUS REPLICATION, HERPESVIRUS- INDUCED DNA POLYMERASE, AND RETROVIRUS REVERSE TRANSCRIPTASE BY PHOSPHONOACETIC ACID AND PHOSPHONOFORMIC ACID By John Marshall Reno A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1980 ABSTRACT INHIBITION OF HERPESVIRUS REPLICATION, HERPESVIRUS- INDUCED DNA POLYMERASE, AND RETROVIRUS REVERSE TRANSCRIPTASE BY PHOSPHONOACETIC ACID AND PHOSPHONOFORMIC ACID By John Marshall Reno Phosphonoacetic acid was an effective inhibitor of both the Marek's disease herpesvirus- and the herpesvirus of turkey-induced DNA polymerase. The apparent inhibition constants were 1 to 3 uM. Using the herpesvirus of turkey-induced DNA polymerase, the results of an enzyme kinetic inhibition analysis demonstrated that phosphono- acetic acid inhibits by interacting with the enzyme at its pyro- phoSphate binding site and functioning as an alternate product inhibitor. Several analogs of pyrophosphate were also tested for inhibition of the herpesvirus-induced DNA polymerase. Only one, phosphonoformic acid, was found to be effective. The apparent inhibition constants were again 1 to 3 uM and the mechanism of inhibition was analogous to that of phosphonoacetic acid. Phosphono- formic acid was able to block the replication in cell culture of Marek's disease herpesvirus, the herpesvirus of turkeys, and herpes simplex virus. Phosphonoformic acid was not an effective inhibitor of a phosphonoacetate-resistant mutant of the herpesvirus of turkeys nor of its induced DNA polymerase. John Marshall Reno Phosphonoformic acid was effective in treating herpesvirus infections in animal model systems. When mice or guinea pigs were inoculated intravaginally with herpes simplex type 2 and treated intravaginally with 10% trisodium phosphonoformate beginning at times up to 24 hours post infection, a significant reduction in virus titer was obtained. Phosphonoformic acid was not effective in mice inocu- lated intraperitoneally or intracerebrally with herpes simplex virus or intraperitoneally with murine cytomegalovirus. ‘ The antiviral effects of phosphonoacetic acid and phosphono- formic acid are not entirely specific. Both compounds were shown to inhibit the DNA polymerase a from HeLa, Wi-38, and phytohemagglutin- stimulated lymphocytes, all human cells, and from Chinese hamster ovary cells and calf thymus. The apparent inhibition constants were about 30 uM. The mechanism of inhibition was analogous to that of the herpesvirus-induced DNA polymerase. The DNA polymerases B and 7 were not sensitive to either phosphonoacetic acid or phosphonoformic acid. Phosphonoformic acid, but not phosphonoacetic acid, was a potent inhibitor of DNA synthesis catalyzed by reverse transcriptase from avian myeloblastosis virus, Rous sarcoma virus, Moloney murine leukemia virus, and feline leukemia virus with poly(A)-oligo(dT) or 2+ activated DNA as substrate. With Mg as the cofactor, 50% inhibition in the rate of DNA polymerization was observed with about 10 uM 2+ as the cofactor, 50% inhibition was phosphonoformic acid. With Mn obtained with about 0.5 uM phosphonoformic acid. The endogenous reactions or reactions in which purified viral RNA was the substrate John Marshall Reno were much less sensitive to phosphonoformate. The RNase H activity of AMV reverse transcriptase wasrxm inhibited by either phosphonate 2+ 2+. Inhibition studies of both in the presence of either Mg or Mn the DNA polymerization and the deoxyribonucleoside triphosphate- pyrophosphate exchange reaction were carried out. The results were different from those obtained with the herpesvirus-induced DNA polymerase and DNA polymerase a and are consistent with phosphono- formic acid interacting with the reverse transcriptase at the pyrophosphate binding site but functioning as a dead-end inhibitor. To Kristine ii ACKNOWLEDGMENTS I would like to express my deepest gratitude to my advisor, John A. Boezi, for his faith in my ability and for his constant guidance and encouragement in this research work. Special thanks also go to my colleagues and friends, Susan Leinbach, Carol Sabourin, Lucy Lee, and Betty Baltzer, not only for their help but also for creating the stimulating atmosphere in the laboratory which made this work possible and the many hours in the laboratory pleasurable. I also want to express my greatest appreciation to my beloved wife, Kristine, for her understanding, devotion, and determination to make a home despite my long hours in the laboratory. TABLE OF CONTENTS LIST OF TABLES . LIST OF FIGURES GENERAL INTRODUCTION . LITERATURE REVIEW . Phosphonoacetic Acid Chemistry. . Industrial Applications . . Phosphonoacetate and Herpesvirus Infections . Tissue Culture Studies with Phosphonoacetate Effect on Uninfected Cells in Tissue Culture Effect of Phosphonoacetate on the Herpesvirus- Induced DNA Polymerase . . . . . Effects on Other DNA Polymerases Animal Studies with Phosphonoacetate . Analogs of Phosphonoacetate . . . Metabolism and Toxicity of Phosphonoacetate. Phosphonoformic Acid . . . . . . Chemistry. . . Effect on the Herpesvirus- -Induced DNA Polymerase . Inhibition of Herpesvirus Replication in Cell Culture . . . Effect on Uninfected Cells in Culture Effect on Other DNA Polymerases Herpesvirus Mutants Resistant to Phosphonoformate Efficacy in Animal Model Herpesvirus Infections . Analogues of Phosphonoformate . . . . Toxicity of Phosphonoformate References ARTICLE 1 Inhibition of Herpesvirus-Induced DNA Polymerase and Herpesvirus Replication by Phosphonoformate. John M. Reno, Lucy F. Lee, and John A. Boezi. Antimicrob. Ag. Chemother. 1;; 188 (1978) iv Page vi vii 25 ARTICLE 2 Mechanism of Phosphonoformate Inhibition of Reverse Transcriptase. John M. Reno, Hsing-Jien Kung, and John A. Boezi . . . . . . . . . APPENDICES A. Mechanism of Phosphonoacetate Inhibition of Herpesvirus-Induced DNA Polymerase. Susan S. Leinbach, John M. Reno, Lucy F. Lee, A. F. Isbell, and John A. Boezi. Biochemistry 15: 426 (1976) B. Treatment of Experimental Herpesvirus Infections with Phosphonoformate and Some Comparisons with Phosphonoacetate. Earl R. Kern, L. A. Glasgow, J. C. Overall, John M. Reno, and John A. Boezi. Antimicrob. Ag. Chemother. 13: 817 (1978) C. Inhibition of Eukaryotic DNA Polymerases by Phosphonoacetate and Phosphonoformate. Carol L. K. Sabourin, John M. Reno, and John A. Boezi. Arch. Biochem. Biophys. 187: 96 (1978) . Page 31 7O 76 84 LIST OF TABLES Table Page 1. Effect of template-primer on phosphonoformate inhibition of AMV DNA polymerase . . . . . . . 43 2. Phosphonoformate inhibition of reverse transcriptase . 44 vi LIST OF FIGURES ARTICLE 1 Structure of Phosphonoacetic Acid . Structure of Phosphonoformic Acid . ARTICLE 2 Inhibition of AMV DNA polymerase by phosphonoformate and phosphonoacetate Effect of phosphonoformate and phosphonoacetate on AMV RNase H activity . . . . . . Double reciprocal plots of the AMV DNA polymerase catalyged reaction with dTTP as variable substrate and MN T as cofactor . . . . . . . Double reciprocal plots of the AMV DNA pol erase catalyzed reaction with pgly(A) oligo(dT as the variable substrate and Mn+ as cofactor . Double reciprocal plots of the dNTP-pyrophosphate exchange reaction catalyzed by AMV DNA polymerase with pyrophosphate as the variable substrates Proposed mechanism of phosphonoformate inhibition of reverse transcriptase . . vii Page 14 41 46 48 .51 53 58 GENERAL INTRODUCTION Herpesviruses, a large family of DNA containing viruses are the etiological agents for many human infectious diseases: herpes simplex type 1 causes skin and eye infections as well as fever blisters, herpes simplex type 2 causes genital herpes and is asso- ciated with a type of cervical carcinoma, varicella—zoster virus causes shingles in adults and chicken pox in children, human cytomegalovirus is associated with fetal damage and is a major cause of birth defects, Epstein-Barr virus causes infectious mono- nucleosis and is associated with Burkitt's lymphoma. There are also a number of herpesviruses that are pathogenic in animals: Marek's disease virus causes tumors in chickens and has been a serious cause of mortality in commercial poultry, pseudorabies virus is the causative agent of mad itch in swine and cattle, and host specific cytomegaloviruses infect many animals. Clearly the need exists for specific agents which have the potential for con- trolling and perhaps eliminating herpesvirus infections. Along with their potential therapeutic value, inhibitors of replication are important for their ability to complement condi- tional mutants in the dissection of the complex events involved in nucleic acid synthesis. A much more efficient use of these inhibitors can be made if the target protein and the mechanism of inhibition is known. Also the pharmacological factors and toxicity are more easily handled. Following productive infection, herpesviruses induce the synthesis of a herpesvirus specific DNA polymerase. Because virus infected cells contain this unique enzyme and other proteins essen- tial for virus reproduction, it should be possible to inhibit virus replication specifically. A compound, phosphonoacetic acid, is now known to specifically inhibit the herpesvirus-induced DNA polymerase. Although its efficacy as an antiviral has been demonstrated in many animal model systems, it has toxicity problems and its clinical usefulness is in doubt. Results contained in this thesis on investi- gations into the mechanism of inhibition showed that phosphonoacetic acid is acting as a pyrophosphate analog and thus provided enough information form the development of another effective antiherpesvirus compound, phosphoformic acid. Phosphonoformic acid may be of suffi- ciently different chemistry that it would be less toxic than phospho- acetic acid and therefore clinically useful. Phosphoformic acid has also been discovered to be an inhibitor of the retrovirus reverse transcriptase. Retroviruses are RNA containing viruses but their intracellular state is as an integrated DNA. There are many different retroviruses and many cause cancer in animals. The retrovirus DNA polymerase, also called reverse transcriptase, is necessary for transcription of viral RNA into DNA from which progeny virus are made. This thesis is organized in a series of articles that either have been published or will be submitted for publication. Much of this work has been the result of several collaborations and will therefore be presented as appendices. Independent research will be presented in the body of the thesis as two articles, each with its own Abstract, Introduction, Methods, Results, Discussion, and References sections. It begins with a literature review in which much of the current information on phosphonoacetic acid is summar- ized. A complete review of the literature on phosphonoformic acid is also presented. The antiherpesvirus effect of phosphonoacetic acid was initially discovered by Abbott Laboratories. Investigations into the mechanisms of inhibition of the herpesvirus-induced DNA poly- merase showed that phosphonoacetic acid was interacting with the enzyme at its pyrophosphate binding site. This work is presented as Appendix A in the form of a reprint from the journal in which it was published. Another analog of pyrophosphate, phosphonoformic acid, was found to be a potent antiherpesvirus agent. Its synthesis, characterization, and mechanism of inhibition of the herpesvirus- induced DNA polymerase are contained in Article 1, also in the form of a reprint. After showing the effectiveness of phosphonoformic acid in cell culture infections with herpesviruses, its efficacy in animal model systems was tested. The results of this collaborative effort are presented in reprint form in Appendix B. The eukaryotic host cells contain three DNA polymerases designated a, B, and 7. Since phosphonoacetic acid and phosphonoformic acid could potentially interact with these enzymes in an analogous manner to the herpes- virus-induced DNA polymerase and be cytotoxic, a study of the effects of these two drugs on the host cell DNA polymerases was carried out. The results are presented in reprint form in Appendix C. Phosphosphonoformic acid and not phosphonoacetic acid was then discovered to be an inhibitor of reverse transcriptase. The results of a detailed study into the scope and mechanism of inhibi- tion are presented in Article 2. This report will be submitted for publication. A preliminary report was submitted to the American Society of Biological Chemists for presentation at the 1980 meetings. Dr. Lucy F. Lee was included as a coauthor on Article 1 in recognition of her assistance with some of the experiments. Dr. Hsing-Jien Kung, Assistant Professor, was included as a coauthor on Article 2 in appreciation for providing the AMV reverse transcriptase. Also, it is hoped that Dr. Kung will continue studying the biology of this problem. LITERATURE REVIEW Phosphonoacetic Acid Since its efficacy as an antiviral agent was discovered in 1973, the literature on phosphonoacetic acid has burgeoned and has been reviewed several times (1-4). A comprehensive review by Boezi has recently been published (5). Therefore only a succinct review of the literature, for the purpose of introduction, will be presented here. This review also will include reports receiving little atten- tion in past reviews as well as a survey of the newest literature. Phosphonoacetic acid will also be referred to as phosphonoacetate, its completely deprotonated salt. Chemistry Phosphonoacetic acid (Figure I), an organophosphorus com- pound, was first synthesized by Nylen in 1924 (6). It is a white solid of molecular weight 140.03 and melting point of 142-143°C. The triacid has pKa values of 2.0 (P-OH), 5.11 (COOH), and 8.69 (P-OH) at zero ionic strength (7). Phosphonoacetic acid also forms stable complexes with a variety of divalent and trivalent cations. It is used in the extraction of rare earths (8) and its complexation 3+ 3+ 3+ ions has been studied (9). Because of its 2+ with Am , Cm , and Pm biological importance, complexes of phosphonoacetic acid with Mg Ca2+, Cu2+, and Zn2+ have been investigated (10, P-H. Heubel and .u_u< ovumomococamoga mo weapoacum--._.mcam_d . :6 won zuumo: O A. Popov, personal communication). It forms 1:1 complexes with magnesium with log Kf values of 3 and 5.6 for the mono- and deprotonated forms, respectively. Industrial Applications The industrial uses of phosphonoacetate are numerous and it has been patented for widely different purposes. It has been used as a flame retardant in thermoplastics (11), as a corrosion inhibitor in boilers and water cooling systems (12), and to prevent the forma- tion of dark spots on photographic plates due to iron and rust (13). Its biological applications are also varied. In addition to its potential use as an anti-herpesvirus agent, phosphonoacetate has been proposed for use as an insecticide (14), as a herbicide, and as a plant growth regulator (15). It has also been proposed as a treatment for warts (16), and phosphonoacyl proline has been patented for use as a hypotensive (l7). Phosphonoacetate and Herpesvirus Infections Tissue Culture Studies with Phosphonoacetate Phosphonoacetate was first discovered to have antiviral properties by Abbott Laboratories in 1973 (18). Using a random testing of compounds with a tissue culture screen revealed that phosphonoacetate consistently inhibited herpes simplex virus at a concentration of 0.5 mM. It was effective in reducing the lesions and mortality of herpes simplex virus skin infections in mice and eye infections in rabbits. Herpes simplex virus infection of human Wi-38 cells results in cell destruction; however in the presence of 0.5 mM phosphonoacetate the infected cells could not be distinguished from uninfected cells (19). Biochemical investigations revealed that phosphonoacetate had no effect on RNA or protein synthesis but was specific in inhibiting viral DNA synthesis. The herpes virus induced DNA polymerase was implicated as the target protein. Since these initial studies by Abbott Laboratories, reports from a large number of laboratories have confirmed and extended these results on the antiviral properties of phosphonoacetate. In tissue culture studies it has been shown that phosphono- acetate is an effective inhibitor of the replication of all herpes- viruses examined and that the inhibition is specific for viral DNA synthesis. A growing contention is that inhibition by phosphono- acetate is a new characteristic of the herpesvirus group. Those herpesviruses studied were: human, murine, and simian cytomegalo- viruses (20, 21, 22), equine abortion herpesvirus (1), herpesvirus of turkeys (23), herpesvirus saimiri (24), Marek's disease herpes- virus, owl herpesvirus (23), varicella-zoster virus (25), and the Epstien-Barr virus (5). The replication of each of these herpes- viruses was consistently and completely repressed by concentrations of phosphonoacetate of 0.5 mM or less. The channel catfish herpesvirus is an exception in that to inhibit its replication in tissue culture some 10-20 times more phos- phonoacetate is required (26). The virus only grows in cold blooded catfish cells which are also 10-20 times more resistant to phosphono- acetate than warm blooded cells. Further investigations are required to determine if the virus itself is responsible for the decreased sensitivity. Phosphonoacetate is clearly an antiviral agent selective for herpesviruses. It does not inhibit the replication of the DNA con- taining viruses simian virus -40 and human adenovirus -12. Insensi- tive RNA containing viruses are poliovirus, rhinovirus, and measles virus (5). Vaccinia virus is inhibited by phosphonoacetate but much higher concentrations are required (1). Effect on Uninfected Cells in Tissue CuTture At concentrations of 0.2-0.5 mM at which it is an effective inhibitor of herpesvirus replication, phosphonoacetate induces no obvious morphological changes and there is no inhibition of cellular DNA, RNA, or protein synthesis (1, 5). At higher concentrations in the range 1-5 mM, phosphonoacetate is cytotoxic. Cell growth is arrested and cellular macromolecule synthesis is inhibited. Effect of Phosphonoacetate on the Herpesvirus-Induced DNA P61ymerase Mao et a1. (27) first observed that phosphonoacetate specifi- cally inhibits the herpesvirus-induced DNA polymerase. When assayed in the presence of 1-2 uM phosphonoacetate, enzyme activity was reduced by 50%. A11 herpesvirus-induced DNA polymerases are sensi- tive to phosphonoacetate at this level (5). Studies of phosphonoacetate resistant mutants of herpesvirus provided conclusive evidence that the inhibition of herpesvirus replication in cell culture is through a specific effect on the 10 herpesvirus-induced DNA polymerase (1, 5). The DNA polymerase induced by mutant viruses are much less sensitive to phosphonoacetate inhibition than is the polymerase induced by the wild type virus. Many of these mutants are temperature sensitive and the isolated DNA polymerases are also temperature sensitive. Revertants of the mutant viruses and their DNA polymerases are neither temperature sensitive nor phosphonoacetate resistant. Effects on Other DNA Polymerases The results of several studies on various eukaryotic cells have shown that DNA polymerase a is inhibited by phosphonoacetate (5). The apparent inhibition constants are about 15-30 times higher than the herpesvirus-induced DNA polymerase. DNA polymerase B and the y- polymerase from eukaryotic cells are both insensitive to phosphono- acetate. Various prokaryotes and viral DNA polymerases have been found to be insensitive to phosphonoacetate (5). They include E. £911 DNA polymerase 1, M, lutgus DNA polymerase, hepatitis B virus DNA poly- merase and the reverse transcriptases of Rous sarcoma virus and avian myeloblastosis virus. The vaccinia virus-induced DNA polymerase is somewhat inhibited. The non-polymerase enzymes are sensitive to phosphonoacetate. Phosphonoacetate is a competitive inhibitor for carbamyl phosphate in the aspartate transcarbamylase catalyzed reaction (28). The apparent inhibition constant is 0.32 mM. For pyruvate carboxylase, phosphono- acetate is noncompetitive with respect to ATP and the apparent inhibition constant is 0.5 mM (29). 11 Animal Studies with Phosphonoacetate Phosphonoacetate has shown good efficicacy in the animal model systems for herpesvirus infections (5). Phosphonoacetate is efficacious for the herpes simplex diseases: herpes dermatitis, herpes keratitis, herpes iritis, herpes genitalis, and herpes encephalitis. Cytomegalovirus infection of mice and Marek's disease infection in chickens are also susceptible to phosphonoacetate. In general, topical administration of the drug shortly after infection gives the best results. Phosphonoacetate is not effective for herpes infections which are latent (30). During latency, infectious virus cannot be detected, but can be recovered when re-activated. The state of the virus during latency is unknown. Analogs of Phosphonoacetate Studies of analogs of phosphonoacetate indicate that the structural requirements for antiviral actively are rather narrowly defined (5). The results indicate that to be an effective inhibitor of the herpesvirus-induced DNA polymerase, the inhibitor should have an unsubstituted phosphono group and an unsubstituted carboxyl group. Phosphonocarboxylic acids having a longer carbon chain length are not inhibitors. The methylene carbon should also be unsubstituted. Some modification is allowed if the drug is used in tissue culture or animal systems. 1,3-Dipalmitoyl-Z-phosphonoacetylglycerol is effective in treating herpes simplex skin infection in mice (31). Amides of phosphonoacetate of the type RZNCOCH2P(O)(OH)2 are also 12 effective (32). Apparently the compounds are readily taken up by the cells in this form and metabolically hydrolized to give free phosphonoacetate which is then available to inhibit the herpesvirus- induced DNA polymerase. Metabolism and Toxicity of Phosphonoacetate Systematic studies on the toxicity of phosphonoacetate have not been carried out. In reports on the efficacy of phosphonoacetate in viral infections of animals there are some statements concerning this question. The LD50 dose for mice is 1500 mg/kg/day (33). At concentrations up to about one-half the L050, phosphonoacetate is well tolerated in mice. In rabbits, when applied to the eyes, the compound is also tolerated at concentrations at which it is effective (5). In animals studies employing disodium [1-14C] phosphonoace— tate, no evidence was obtained for its conversion to other compounds (34). Most of the administered drug was excreted rapidly in the urine in an unchanged form. Whole-body autoradiography of treated animals demonstrated that the absorbed drug is accumulated in bone. After administration has ceased, phosphonoacetate is released from bone but very slowly. Toxicity problems associated with this deposi- sition in bone have not been evaluated. In tissue culture studies monitoring calcification of tendon matrix it was found that phosphono- acetate inhibited the uptake of Ca2+ and inorganic phosphate by both uncalcified and previously calcified matrices (35). The inhibition is completely reversed when phosphonoacetate is washed off. 13 Severe toxic reactions have been observed in rabbits and patas monkeys. An intravenous concentratkulof 300 mg/kg phosphono— acetate produced severe tetanic muscular spasms in rabbits, often resulting in death; the same dose given to mice orally or intra- peritoneally was well tolerated (5). In patas monkeys receiving a daily dose of 800 mg or higher there was liver degeneration, severe dermatitis, and a change in skin and hair color (36). Phosphonoacetate was not mutagenic in the Salmonella typhimurium test (37). Animal carcinogenic studies have not been reported. Abbott Laboratories has applied to the Food and Drug Admini- stration for permission to classify phophonoacetate as an investiga- tive drug for the purpose of initiating clinical studies. The FDA did not approve their request and has asked for further toxicity studies. Therefore, the prospect of phosphonoacetate being a clinically useful drug remains uncertain. Phosphonoformic Acid The synthesis, characterization, and assessment of the bio- logical effects of phosphonoformic acid (Figure 2) are contained in Article 1 and Article 2 of this thesis. These results have been con- firmed and extended by researchers at Astra Laboratories in Sodertalje, Sweden and at the University of Uppsala. Inhibition of herpesvirus replication by phosphonoformic acid has been included in reviews by Boezi (5) and Newton (39). A review of the Swedish effort has also appeared (40). A comprehensive review of the literature to date on phosphonoformic acid will be presented here. .uwo< uwscococognmozm mo mczuuacum .N mczmwu o :0 dunno: 15 Chemistry Trisodhnnphosphonoformate hexahydrate was first synthesized by Nylen in 1924 (6)--the same paper which describes the synthesis of phosphonoacetic acid. Zn, Mn, Cu, Pb, and Ag salts of phosphono- formate were also prepared. Upon converion to the completely proto- nated form, phosphoformic acid decomposes to form phosphorous acid and CO2 (41, 42). The acidity constants at 25°C and ionic strength 0.05 are pK1 of 1.7 (P-OH), pK2 of 3.59 (COOH), and pK3 of 7.56 (P-OH) (7). Phosphonoformate also forms stable complexes with magnesium with log Kf of 1.7 for the monoprotonated form and log Kf of 3.6 for the completely deprotonated form of phosphonoformate (P-H. Heubel and A. Popov, personal communication). The crystal structure of trisodium phosphonoformate hexa- hydrate has been determined (43, 44). It consists of sodium ions surrounded octahedrally by six oxygen molecules, some from water molecules and from the phosphonoformate ions. Esters of phosphonoformate are used in lubricating oils and hydraulic fluids because they have extreme pressure lubricating ability (45). Effect on the Herpesvirus- Induced DNA Polymerase The anti-herpesvirus effect of phosphonoformate was initially found by screening analogues of pyrophosphate as inhibitors of the herpesvirus-induced DNA polymerase (Article 1). The DNA polymerases induced by Marek's disease herpesvirus, the herpesvirus of turkeys, and herpes simplex virus are inhibited 50% by 1-3 uM 16 phosphonoformate (Article 1, 38). The molecular mechanisms by which phosphonoformate and phosphonoacetate inhibit the herpesvirus- induced DNA polymerase are analogous (Article 1). Inhibition of Herpesvirus Replication in Cell Culture Phosphonoformate is an effective inhibitor of herpesvirus replication in infected cells (Article 1). The addition of 60-70 uM phosphonoformate brought about a 50% reduction in the number of plaques observed in cell cultures infected with Marek's disease herpesvirus, the herpesvirus of turkeys and herpes simplex type 1. Herpes simplex type 2 and pseudorabies herpesvirus replication is inhibited by more than 90% with 100 uM phosphonoformate (38). Vaccina virus, adenovirus type 2, and poliovirus are all insensitive to phosphonoformate (38). Influenza virus replication is inhibited by high concentrations of phosphonoformate. At 500 uM phosphonoformate influenza virus replication was inhibited by over 90% where the same concentration of phosphonoacetate had no effect. In each of the other viral systems the efficacy of phosphonoformate and phosphonoacetate were parallel (Article 1, 38). Effect on Uninfected Cells in Culture At the concentrations which are effective in blocking the replication of herpesviruses in cell culture, phosphonoformate has no obvious cytotoxic effects on duck embryo fibroblasts (Article 1), nor on African green monkey kidney cells, HeLa, and human lung cells (38, 46). Cell proliferation and cellular DNA synthesis is inhibited 50% by 1 mM phosphonoformate and RNA and protein synthesis could be 17 inhibited at concentrations exceeding 2.5 mM (46). HeLa cell division can be completely blocked with 10 mM phosphonoformate for 24 hours but is rapidly reversed after the drug is removed (46). Little cell death occurred during this time. Effect on Other DNA Polymerases The inhibition of herpesvirus-induced DNA polymerases by phosphonoformate is not entirely specific. DNA polymerase a from HeLa cells, Wi-38 cells, and phytohemaglutinin-stimulated lymphocytes are inhibited 50% by 30 uM phosphonoformate. DNA polymerase B from these sources is relatively insensitive (47). These results are analogous to those obtained with phosphonoacetate. Influenza RNA polymerase (38, 59) and the hepatitis 8 Dane particle DNA polymerase (48) are inhibited 50% by 20 uM phosphonoformate but not by phosphono- acetate. Replication of visna virus, an RNA virus, is also inhibited by phosphonoformate and this effect is ascribed to inhibition of the viral reverse transcriptase (49). Inhibition of reverse transcript- ases from the RNA tumor viruses has been studied in depth (Article Ii, 50). A 50% reduction in the DNA polymerase activity is observed at about 10 pH. The mechanism of inhibition is different with reverse transcriptases than that for herpesvirus-induced DNA poly- merases. The RNA dependent RNA polymerase of vesicular stomatitis virus is inhibited by high concentrations of phosphonoformate (51). A concentration of 5 mM is required to give 90% inhibition. 18 Herpesvirus Mutants Resistant to Phosphonoformate Mutants of herpes simplex virus and Marek's disease herpes- virus resistant to phosphonoformate are easily obtained in cell culture (52, 53, 54). The induced DNA polymerases isolated from cells infected with these mutants are also resistant to phosphono- formate as well as phosphonoacetate. This provides evidence that the target protein for phosphonoformate is in fact the herpesvirus- induced DNA polymerase and that the merchanism of inhibition is the same for both phosphonates. However, the ease of which these mutants were obtained may indicate a limited clinical value for this com- pound. Efficaqy in Animal Model Herpesvirus Infections Four reports on phosphonoformate efficacy in animal model studies are available and the results so far parallel those obtained with phosphonoacetate (55, 56, 57, 58). Topically applied phosphono- formate has a good therapeutic activity against established herpes simplex virus skin infections in guinea pigs (38, 55). Intra- vaginal treatment of herpes simplex virus genital infections in mice and guinea pigs with phosphonoformate reduces virus titer in the genital tract and the mortality rate but treatment must begin within 24 hours of infection (56, 57). Phosphonoformate has only minimal effectiveness in mice inoculated intra-cerebrally with herpes simplex virus or intraperitoneally with cytomegalovirus (56). Treat- ment of skin infections of herpes simplex virus in hairless mice 19 topically with phosphonoformate was very effective, even when delayed 24 hours (58). However, phosphonoformate could not prevent the establishment of latent infections in the nervous system of treated mice even when treatment was started at 3 hours post infec- tion, whereas phosphonoacetate was effective. Analogues of Phosphonoformate Various esters of phosphonoformate have been synthesized and tested for their ability to inhibit the herpesvirus-induced DNA polymerase, herpesvirus replication in cell culture, and herpesvirus skin infection in guinea pigs (Helgstrand, E., N. G. Johansson, S. Stridh, and B. Oberg. Personal Communication, October 1979). None were effective in inhibiting the herpesvirus-induced DNA polymerase. Aryl esters of the phosphonate moiety were effective in cell culture at concentrations of 3-15 uM. It is suggested that these esters are more readily taken up into cells and then enzymatically hydrolyzed to yield free phosphonoformate. All were about as effective as free phosphonoformate in the animal model system. Toxicity of Phosphonoformate Systematic studies on the toxicology of phosphonoformate have not been reported. Summary statements of reports give some observa- tions on the toxicity. Phosphonoformate does not cause the skin irritation seen with phosphonoacetate treatment (40, 56). Phosphono- formate is reported to be non-toxic to rats and dogs subcutaneously treated with the drug daily for 1 month at levels up to 195 mg/kg (40). Phosphonoformate is deposited in bone and cartilage tissues 20 from which it is only slowly released. No apparent toxicity is associated with the deposition in bone (40). 10. 11. 12. 13. 14. 15. 16. 17. REFERENCES Overby, L. R., R. G. Duff, and J. C-H. Mao. 1977. Ann. N.Y. Acad. Sci. 284: 310-320. Hay, J., S. M. Brown, A. T. Jamieson, F. J. Rixon, H. Moss, D. A. Dargan, and J. H. Subak-Sharpe. 1977. J. Antimicrob. Chemother. 3A: 63-70. Leinbach, S. S. 1976. Ph.D. Thesis. Michigan State University. Sabourin, C. O. K. 1977. Ph.D. Thesis. Michigan State University. Boezi, J. A. 1979. International Encyclopedia of Pharmacology and Therapeutics. Volume on Antiviral Chemotherapy. (Shugar, 0., ed.), 4: 231-243. Nylen, P. 1924. Chem. 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U.S. Patent #4,087,522. Fitzwilliam, J. R. and J. F. Griffith. 1976. J. Infect. Dis. .155: A221-A225. BopP. B. A., C. B. Estep, and D. J. Anderson. 1977. Fed. Proc. 25: 939. Wadkins, C. L. and R. A. Luben. 1978. Calcif. Tissue Res. 25: 51-60. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 23 Felsenfeld, A. D., C. R. Abee, P. J. Gerone, K. F. Soike, and S. R. Williams. 1978. Antimicrob. Ag. Chemother. 44: 331-335. Becker, B. A., B. A. Bopp, D. J. Brusick, and S. B. Lehrer. 1976. Fed. Proc. 55: 533. Helgstrand, E., B. Eriksson, N. G. Johansson, 8. Lannero, A. Larsson, A. Misiorny, J. O. Noren, B. Sjoberg, K. Stenberg, G. Stening, S. Stridh, B. Oberg, S. Alenius, and L. Philipson. 1978. Science 254: 819-821. Newton, A. A. 1979. Adv. Ophthal. 55: 267-275. Helgstrand, E., B. Oberg, and S. Alenius. 1979. Adv. Ophthal. 55: 276-280. Nylen, P. 1937. Z. Anorg. Chem. 255: 33. Warren, S. and M. R. Williams. 1971. J. Chem. Soc. 1971B: 618-621. Naqui, R. R., P. J. Wheatley, and E. Forest-Serantoni. 1971. J. Chem. Soc. 1971A: 2751-2754. Abrahams, S. C. 1972. Acta Cryst. 1525: 2886-2887. Denham, H. 1953. U.S. Patent #2,629-73l. Stenberg, K. and A. Larsson. 1978. Antimicrob. Ag. Chemother. 44: 727-730. Sabourin, C. L. K., J. M. Reno, and J. A. Boezi. 1978. Arch. Biochem. Biophys. 454: 96-101. Nordenfelt, E., E. Heigstrand, and B. Oberg. 1979. Acta Path. Microbiol. Scand. 554: 75-76. Sundquist, B. and E. Larner. 1979. J. Virol. 55: 847-851. Sundquist, B. and B. Oberg. 1979. .1. Gen. Virol. 45; 273-281. Chanda, P. K. and A. K. Banerjee. 1979. Fed. Proc. 55: 3. Lee, L. F., J. M. Reno, and J. A. Boezi. 1978. In Proceedings of Mechanism of Genetic Resistance to Marek's Disease. (Biggs, P., ed.). Berlin, accepted for publication. Eriksson, B. and Oberg, B. 1979. Antimicrob. Ag. Chemother. 45: 758-762. 54. 55. 56. 57. 58. 59. 24 Svennerhold, B., A. Vahlne, and E. Lycke. 1979. Proc. Soc. Exp. Bio. Med. 454: 115-118. Alenius, 5., Z. Dinter, and B. Oberg. 1978. Antimicrob. Ag. Chemother. 44: 408-413. Kern, E. R., L. A. Glasgow, J. C. Overall, J. M. Reno, and J. A. Boezi. 1978. Antimicrob. Ag. Chemother._44: 817-823. Alenius, S. and H. Nordlinder. 1979. Arcy. Virol. 55: 197-206. Klein, R. J., E. DeStefano, E. Brady, and A. E. Friedman-Kien. Antimicrob. Ag. Chemother. 45: 266-270. Stridh, S., E. Helgstrand, B. Lannero, A. Misiorny, G. Stening, and B. Oberg. 1979. Arch. Virol. 54: 245-250. ARTICLE 1 INHIBITION OF HERPESVIRUS-INDUCED DNA POLYMERASE AND HERPESVIRUS REPLICATION BY PHOSPHONOFORMATE By John M. Reno Lucy F. Lee and John A. Boezi Reprinted from Antimicrobial Agents and Chemotherapy 45, 188 (1978). 25 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Feb. 1978. p. 188-192 (X)66—4804/78/1302-OlSB$02.00/O Copyright © 1978 American Society for Microbiology Vol. 13. No. 2 Printed in U. S. A. Inhibition of Herpesvirus Replication and Herpesvirus- Induced Deoxyribonucleic Acid Polymerase by Phosphonoformatei' JOHN M. RENO,‘ LUCY F. LEE,” AND JOHN A. BOEZI“ Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824,1 and U.S. Department of Agriculture Agricultural Research Service, Regional Poultry Research Laboratory, East Lansing, Michigan 488232 Received for publication 24 October 1977 Phosphonoformate was found to be an inhibitor of the deoxyribonucleic acid polymerase induced by the herpesvirus of turkeys. The apparent inhibition constants were 1 to 3 11M. Phosphonoformate was also able to block the replication in cell culture of Marek’s disease herpesvirus, the herpesvirus of turkeys, and herpes simplex virus. It was as effective as phosphonoacetate. Phosphonoformate was not an effective inhibitor of a phosphonoacetate-resistant mutant of the herpesvirus of turkeys nor of its induced deoxyribonucleic acid polymerase. Phosphonoacetate is an effective inhibitor of the replication of herpesviruses (11, 13, 23, 25). The inhibition of herpesvirus replication is through an effect on the viral-induced deoxyri- bonucleic acid (DNA) polymerase (9, 14-16, 18). In animal model studies, the efficacy of phos- phonoacetate as an antiherpesvirus drug has been clearly demonstrated (7, 8, 12). Its clinical use, however, may be limited because it is some- what toxic to test animals and because it is accumulated in bone (4). Other phosphonate compounds are of interest as inhibitors of herpesvirus replication because they might exhibit an improved therapeutic ra- tio over phosphonoacetate either by being more effective inhibitors of virus replication or by being less toxic to animals. These compounds are also of interest at the enzymological level for the information that they might provide about the binding site on the herpesvirus-in- duced DNA polymerase. Consequently, using as an assay procedure the ability to inhibit the herpesvirus-induced DNA polymerase or the ability to block herpesvirus replication in cell culture or in animals, many other phosphonates have been looked at (10, 13, 14, 23). Only the low-molecular-weight carboxyl esters of phos- phoacetate have proven to be effective inhibi- tors. In this report, we demonstrate that phosphon- oformate, a compound first synthesized in 1924 (17), is an effective inhibitor of the DNA polym- erase induced by the herpesvirus of turkeys (HVT) and blocks the replication of Marek’s disease herpesvirus, HVT, and herpes simplex virus (HSV). Further, we compare the effective- ‘I Michigan Agricultural Station article no. 8188. 188 ness of phosphonoformate with phosphonoace- tate. We also report the effect that phosphono- formate has on a phosphonoacetate-resistant mutant of HVT and point out that phosphono- formate inhibits herpesvirus replication through an effect on the DNA polymerase in an analo- gous manner to phosphonoacetate. EXPERIMENTAL PROCEDURE Reagents. Phosphonoacetate, disodium salt, was a gift from Abbott Laboratories. Triethyl phosphite and ethyl chloroformate were pur- chased from Aldrich Chemical Co. Triethyl phosphite was redistilled before use. Other re- agents were from sources previously described (3) or were from the usual commercial sources. Synthesis of triethyl phosphonoformate and trisodium phosphonoformate. Both triethyl phosphonoformate and trisodium phos- phonoformate were prepared following the pro- cedure of Nylen (17) as described by Warren and Williams (26). In brief, triethyl phosphono- formate was prepared by an Arbuzov reaction with ethyl chloroformate and triethyl phosphite and was purified by vacuum distillation. The infrared spectrum and proton nuclear magnetic resonance spectrum agreed with published spec- tra (21, 22). The 13C-nuclear magnetic resonance proton-decoupled spectrum gave resonances at 14.6 ppm (COgCHgCHa), 16.8 ppm (doublet, Jp. c = 5.9 Hz, POCH2CHa), 62.6 ppm (doublet, J p. c = 4.4 Hz, CO2CH2CH3), 64.9 ppm (doublet, J p.c = 6.6 Hz, POCHzCHa), and 168.2 ppm (dou- blet, Jp.c = 265.4 Hz, C = 0). The spectrum was obtained using a Bruker WP-60 spectrome- ter, and all chemical shifts are relative to tetra- methylsilane. The triethyl ester was saponified VOL. 13. 1978 with NaOH, and the product was recrystallized several times from water to give trisodium phos- phonoformate hexahydrate. Phosphorus analy- sis (1) for CN8305P'6H20 gave a molecular weight of 299 (in theory, 300). The 13C-nuclear magnetic resonance proton-decoupled spectrum gave a resonance at 180 ppm (doublet, Jp-c = 229.3 Hz). Trisodium phosphonoformate was further characterized by descending paper chro- matography with the following solvent systems: (i) isopropyl alcohol-water-concentrated ammo- nia (7:2:1), R, = 0.03; (ii) methanol-water-con- centrated ammonia (6:1:3), R; = 0.63; (iii) ethanol-l M ammonium acetate, pH 7.5 (5:2), Rf = 0.06. The chromatograms were sprayed as described by Bandurski and Axelrod (2), and only a single blue spot was detected with each solvent system. Virus strains. Marek's disease herpesvirus strain GA (MDHV) (19) was propagated in pri- mary duck embryo fibroblasts as previously de- scribed (24). HVT strain FC-126 (HVth) (28) and a phosphonoacetate resistant mutant of this strain (HVTN) (L. F. Lee, K. Nazerian, R. Wit- ter, S. Leinbach, and J. Boezi, manuscript in preparation) were also propagated in primary duck embryo fibroblasts as previously described. The HSV type 1 used was the MP strain (20) adapted to duck embryo fibroblasts. Assessment of phosphonate effect on herpesvirus replication. Triplicate duck em- bryo fibroblasts culture samples were inoculated with MDHV and incubated with various concen- trations of phosphonoformate or phosphonoac- etate according to the following regimen: with 0.035 mM phosphonate, cultures were inocu- lated with 50, 100, 500, and 1,000 plaque-forming units (PFU); with 0.07 mM phosphonate, cul- tures were inoculated with 100, 500, 1,000, and 5,000 PFU; at 0.14 mM phosphonate, with 5,000 and 10,000 PFU; and at 0.28 mM, with 50,000 and 100,000, and 500,000 PFU. Cultures were incubated, and plaques were counted 6 or 7 days postinfection. Relative numbers of plaques were determined by dividing the observed number of plaques formed by the input PFU. Identical culture conditions were used for HVT," and HVTp. except that triplicate cultures were all inoculated with 100 PFU in various concentrations of either phosphonate, and plaques were counted 5 days postinfection. The percentage of plaques surviving was calculated from cultures containing no phosphonate. Identical culture conditions were also used for HSV except that it was inoculated at about 1,000 PFU/ plate into cultures containing various con- centrations of either phosphonate. Preparation of HVT-induced DNA po- lymerase. Both HVT,“ and HVT,. were treated PHOSPHONOFORMATE INHIBITION OF HERPESVIRUS 189 in an identical manner. The preparation and growth of duck embryo fibroblasts and infection with virus was as previously described (3). The partial purification of the HVT-induced DNA polymerase was from the nuclear fraction of infected cells by phosphocellulose chromatog- raphy as described by Leinbach et al. (14). The specific enzymatic activity of the preparation used in the kinetic studies reported here was about 1.000 nmol of deoxynucleoside monophos- phate incorporated into DNA/ 30 min per mg of protein. This enzyme fraction, when tested using the standard assay conditions for DNA polym- erization, contained no detectable deoxyribonu- clease activity, deoxyribonucleoside triphospha- tase activity, or inorganic pyrophosphatase ac- tivity. The kinetic experiment with HVT,..-in- duced DNA polymerase was also performed with a more highly purified preparation which had been purified by phosphocellulose and hydrox- ylapatite chromatography. No differences in the results were seen. Inhibition patterns. Inhibition patterns and kinetic constants were defined according to the nomenclature of Cleland (5, 6). The data for the double reciprocal plots were evaluated using a computer program based on the method of Wilk- inson (27). For evaluation of the apparent inhi- bition constants, replots of the intercepts and slopes of the double reciprocal plots were ana- lyzed by the method of least squares. RESULTS Phosphonoformate inhibition of the DNA polymerization reaction catalyzed by HVTm-induced DNA polymerase. In the course of a study examining phosphonate com- pounds for their ability to inhibit the herpesvi- rus-induced DNA polymerase, it was discovered that phosphonoformate was an effective inhibi- tor of the HVTm-induced DNA polymerase. The addition of 2 to 3 11M phosphonoformate to the standard assay mixture resulted in a decrease in the rate of the DNA polymerization reaction by about 50%. The inhibition patterns produced by phosphonoformate were examined, and as shown in Fig. 1, phosphonoformate gave linear noncompetitive inhibition with the four deox- ynucleoside triphosphates (dNTP’s) as variable substrate and activated DNA at a saturating concentration of 200 ug/ml. The apparent inhi- bition constant determined from the replot of the vertical intercepts against phosphonofor- mate concentration (Kn) was 1.1 uM. The ap- parent inhibition constant determined from the replot of the slopes against phosphonoformate concentration (K.,) was 0.9 uM. With activated DNA as the variable substrate and the four dNTP’s at their apparent Michaelis constant 190 RENO, LEE, AND BOEZI a B/ L. g/ 0 K /e/°/o P” O Intercept o .-——s—-""" [Pnosohonoto:mote],(nM I 1 I 1 I 1 0| 02 03 04 OS [dNTp]",(,IMI" FIG. 1. Double reciprocal plots of the H VTWin- duced DNA polymerase-catalyzed reaction with the four dNTP's as the variable substrate and phosphon- oformate as inhibitor. Activated DNA was at a con- centration of 200 pg/ml. The initial velocities were expressed as picomoles of [JHIthymidine 5'-mono- phosphate incorporated into DNA/30 min. Phos- phonoformate concentrations were 0 (O), 0.7 ILM (O), 1.5uM (Cl), and 3.0;LM (A). Equimolar concentrations of each of the four dNTP's were present in the differ- ent reaction mixtures. The replots of the slopes (O) and intercepts (O) as a function of phosphonoformate concentration are shown in the left panel. concentration of 2.5 uM each, phosphonofor- mate gave linear noncompiatitive inhibition (data not shown). A replot of the vertical inter- cepts yielded a K., of 2.6 11M, and a replot of the slopes yielded a K., of 2.3 uM. The inhibition patterns and the apparent inhibition constants are similar to those obtained with phosphonoac- etate (14). Effect of phosphonoformate on the rep- lication of herpesviruses in cell culture. After this discovery, phosphonoformate was tested for its ability to block the replication of HVT,... MDHV, and HSV in cell culture. Again, phosphonoformate was an effective inhibitor. With HVT,“, phosphonoformate was as effective an inhibitor as phosphonoacetate (Fig. 2). The addition of 0.06 to 0.07 mM of either phosphon- ate to the culture medium brought about a 50% reduction in the number of plaques observed. Essentially no plaques were observed at concen- trations above 0.3 mM. Phosphonoformate and phosphonoacetate also exhibited a parallel ability to block the replication of MDHV. The addition of either phosphonate to a final concentration of about 0.02 mM brought about a 50% reduction in the number of plaques produced. Either phosphon- ate at 0.14 mM reduced the number of plaques by more than three orders of magnitude (Fig. 3). At 0.28 mM, no plaques at all were observed (data not shown). Phosphonoformate also inhibited the replica- tion of HSV type 1 in cell culture (data not shown). Again, phosphonoformate was about as effective an inhibitor as phosphonoacetate. ANTIMICROB. AGENTS CHEMOTHER. I T I I °/o Ploques Dr‘- 1 - 0. I 0.2 0.3 0.4 0.5 [Phosphonote] ,(rnM) FIG. 2. Effect of phosphonoformate and phosphon- oacetate on the replication of HVT,... and HVT,... A known titer of each virus preparation was used to infect secondary duck embryo fibroblast cultures in growth media without and with different concentra- tions of phosphonate. Plaques were enumerated 5 days postinfection. Each point represents the average of three experiments, and the number of plaques enu- merated in cultures without phosphonate was consid- ered as 100%. Viruses and inhibitors were H VT,“ with phosphonoformate (O), H VT.“ with phosphon- oacetate (O), H VT,” with phosphonoformate (I),and H VT,” with phosphonoacetate (Cl). IO 1 l j I O o “I 10 >- 'i 11‘ U 3 U .9 a ‘6 2 § 10L 1 0 3 E 0 a. 103— -< o O I l i 1 0035 0070 OIOS 0140 [Phosphonate] .(mMI FIG. 3. Semilog plot of the effect of phosphonofor- mate I.) andphosphonoacetate (O) on the replication of MDH V. A known titer of virus was used to infect secondary duck embryo fibroblast cultures in growth media without and with different concentrations of phosphonate. Plaques were enumerated 6 to 7 days postinfection. Each point represents the average of three experiments. The number of plaques observed in each experiment was normalized to the input num- ber of PF U. VOL. 13, 1978 At concentrations up to 0.55 mM, phosphon- oformate had no obvious cytotoxic effect on the growth of normal duck embryo fibroblasts. The treated cells formed a confluent monolayer in the usual time, and maintenance of the mono- layer was normal. In the above studies, phosphonoformate was an effective inhibitor of the replication of MDHV, HVT,", and HSV in cell culture. Phos- phonoformate, however, was not an effective inhibitor of the replication of HVT,. in cell culture. As seen in Fig. 2, the replication of HVT,”. is less sensitive to phosphonoformate than is the replication of HVT,“. This indicates that the mutation to phosphonoacetate resis- tance also leads to phosphonoformate resistance and suggests that the DNA polymerase of HVT,“, is altered so as to be less sensitive to phosphonoformate as well as phosphonoacetate. Phosphonoformate inhibition of the DNA polymerization reaction catalyzed by the HVTpa-induced DNA polymerase. Indeed, phosphonoformate, like phosphonoacetate, was not an effective inhibitor of the DNA polymer- ase of this phosphonoacetate-resistant mutant. With the four dNTP’s as the variable substrate, phosphonoformate again gave linear noncom- petitive inhibition (Fig. 4). The K., was 8 11M and K., was 18 M. This represents an increase of 10 to 20 times over the values obtained for the HVTM-induced DNA polymerase and is sim- ilar to the increase seen with phosphonoacetate (L. F. Lee et al., manuscript in preparation). With activated DNA as the variable substrate and the four dNTP’s at their apparent Michaelis O Slopeo N (l— <’\ Intercept. 9 / gr" I I I 0 l 0.2 0 3 0,9 0.5 [dNTP]—', (,IM)" 30 ‘0 [Phosphonoformate] ,( 1.1M) IO 20 FIG. 4. Double reciprocal plots of the HVTpa-in- duced DNA polymerase-catalyzed reaction with the four dNTP’s as the variable substrate and phosphon- oformate as inhibitor. Activated DNA was at a con- centration of 200 ug/ml. The initial velocities were expressed as picomoles of [‘H]thymidine 5’-mono- phosphate incorporated into DNA/30 min. Phos- phonoformate concentrations were 0 (0), [GM (0), 25 11M ([3), and 40 W (A). Equimolar concentrations of each of the four dNTP’s were present in the reaction mixtures. The replots of the slopes (O) and intercepts (O) as a function of phosphonoformate concentration are shown in the left panel. PHOSPHONOFORMATE INHIBITION OF HERPESVIRUS 191 concentration of 5 uM, phosphonoformate gave linear noncompetitive inhibition, a Kit of 13 uM, and a K., of 43 uM. Again, this represents an increase of about 10 to 20 times over the same values obtained for the HVTm-induced DNA polymerase, as was also seen with phosphono- acetate. DISCUSSION The replication of MDHV, HVT,“, and HSV in cell culture was effectively inhibited by phos- phonoformate. This inhibition was as effective as the inhibition by phosphonoacetate. The in- hibition patterns seen in the steady-state en- zyme kinetic analysis of the HVTm-induced DNA polymerase with phosphonoformate as in- hibitor were identical to those reported for phos- phonoacetate (14). Both phosphonates showed noncompetitive inhibition with the four dNTP’s as variable substrate. With activated DNA as the variable substrate and the four dNTP’s at their Michaelis concentration, noncompetitive inhibition was also observed. The apparent in- hibition constant values were similar. These ob- servations, taken together with the resistance of HVTp. replication to the effect of phosphonofor- mate and the much higher inhibition constant values obtained for the HVTm-induced DNA polymerase, indicate that the inhibition of her- pesvirus replication by phosphonoformate is through an effect on the herpesvirus-induced DNA polymerase in a manner analogous to the inhibition by phosphonoacetate (9, 14-16). Although phosphonoformate is a potent inhib- itor of the herpesvirus-induced DNA polymer- ase, it is not entirely specific. Recent experi- ments in this laboratory have shown that the a-polymerase of Hela, KB, and Wi-38 cells was inhibited by phosphonoformate (C. L. K. Sa- bourin, J. Reno, and J. Boezi, manuscript in preparation). Phosphonoacetate also inhibits these enzymes. The apparent inhibition constant values for either phosphonate were about 30 11M. The B and y polymerases are not effectively inhibited by phosphonoformate or phosphon- oacetate. The results of previous studies on analogs of phosphonoacetate demonstrated that the struc- tural requirements for inhibition were rather narrowly defined (10, 13, 14, 23). For example, analogs containing a mono- or diester on the phosphono group or containing a carboxyl or sulfo substitution for the phosphono group were not inhibitors. Analogs that contained a methyl- amino- or phenyl-substituted methylene carbon also were not inhibitors. Apparently some mod- ification at the carboxyl end of phosphonoace- tate is permissible. Low-molecular-weight car- boxyl esters are reported to be effective inhibi- 192 RENO, LEE, AND BOEZI tors (10). Aldehyde, amide, and acetonyl substi- tutions for the carboxyl group of phosphonoac- etate, however, did not yield effective inhibitors (J. A. Boezi, unpublished results). Phosphonates having longer carbon chain length than phos- phonoacetate (for example, phosphonopropio- nate or phosphonobutyrate) were not inhibitors. This report demonstrates that the shorter chain length of phosphonoformate yielded an effective inhibitor. Now that phosphonoformate has been shown to be an effective inhibitor of herpesvirus repli- cation, its efficacy as an antiherpesvirus drug in animals must be determined. Recent results have shown that phosphonoformate is as effec- tive as phosphonoacetate against HSV types 1 and 2 in mice and guinea pigs (E. R. Kern, J. Overall, L. Glasgow, J. Reno, and J. Boezi, man- uscript in preparation). Additional animal model systems will be tested, and toxicity studies will follow. Phosphonoformate may be of sufficiently different chemistry that it would be less toxic, would not be accumulated in bone, and might become a useful drug in the treatment of her- pesvirus infections. ACKNOWLEDGMENTS This work was supported by Public Health Service grant 17554 from the National Cancer Institute. LITERATURE CITED 1. Amen. B. N., and D. T. Dubin. 1960. The role of polya- mines in the neutralization of bacteriophage deoxyri- bonucleic acid. J. Biol. Chem. 235:769-775. 2. Bandurski, R. S., and B. Axelrod. 1951. The chromato- graphic identification of some biologically important phosphate esters. J. Biol. Chem. 193:405-410. 3. Boezi, J. A., L. F. Lee, R. W. Blakesley, M. Koenig, and H. C. Towle. 1974. Marek's disease herpesvirus- induced DNA polymerase. J. Virol. 14:1209-1219. 4. Bopp, B. A., C. B. Estep, and D. J. Anderson. 1977. Disposition of disodium phosphonoacetate-"C in rat, rabbit. dog and monkey. Fed. Proc. 36:939. 5. Cleland, W. W. 1963. The kinetics of enzyme-catalyzed reactions with two or more substrates or products. I. Nomenclature and rate equations. Biochim. Biophys. Acta 67:104-137. . Cleland, W. W. 1963. The kinetics of enzyme-catalyzed O) reactions with two or more substrates or products. 11. Inhibition: nomenclature and theory. Biochim. Biophys. Acta 67:173-187. 7. Fitzwilliam, J. F., and J. F. Griffith. 1976. Experimen- tal encephalitis caused by herpes simplex virus: com- parison of treatment with tilorone hydrochloride and phosphonoacetic acid. J. Infect. Dis. 133:A221-A225. 8. Gerstein. D. D., C. R. Dawson, and J. 0. Oh. 1975. Phosphonoacetic acid in the treatment of experimental herpes simplex keratitis. Antimicrob. Agents Chemo- ther. 7:285—288. 9. Hay, J., and J. H. Subak-Sharpe. 1976. Mutants of herpes simplex virus types 1 and 2 that are resistant to phosphonoacetic acid induce altered DNA polymerase activities in infected cells. J. Gen. Virol. 31:145-148. 10. Herrin, T. R., J. S. Fairgrieve. R. R. Bower, N. L. 13. 14. 15. 16. 17. 18. 19. 20. 21. 23. 24. ANTIMIcnon. AGENTS CHEMOTHER. Shipkowitz, and J. C-H. Mao. Synthesis and anti- herpes simplex activity of analogues of phosphonoacetic acid. J. Med. Chem. 20:660-663. . Huang. E-S. 1975. Human cytomegalovirus. IV. Specific inhibition of virus-induced DNA polymerase activity and viral DNA replication by phosphonoacetic acid. J. Virol. 16:1560-1565. . Kern, E. R., J. T. Richards, J. C. Overall, and L. A. Glasgow. 1977. Genital Herpesvirus hominis infection in mice. II. Treatment with phosphonoacetic acid, ad- enine arabinoside, and adenine arabinoside 5'-mono- phosphate. J. Infect. Dis. 135:557-567. Lee, L. F ., K. Nazerian, S. S. Leinbach, J. M. Reno, and J. A. Boezi. 1976. Effect of phosphonoacetate on Marek's disease virus replication. J. Nat. Cancer Inst. 56:823-827. Leinbach, S. 8., J. M. Reno, L. F. Lee. A. F. Isbell, and J. A. Boezi. Mechanism of phosphonoacetate inhibition of herpesvirus-induced DNA polymerase. Biochemistry 15:426-430. Mao. J. C-H.. and E. E. Robishaw. 1975. Mode of inhibition of herpes simplex virus DNA polymerase by phosphonoacetate. Biochemistry 14:5475-5479. Mao, J. C-H., E. E. Robishaw, and L. R. Overby. 1975. Inhibition of DNA polymerase from herpes sim- plex virus-infected Wi-38 cells by phosphonoacetic acid. J. Virol. 15:1281-1283. Nylen, P. 1924. Beitrag zur Kenntnis der organischen Phosphorverbindungen. Chem. Ber. 578:1023-1038. Overby, L. R., E. E. Robishaw, J. B. Schleicher, A. Rueter, N. L. Shipkowitz, and J. C-H. Mao. 1974. Inhibition of herpes simplex virus replication by phoe- phonoacetic acid. Antimicrob. Agents Chemother. 6:360-365. Purchase, H. G. 1969. Immunofluorescence in the study of Marek's disease. I. Detection of antigen in cell culture and antigenic comparison of eight isolates. J. Virol. 3:557-565. Roizman, B., and L. Aurelian. 1965. Abortive infection of canine cells by herpes simplex virus. I. Characteri- zation of viral progeny from co-operative infection with mutants differing in capacity to multiply in canine cells J. Mol. Biol. 11:528-538. Sadtler Standard Spectra. 1976. Researchers, editors, and publishers. spectrum no. 11649K. Sadtler Research Laboratories. Inc., Philadelphia. . Sadtler Standard Spectra. 1976. Researchers, editors. and publishers. spectrum no. 5481M. Sadtler Research Laboratories, Inc., Philadelphia. Shipkowitz, N. L., R. R. Bower, R. N. Appell, C. W. Nordeen, L. R. Overby, W. R. Roderick, J. B. Schleicher, and A. M. Von Each. 1973. Suppression of herpes simplex virus infection by phosphonoacetic acid. Appl. Microbiol. 26:264-267. Solomon, J. J., P. A. Long, and W. Okazaki. 1971. Procedures for the in vitro assay of viruses and antibody of avian lymphoid leukosis and Marek's disease. Agri- cultural handbook no. 404, Agricultural Research Ser- vice. U.S. Department of Agriculture, Washington, DC. . Summers, W. C., and G. Klein. 1976. Inhibition of Epstein-Barr virus DNA synthesis and late gene expres- sion by phosphonoacetic acid. J. Virol. 18:151-155. . Warren, S., and M. R. Williams. 1971. The acid-cata- lyzed decarboxylation of phosphonoformic acid. J. Chem. Soc. lO71(B):618-621. . Wilkinson, G. N. 1961. Statistical estimations in enzyme kinetics. Biochem. J. 80:234-332. . Witter, R. L., K. Nazerian, H. G. Purchase. G. H. Burgoyne. 1970. Isolation from turkeys of a cell-asso- ciated herpesvirus antigenically related to Marek's dis- ease virus. Am. J. Vet. Res. 31:525-538. ARTICLE 2 MECHANISM OF PHOSPHONOFORMATE INHIBITION OF REVERSE TRANSCRIPTASE By John M. Reno Hsing-Jien Kung and John A. Boezi 31 ABSTRACT Phosphonoformate, and not phosphonoacetate, was a potent inhibitor of DNA synthesis catalyzed by reverse transcriptase from avian myeloblastosis virus, Rous sarcoma virus, Moloney murine leukemia virus, and feline leukemia virus with poly(a)-oligo(dT), activated DNA or poly(A)-containing RNA°oligo (dT), as substrate, 2+ With Mg as the cofactor, 50% inhibition in the rate of polymeriza- tion was observed with about l0 pM phosphonoformate. With Mn2+ as the cofactor, 50% inhibition was obtained with about 0.5 uM phosphono- formate. The endogenous reactions or reactions in which purified viral RNA was the substrate were much less sensitive to phosphono- formate. The RNase H activity of AMV reverse transcriptase was not inhibited by either phosphonoate in the presence of either Mg2+ or Mn2+. A steady-state enzyme inhibition kinetic analysis of both the DNA polymerization and the deoxyribonucleoside triphosphate- pyrophosphate exchange reaction was carried out. The results are consistent with phosphonoformate interacting with the reverse tran- scriptase at the pyrophosphate binding site and functioning as a dead-end inhibitor. 32 INTRODUCTION Inhibitors of nucleic acid synthesis that interact directly with a target enzyme are few in number (1). Phosphonoacetate (2, 3, 4), one such compound, has been the subject of several reviews (5, 6, 7). It is an effective inhibitor of herpesvirus replication in infected cell cultures at concentrations at which cell proliferation is unaffected and is efficacious as an antiherpesvirus agent in animal model systems. Its target protein is the herpesvirus-induced DNA polymerase and it inhibits by interacting with the enzyme at its pyrophosphate binding site with an apparent inhibition constant of 1 uM (8, 9). Phosphonoacetate also inhibits eukaryotic DNA poly- merase a in an analogous manner but with an apparent inhibiton constant of about 30 uM (9, 10). Eukaryotic B- and v-polymerases are not inhibited. 0f the many phosphonoacetate analogs tested, only phosphonoformate was found to be an effective inhibitor of the herpesvirus-induced DNA polymerase (ll, l2). Its mechanism of inhibition and effectiveness are similar to that of phosphonoacetate. Like phosphonoacetate, phosphonoformate blocks herpesvirus replica- tion in infected cell cultures and in animal model systems (13, l4, 15, 16). 33 34 Sundquist and Larner (l7) reported that phosphonoformate, but not phosphonoacetate, inhibited the replication of visna virus in cultures of sheep choroid plexus cells. Visna virus is a retro- virus that causes a slow degenerative disease of the central nervous system of sheep (l8). In contrast to the oncornaviruses which transform cells in culture, visna virus causes a productive lytic infection in cultures of sheep cells (l9). At lOO pM, the lowest concentration tested, phosphonoformate but not phosphonoacetate inhibited polymerization by the reverse transcriptase of this virus (l7). In this report, we examine the phosphonoformate inhibition of reverse transcriptase of some oncogenic retroviruses from avian and mammalian sources. With purified avian myeloblastosis virus DNA polymerase, we report the results of our enzyme kinetic inhibition studies and propose a mechanism by which phosphonoformate inhibits polymerization. We also look into the effect of phosphonoformate on the RNase H activity of reverse transcriptase. METHODS Avian myeloblastosis virus and purified AMV DNA polymerase (specific enzymatic activity of 65.3 umoles of dTMP incorporated per 10 min per mg of protein) were obtained from Dr. J. Beard, Life Science Research Laboratories. Rous sarcoma virus and RSV RNA were purified as previously described (20), Feline leukemia virus and Moloney murine leukemia virus clone l were gifts from Dr. L. Velicer, Department of Microbiology, Michigan State University. g, £911_RNA polymerase and poly(A)-containing RNA from bovine pituitary glands were gifts from Dr. A. Revzin and Dr. J. Nilson, respectively, of the Department of Biochemistry. Phosphonoacetic acid was from Richmond Organics. Trisodium phosphonoformate hexahydrate was pre- pared following the procedure of Nylen (2l). Aphidicolin was obtained from the Developmental Therapeutics Program, Chemotherapy, National Cancer Institute. All other reagents were from sources previously described (9). Assay of the DNA Polymerization Reaction Catalyzed by Reverse Transcriptase. The standard reaction mixture contained in 200 pl: 50 mM Tris-HCl, pH 8, 1 mM dithiothreitol, 60 mM KCl, l00 pg per ml bovine serum albumin, either l0 mM MgCl2 or 0.5 mM MnClz, l8 mg per ml poly(A)-oligo(dT)]2_]8, 100 uM [3HJdTTP (specific radioactivity 30-250 cpm per pmol), and DNA polymerase. With l8 pg. 35 36 per ml poly(C)-oligo(dG)]2_]8 as template-primer, 100 uM [3H]dGTP (specific radioactivity 40-l60 cpm per pmol) was used. When 200 pg per ml activated calf thymus DNA was the substrate, the three unlabeled dNTPs were at l00 pM and [3HJdTTP (specific radioactivity 100-400 cpm per pmol) was at 20 uM. With 4.2 pg per ml poly(A)- cOntaining RNA as substrate, oligo(dT) was added at 5 pg per ml, the three unlabeled dNTPs were at l00 uM and [3H]dTTP (specific radio- activity 900-l600 cpm per pmol) was at 20 uM. For experiments with 55 pg per ml RSV RNA as substrate, the three unlabeled dNTPs were at l00 uM and [a-32PJdCTP (specific radioactivity 2100 cpm per pmol) was at 20 uM. For reactions in which purified virus was used as the source of DNA polymerase and substrate RNA, 0.05% NP-40 was used, the three unlabeled dNTPs were at l00 DM and [3H]dTTP (specific radioactivity 300-650 cpm per pmol) was at 20 uM. For experiments when aphidicolin was evaluated as an inhibitor, it was dissolved in dimethylsulfoxide. The concentration of dimethylsulfoxide in the reaction mixtures, including controls to which no aphidicolin was added, was kept constant at 6%. The amounts of DNA polymerase or purified virions added to the reaction mixtures were within the activity range which gave direct proportionality with the rate of DNA synthesis and with time. The stock enzyme solution was diluted with buffer containing 50 mM Tris-HCl, pH 8, l mM dithiothreitol, and 1 mg per ml bovine serum albumin. For the kinetic studies, changes in concentration of assay components were as noted in the Legends to the Figures. Incubation 37 was for 30 min at 37°C. The reactions were terminated by addition of 2 ml of a cold solution of 10% trichloroacetic acid-1% socium pyro- phosphate. After 5 min at 0°C, the reaction mixtures were filtered onto glass fiber filters (GF/C). The filters were washed, dried, and monitored for radioactivity by liquid scintillation spectrometry. Assay of the RNase H Reaction Catalyzed by AMV DNA Polymerase. The substrate [3H]poly(A)-poly(dT) was synthesized using g, ggli_RNA polymerase with poly(dT) as template and [BHJATP (specific radio- activity 400 cpm per pmol) using the conditions described by Watson et al. (22). After 2.5 hr at 37°C the reaction mixture was concen- trated with n-butanol and the product purified on a Sephadex G-50 column. The RNase H assay mixture contained in 200 pl: 50 mM Tris- or 0.5 mM MnCl HCl, pH 8, either l0 mM MgCl 1 mM dithiothreitol, 2 2’ l00 pg per ml bovine serum albumin, 2 nmol [3H]poly(A)-poly(dT) as AMP residues, and 9.6 ng AMV DNA polymerase. The reaction was stopped on ice with the addition of 250 pl of cold 10% HCl04 and 50 pl of 5 mg per ml bovine serum albumin. After standing 5 min at 0°C, the reaction mixtures were centrifuged at 9000 rpm for 5 min and an aliquot of the supernatant was counted in Triton X-l00 scintillation cocktail. The assay was linear with the amount of DNA polymerase added over the time course of the reaction. Assay of the dNTP-Pyrophosphate ExchangeReaction Catalyzed by AMV DNA Polymerase. The exchange reaction was routinely assayed in 100 pl with 50 mM Tris-HCl, pH 8, 10 mM MgClz, 60 mM KC], 1 mM 38 dithiothreitol, l00 pg per ml bovine serum albumin, 100 pM dTTP, l8 pg per ml poly(A)-oligo(dT), 2 mM sodium [32Pprrophosphate (specific radioactivity 200 cpm per pmol), and AMV DNA polymerase. The kinetic experiment (Figure 5) was performed in 200 pl with 50 mM Tris-HCl, pH 8, l0 mM MgCl2, 60 mM KCl, l mM dithiothreitol, 100 pg per ml bovine serum albumin, 1 mM each of the four dNTPs, 200 pg per ml of activated calf thymus DNA, l06 ng AMV DNA polymerase, and various concentrations (0.82-2.0 mM) of sodium [32P]pyrophosphate (specific radioactivity 100 cpm per pmol). The stock solution of unlabeled sodium pyrophosphate was treated with Chelex 100 before use. In addition to the l0 mM MgCl2 in the reaction mixture, supplementary MgClz, in an amount equimolar to the sodium pyro- phosphate in the reaction, was used. Incubation was for 30 min at 37°C. Reactions were stopped on ice with 1 ml of 2 N HClO4-0.4 M sodium pyrophosphate. After addition of 0.2 ml of 25% acid-washed Norit, the reaction mixtures were centrifuged, the supernatant removed, and the Norit collected on glass fiber filters (GF/C). The filters were washed with 50 ml of l0 mM sodium pyrophosphate, pH 6, dried, and counted in a Nuclear Chicago low background flow counter. The pyrophosphate exchange reaction was shown to be dependent on added enzyme, template-primer, MgClz, and dNTPs. The reaction was linear with time through the course of the reaction and was directly proportional to the amount of AMV DNA polymerase added to the reaction mixture. 39 Assay of the DNA Polymerization Reaction Catalyzed by the a- and B-Polymerases from Chinese Hamster Ovary Cells (CHO). The CHO a— and B-polymerases were purified and assayed as described pre- viously (l0). Inhibition Patterns. Inhibition patterns and kinetic con- stants were defined according to the nomenclature of Cleland (23, 24). Analysis of each reaction mixture was done in duplicate. The data for the double reciprocal plots were evaluated using a computer program based on the method of Wilkinson (25). For evaluatin of the apparent inhibition constants, replots of the intercepts and slopes of the double reciprocal plots were analyzed by the method of least squares. RESULTS Inhibition of DNA Synthesis Catalyzedgby Reverse Transcript- .asg. Phosphonoformate is a potent inhibitor of DNA synthesis catalyzed by AMV DNA polymerase (Figure 1, upper and lower panels on left). With poly(A)~oligo(dT) as the template-primer and Mn2+ as the cofactor, the rate of dTTP polymerization was decreased 50% by 0.25 pM phosphonoformate. With Mg2+ as the cofactor, the polymeriza- tion rate was decreased 50% by 7 uM phosphonoformate. With phosphono- acetate, a 50% inhibition in the rate of dTTP polymerization was observed at 180 pM with Mn2+ as the cofactor and no inhibition was observed with Mg2+ at concentrations up to 500 pM (Figure l, upper and lower panels on right). The concentration of phosphonoformate required to inhibit DNA polmerization by 50% was found to be dependent on the template- primer (Table l). Phosphonoformate was essentially equally effective in inhibiting DNA polymerization with poly(A)~oligo(dT), activated DNA, and poly(A)-containing RNA-oligo(dT) as template-primers. With poly(C)-oligo(dG) and with RSV RNA, however, higher concentrations of phosphonoformate were required to yield 50% inhibition. The phosphonoformate sensitivity of reverse transcriptase from some different viral sources was examined (Table 2). Using poly(A)-oligo(dT) as the template-primer and detergent-treated virions, no significant differences in phosphonoformate sensitivity 40 FIGURE 1. 4] Inhibition of AMV DNA polymerase by phosphonoformate and phosphonoacetate. Vo represents pmol [3H]dTMP incorporated into poly(A)-oligo(dT) per 30 min in the absence of phosphonate. V represents dTMP incorporation in the presence of phosphonate. The phosphonate concen- tration which inhibits the enzyme activity by 50% is that concentration for which Vo/V is 2. upper and lower panels on left: Inhibition by phosphonoformate in the presence of Mg2+ (o) for which VO 2+ ( was 430 pmol per 30 min and in the presence of Mn 0) for which Vo was 200 pmol per 30 min. Upper and lower panels on right: Inhibition by phosphonoacetate in the presence of Mg2+ (o) for which VO was 360 pmol per 30 min and in the presence of Mn2+ (o) for which Vo was 180 pmol per 30 min. 42 :24: . 763808535”— OON 00. _ _ p P mgamwu 22.3 . HEoEooocozamosaH— CON 00. _ :21 v . 765835885”— O. 0.x. Cd n.N _ _ _ _ 4 l nfim T nflc A%N >< \Jv 4, t 06 > I nfim 0A” 1 . _ _ _ 0.0. A 2.3 . 78658203805“— ON. om cm on _ :g‘ o .1. ~.o t ++ .2 No I «no 0 5x7 T ogu l mgu 43 .H menu,“ cw mo vmcmELmumu mew: cowpwnwccw Rom comatose was» mnosgotocogamosa we mconmcpcmocou msp .muosumz Love: umnwsummv mm tom: mo: Louuowoo cow Poppa new .mmumgamozn -mcp muwmompuzconwcxxowu .stpgnumumpasmg mumwsgognao mnu saw: mcawxwe cowuummc vcoucoum och ate 5% om m.N ahmm shoe .shoa Ahevomw_o._oo< apes .apuo e m.o ahroo< co, m ahoeHIMQ “wavama_o.fiovzpaa N.“ mN.o appomxmg Aeevomapo.fiz< to cowuwawccw oposaotococamoga co cmewgatmvmpaemp we uummwmit.p m4m mmpmmsm mcmmsmmmu mcwm: cmEcowema msmz mxmmm< om_ N.“ mm mp m.o Amevomwpo.fimm m, e.o Abovom_mo.fioom mm k.o Akevomm_o.fio=z-z m «.0 Apevomw_o.fiz< z: +wm2 +~cz gmevgmimpopasmh mmmmmsapom <2: cowmmNPgmezpoa mo mums mzu cm cowuwnwgcw Rom mmmsuosa «on» mpmscowocozmmozm mo cowpmsmcmucou .mmmuawgomcmgu mmsm>mg mo cowmwnwgcm mmmsgowocozamosmtu.m mmm PA PFfl o 6 — - V . /V0 0.4 — - 0.2 — ~ 1 1 1 1 I '00 200 300 400 500 [Phosphonate] , (LLM) I C) T T I l I PF 0.8- - '- v/ 0.6 — — Vo 0.4 - — 0.2 - '- J 1 l l L ICC 200 300 400 500 [Phosphonate] , (uM) Figure 2 48 .chmm ammp mg» cw czogm mgm mpmsgomocozamoga mo cowuocam m mm A-V mmmmmcmpcw ucm on mmmopm ms» mo muopmmg mg» .A-e z: u.o new .on z: e.o .Anu z: ~.o .Amv o msmz mcomumsucmucom mumsgowocogamogm .cws om cw um~wsmezpoa mpkomxmu mo Foam mm vmmmmcaxm mcmz mmwuwmopm> mepwcw mch .FE Lma m: mp mm mm: Ahuvomwpo.fi mm N chem saw: cowmmmmc um~xpmumm mmmgmsapom z< ms» mo myopm meosnpmmc mpnzoo .m umstu 49 .023 “but oH_ 00.0 00.0 v0.0 No.0 CDC) m mczmwu .0.0 _|> o adols No.0 0.0 A .2 i V . TBEBBcofimoch— 0.0 #0 N0 _.0 N0 a _ fl N00.0 V000 000.0 000.0 0.0.0 GOJSIUI AU 04 50 With poly(A)-oligo(dT) as the variable substrate, Mn2+ as the cofactor and dTTP at 20 pM, phosphonoformate gave linear uncompe- titive inhibition (Figure 4). A replot of the vertical intercepts yielded a Kii of 0.l0 uM. The apparent Km for poly(A)-oligo(dT) was 3.5 pg per ml. Phosphonoformate also gave linear uncompetitive inhibition with dTTP at concentrations of 2 HM and 100 pM. With Mg2+ as the cofactor and with dTTP at l00 pM, again linear uncompetitive inhibition was observed. Phosphonoformate Inhibition of the dNTP-Eyrophosphate Exchange Reaction Catalyzed by AMV DNA Polymerase. The effect of the phosphonoates on the AMV DNA polymerase catalyzed pyrophosphate exchange reaction was studied. With poly(A)-oligo(dT) as the template-primer and Mg2+ as the cofactor, phosphonoformate gave 50% inhibition in the rate of dTTP-pyrophosphate exchange reaction at a concentration of 9 pM. Under the same conditions, phosphonoacetate did not inhibit the exchange reaction at concentrations up to 300 pM. Because AMV DNA polymerase is more active in catalyzing the pyrophosphate exchange reaction with activated DNA and the four dNTPs than with poly(A)-oligo(dT) and dTTP (26), activated DNA and the four dNTPs with Mg2+ as the cofactor was used to determine the phosphonoformate inhibition pattern for the pyrophosphate exchange reaction. Phosphonoformate was a competitive inhibitor of pyro- phosphate (Figure 5). The apparent Kis was l2 pM and the apparent Km for pyrophosphate was 2.7 mM. 51 .20 z: 50 2e .3 2: mm .on z: m.o .on o msmz mcowumspcmucom mumELomocognmocm .2: cm mm mm: mhhu .sopmmmom mm + :2 use mumsumnzm mFDmpcm> ms» mm Ahuvomw_o.fiz< mcp 0o mmopm meogmwums mpnaoc .e mmstu 52 72.53.. _._H....Boo__o.._o& 0.~ 0.. 0.. 0.0 _ ~ _ _ 000.0 0.0.0 0.0.0 0N0.0 0N0.0 v ms=m_. =23. TEESESEmoznm 0.0 0.0 0.0 l D 000.0 0.0.0 0.0.0 o IdGOJGIUI 53 .Ans 2: cm mam .on z: m .A.. o msm: meowummmcmmcom mumscowocosamosm .cwe om gmm stay mpnmnsommm uwgoz m on vmpcm>com mumcnmosaogxa ”mum. Foam mm mmmmmgmxm mcmz mmwuwmopm> meuwcw mgh .mmmsumnam mpnmwsm> mg» mm mumgmmogqogzm saw: mmmcmexpom z< an mmuapmmmm :owuummg mmcmnmxm mpogamozaomxmtmhzu mg» mo muopa meosawmms mpnzoa .m umzwmu 54 m mcamw. :22 E . mimmBzgmozgoina N._ 0.. . m. .0 0 .0 c .0 N0 q _ _ _ . _ I -\a O\ .\O m i o \ D I D m\ D D l _ _ _ _ _ _ :24. v. mmfiecopocozamozau 6.0 cm 0. A _ «00.0 . IdaOJaIm v8.0 .1 00.0 55 2+ Effect of Mn on Phosphonate Inhibition of Eukaryotic DNA Polymerases a and B. In studies utilizing Mg2+ as the cofactor, DNA polymerase a, but not B, was found to be inhibited by phosphono- 2+ as the cofactor and formate and phosphonoacetate (9, l0). With Mg activated DNA as the template-primer, a 50% decrease in the rate of DNA synthesis by o-polymerase was observed at a concentration of approximately 30 pM with either phosphonate. No inhibition by phosphonoformate or phosphonoacetate for the B-polymerase was observed at concentrations up to 300 pM. Since in the presence of Mn2+, ii l5 to 30-fold greater sensitivity to inhibition by the phosphonates was observed for AMV DNA polymerase, the sensitivity of the a- and B-polymerases with Mn2+ as the cofactor was examined. For CHO a-polymerase with activated DNA as the template-primer, a 50% inhibition in the rate of DNA synthesis was observed in the presence of 0.4 pM phosphonoformate and 0.9 pM phosphonoacetate. These values are some 30-times lower than those observed with Mgz+ as the cofactor. For CHO B-polymerase with Mn2+ as the cofactor, neither phosphonate inhibited DNA synthesis at concentrations up to 300 pM. Effect of Aphidicolin on DNA Synthesis Catalyzed by AMV DNA Polymerase. Aphidicolin, an inhibitor of eukaryotic DNA polymerase u, did not inhibit AMV DNA polymerase with poly(Cm)-oligo(dG) as template-primer (27). However, aphidicolin was subsequently shown to be a competitive inhibitor of dCTP, a substrate not required for poly(Cm)-oligo(dG) directed polymerization (28). In the course of 56 our experiments with phosphonoformate, aphidicolin was tested as an inhibitor of AMV DNA polymerase using activated DNA as the template- primer and all four dNTPs as substrates. Aphidicolin did not inhibit at concentrations up to 600 pM. Under identical conditions, a 50% decrease in the rate of DNA synthesis catalyzed by CHO DNA polymerase a was observed at 5 pM aphidicolin. DISCUSSION The results of our study are consistent with the mechanism of phosphonoformate inhibition of reverse transcriptase presented in Figure 6. We propose that phosphonoformate binds to the enzyme at the pyrophosphate binding site and functions as a dead-end inhibitor. For such a reaction scheme, the rate equation describing DNA synthesis in the presence of template-primer, dNTP, and phosphono- formate is given by Equation l,* where KT-P’ KiT-P and KT-P" KiT-P' refer to template-primer binding as substrate and product, respectively. Under the experimental conditions that we have used, all terms containing the product, inorganic pyrophosphate, can be eliminated. The reciprocal of this equation arranged with dNTP as the variable substrate is given by Equation 2; the reciprocal arranged with the template-primer as the variable substrate is given by Equation 3. With dNTP as the variable substrate, both the slope and intercept terms are a function of phosphonoformate concentration and the inhibition pattern is noncompetitive. With the template- primer as the variable substrate and dNTP as the fixed substrate only the intercept is a function of phosphonoformate concentration; * The rate equation was first written in terms of individual rate constants by the method of King and Altman (38). It was then transformed into a rate equation in terms of kinetic constants as described by Cleland (23). 57 58 .mumsgo0ocosmmogm mp mm ucm .Lmewcaimampasmu m. nth .mmmumwcumcmcp mmsm>ms mmuocmu m .Anm .mm .NP .m. Emmcmsmme we 03 umgmmgo nmw$wmos m mp mumssomocogamozm mo mucmmnm mgm cw Empcmgums :owummmc mwmmn mmum_:umom mgh .mmmmawgmmcosu mmcm>ms mo cowuwnwgcw mumssomocogamoga we Emwcmzmma ummomocm .o mgamr. 59 .aa 2+5n...u._. oPZUnx ex m mgsmmu n.._.z U m. Em . 0v..p 4 _n_Q0v. Aw+cvalfim coax s 0.2 ma. $5.3m. “:3. .3. V NV. V m ou»_x 6O 3. A VA v HA VA max.aihwxmawx V max.mihw¥H um nth QHZU i i + i + umwxm by + mm¥.m hwy a waahzux wamwx waaxmhszx.aihw¥ AwQQVAQPZUVAQIPv¢IIflII_+ QIHP QPZU V + x x nib? Pam .mihw ¥ .QIF ¥ M _. I III A.va fiAm EVA.QIF + FV + XH QIFw QHZU + x x x .QIPP .thw i x i x ahzu nth ghzc nib? p .aezu. .a P. eihmhmw_+ .. + .a F. eimuHHm + _. x + .akzo. g + x x . .ii. .aa¥.a-P. > ..aa..a-h. . x N - - . a-k. apzox. > .apzo... H. > x 61 max .QIFF x .aa. H a-.. . ma.¥ ma¥.a-h.¥. .apza. .a-epx .a-e. .ahza. a-hg _> > .m. . + a-h.¥ahzeg + .a-h.¥ apze + . a-k¥ + P. + _ Hapzega-».¥ + . ..i_..--- . - + _. g P m a 0.x ma .a-m. me. x x g .ma.. - + . + a #5 P .a-m. .a-h. .apzo. ma a-p_ .a-k. a-k. P> > .iiii. x x. x . .x maze .N. - + . - + F. + H.... - + - + . - + F. x0 .11. ".11. a my a #2 _ a pmxaezex aezexa 0.x a 0.x . m 62 the slope is independent of phosphonoformate concentration and the pattern is uncompetitive. Experimentally, these inhibition patterns were observed (Figures 3 and 4). Furthermore, the competitive inhibition pattern observed for phosphonoformate with pyrophosphate as the variable substrate in the exchange reaction (Figure 5) is consistent with the two compounds binding to the same site on the enzyme. Phosphonoacetate and phosphonoformate inhibit the herpesvirus- induced DNA polymerase by binding to the enzyme at its pyrophosphate binding site (9, 12). In this case, however, the phosphonates may be acting as alternate product inhibitors because, as predicted by the rate equation for an alternate product inhibitor, noncompetitive inhibition is observed with activated DNA as the variable substrate. Phosphonoformate, although an effective inhibitor of the polymerization reaction catalyzed by AMV DNA polymerase, does not inhibit the RNase H reaction. For AMV DNA polymerase, polymerization activity and RNase H activity are present on the same polypeptide chain (29) and are mechanistically independent (30) although the same template-primer binding site may be used for both activities (31). The phosphonoformate results may be interpreted to mean that E;;P(n+1), a form of the enzyme that is inactive in polymerization, still has an active RNase H function. Like phosphonoformate, inorganic pyrophosphate inhibits polymerization but not the RNase H 63 activity of'AMV DNA polymerase (32). Pyridoxal 5' phosphate islanother compound that inhibits polymerization but not RNase H activity (33, 34). Since pyridoxal 5'-phosphate is a competitive inhibitor of the T-P dNTPs, the EB P form of the enzyme, where B6P is pyridoxal 5'- 6 phosphate, must also have an active RNase H function. Knopf (35) recently has reported that herpes simplex virus DNA polymerase has an associated 3' to 5' exonuclease activity which is inhibited by phosphonoacetate. For this enzyme, both the DNA polymerase and exonuclease activities are inhibited by phosphonoacetate. In the Mn2+-containing DNA synthesis reactions catalyzed by AMV DNA polymerase, phosphonoformate was a much more potent inhibitor than in the MgZ+-containing reactions. The apparent inhibition con- stants were lower by 50 to 100 times. The apparent Kms for poly(A)-oligo(dT) and dTTP were also lower, but only by 5 to 10 times. This greater sensitivity to inhibition by phosphonoformate with Mn2+ than with Mg2+ was seen with all the template-primers tested with AMV DNA polymerase (Table 1), with four different reverse transcriptases (Table 2), and with DNA polymerase a. Eukaryotic DNA polymerase a, but not B or y, is inhibited by phosphonoformate (10). The phosphonoformate apparent inhibition constants for the a-polymerase were about 30 pM with activated DNA as the variable substrate and Mg2+ as the cofactor. When tested under identical conditions the phosphonoformate apparent inhibition constants for AMV DNA polymerase were 5 to 10 pM. For the herpes- virus of turkeys induced DNA polymerase, the apparent inhibition 64 constants were 1 to 3 pM (12). AMV DNA polymerase, however, was considerably less sensitive to phosphonoformate inhibition with purified RSV RNA as the template-primer or when the endogenous reaction was monitored with detergent-treated virions (Tables 1 and 2). Since the reaction catalyzed by reverse transcriptase in yiyg might not be as sensitive to phosphonoformate inhibition as that catalyzed by the a-polymerase, the selective inhbition of retrovirus replication by phosphonoformate at concentrations where cell pro- liferation would not be affected might not be realized. Sundquist and Larner (17) however, reported that phosphonoformate inhibited replication of visna virus at concentrations of 20 to 80 pM where cell growth was not affected. In contrast, RSV replication is not inhibited by phosphonoformate when tested at 500 pM (H-J. Kung, unpublished results). Through the use of selective inhibitors, we would like to study the jg_yjyg_roles that reverse transcriptase and cellular DNA polymerases play in the synthesis of viral DNA and its integration into cellular DNA. Perhaps phosphonoformate will not be selective enough in its inhibition to enable us to differentiate the role of reverse transcriptase from that of the a-polymerase. Experiments are underway to decide. Aphidicolin, however, should be useful in defining the role of the a-polymerase in retrovirus replication. Aphidicolin is an effective inhibitor of the a-polymerase but not the B- or v-polymerases (28), and as described in the Results, aphidicolin does not inhibit reverse transcriptase. 65 While this manuscript was in preparation, but after an abstract of our studies had been submitted to the American Society of Biological Chemists for presentation at the 1980 meetings, a report by Sundquist and Oberg (39) appeared describing their studies on the phosphonoformate inhibition of reverse transcriptase of avian and mammalian retroviruses. While their study emphasized the general aspects of phosphonoformate inhibitinn, our study emphasized mechanism. TO. 11. 11. REFERENCES Kornberg, A. 1980. DNA Replication. PP. 434-441, W. H. Freeman, San Francisco. Shipkowitz, N. L., R. R. Bower, R. N. Appell, C. W. Nordeen, L. R. Overby, W. R. Roderick, J. B. Schleicher, and A. M. Von Esch. 1973. Appl. Microbiol. 26; 264-268. Overby, L. R., E. E. Robishaw, J. B. Schleicher, A. Rueter, N. L. Shipkowitz, and J. C-H. Mao. 1974. Antimicrob. Ag. Chemother. g: 360-365. Mao, J. C-H., E. E. Robishaw, and L. R. Overby. 1975. J. Virol. 15: 1281-1283. Boezi, J. A. 1979. International Encyclopedia of Pharmacology and Therapeutics. Volume on Antiviral Chemotherapy. (Shugar, Do, Ed.), 1: 231-243. Overby, L. R., R. G. Duff, and J. C-H. Mao. 1977. Ann. N.Y. Acad. Sci. 284: 310-320. Hay, J., S. M. Brown, A. T. Jamieson, F. J. Rixon, H. Moss, 0. A. Dargan, and J. H. Subak-Sharpe. 1977. J. Antimicrob. Chemother. 3A: 63-70 Mao, J. C-H. and E. E. Robishaw. 1975. Biochemistry 14: 5475-5479. Leinbach, S. S., J. M. Reno, L. F. Lee, A. F. Isbell, and J. A. Boezi. 1976. Biochemistry 15, 426-430. Sabourin, C. L. K., J. M. Reno, and J. A. Boezi. 1978. Arch. Biochem. Biophys. 181: 96-101. Helgstrand, E., B. Eriksson, N. G. Johansson, 8. Lanner6, A. Larsson, A. Misiorny, J5 0. Norén, B. Sjfiberg, K. Stenberg, G. Stening, S. Stridh, B. Oberg, S. Alenius, and L. Philipson. 1978. Science 291: 819-821. Reno, J. M., L. F. Lee, and J. A. Boezi. 1978. Antimicrob. Ag. Chemother. 13: 188-192. 66 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 67 Alenius, 5., Z. Dinter, and B. Oberg. 1978. Antimicrob. Ag. Chemother. 14: 408-413. Kern, E. R., L. A. Glascow, J. C. Overall, J. M. Reno, and J. A. Boezi. 1978. Antimicrob. Ag. Chemother. 14: 817-823. Alenius, S. and H. Norlinder, 1979. Arch. Virol. 60: 197-206. Klein, R. J., E. DeStefano, E. Brady, and A. E. Friedman-Kien. 1979. Antimicrob. Ag. Chemother. 16: 266-270. Sundquist, B. and E. Larner. 1979. J. Virol. 39: 847-851. Haase, A. T. 1975. Curr. Top. Microbiol. Immunol. 12; 101-156. Sigurdsson, B., H. Thorman, and P. A. Palsson. 1960. Arch. Virusforsch 19: 368-381. Kung, H-J., J. M. Bailey, N. Davidson, P. K. Vogt, M. 0. Nicolson, and R. M. McAllister. 1974. Cold Spring Har. Symp. Quant. Biol. 39: 827-834. Nylen, P. 1924. Chem. Berichte 518; 1023-1035. Watson, K. F., P. L. Schendel, M. J. Rosok, and L. R. Ramsey. Biochemistry 15; 3210-3218. Cleland, W. W. 1963. Biochem. Biophys. Acta 61; 104-137. Cleland, W. W. 1963. Biochem. Biophys. Acta 61: 173-187. Eilkinson, G. N. 1961. Biochem. J. ‘89: 324-332. . Seal, 0. and L. A. Loeb. 1976. J. Biol. Chem. 254:975-981. Ohashi, M., T. Taguchi, and S. Ikegami. 1978. Biochem. Biophys. Res. Commun. 82: 1084-1090. Oguro, M., C. Suzuki-Hori, H. Nagano, Y. Mano, and S. Ikegami. 1979. Eur. J. Biochem. 91: 603-607. Verma, I. M. 1975. J. Virol. 15; 121-126. Brewer, L. C. and R. 0. Wells. 1974. J. Virol. 14: 1494-1502. Modak, M. J. and S. L. Marcus. 1977. J. Virol. 22; 243-246. Srivastava, A. and M. J. Modak. 1979. Biochem. Biophys. Res. Comm. 21: 892-899. Modak, M. J. 1976. Biochemistry 15: 3620-3626. 34. 35. 36. 37. 38. 39. 68 Papas, T. S., T. W. Pry, and D. J. Marciani. 1976. J. Biol. Chem. 252: 1425-1430. Knopf, K. W. 1979. Eur. J. Biochem. 28: 231-244. McClure, W. R. and T. M. Jovin. 1975. J. Biol. Chem. 250: 4073-4980. Tanabe, K., E. W. Bohn, and S. H. Wilson. 1979. Biochemistry 18: 3401-3406. King, E. L. and C. Altman. 1956. J. Phys. Chem. 60: 1375-1378. Sundquist, B. and B. Oberg. 1979. J. Gen. Virol. 4i: 273-281. APPENDICES 69 APPENDIX A MECHANISM OF PHOSPHONOACETATE INHIBITION OF HERPESVIRUS-INDUCED DNA POLYMERASE By Susan S. Leinbach John M. Reno Lucy F. Lee A. F. Isbell and John A. Boezi Reprinted from Biochemistry 15: 426 (1976) 7O [Reprinted from Biochemistry, (1976) 15, 426.] Copyright 1976 by the American Chemical Society and reprinted by permission of the copyright owner. Mechanism of Phosphonoacetate Inhibition of Herpesvirus-Induced DNA Polymerase'r Susan S. Leinbach, John M. Reno, Lucy F. Lee, A. F. Isbell, and John A. Boe2i* ABSTRACT: Phosphonoacetate was an effective inhibitor of both the Marek’s disease herpesvirus- and the herpesvirus of turkey-induced DNA polymerase. Using the herpesvirus of turkey-induced DNA polymerase, phosphonoacetate in- hibition studies for the DNA polymerization reaction and for the deoxyribonucleoside triphosphate-pyrophosphate exchange reaction were carried out. The results demon- strated that phosphonoacetate inhibited the polymerase by interacting with it at the pyrophosphate binding site to create an alternate reaction pathway. A detailed mecha- nism and rate equation for the inhibition were developed. For comparison to phosphonoacetate, pyrophosphate inhibi- tion patterns and apparent inhibition constants were deter- mined. Twelve analogues of phosphonoacetate were tested Using a random testing of compounds with a cell culture screen, workers at Abbott Laboratories discovered that phosphonoacetate was an effective inhibitor of the replica- tion of herpes simplex virus types 1 and 2. Three reports, all from Abbott Laboratories, have been published on studies with phosphonoacetate. Shipkowitz et al. (1973) reported that phosphonoacetate, when administered orally or topical- Iy to mice experimentally infected with herpes simplex virus, was able to significantly reduce the mortality associ- ated with the viral infection. Overby et al. (1974) reported that the mode of inhibition of the replication of herpes sim- plex virus by phosphonoacetate appeared to be a result of the inhibition of herpes simplex viral DNA synthesis. Mao et al. (1975) reported that phosphonoacetate was an effec- tive inhibitor of the herpes simplex-induced DNA polymer- ase. The major and minor DNA polymerase activities, pre- sumably a and 13. from the uninfected cells (Wi-38) were not inhibited by phosphonoacetate. Thus, phosphonoacetate probably inhibits herpes simplex DNA synthesis by specific inhibition of the viral induced DNA polymerase. Phosphonoacetate also inhibits the replication of Marek’s disease herpesvirus (MDHV)l and the herpesvirus of tur- keys (HVT) (L. F. Lee et al., manuscript in preparation). MDHV is an oncogenic herpesvirus that causes a highly contagious malignant lymphoma of chickens (Marek, 1907; Churchill and Biggs, 1967; Nazerian et al., 1968; Solomon I From the Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824 (S.S.L., J.M.R., and .I.A.B.), U.S.D.A., Agricultural Research Service, Regional Poultry Research Laboratory, East Lansing. Michigan 48823 (L.F.L.), and the Department of Chemistry, Texas A & M University, College Station, Texas 77843 (A.F.l.). Received August 2], [975. This work was supported in part by National Institutes of Health Research Grant ROICA 17554-01 and National Institutes of Health Training Grant GM-IO91. Michigan Agricultural Station Article No. 7365. ‘ Abbreviations used are: MDHV, Marek‘s disease herpesvirus; HVT, herpesvirus of turkeys. 47A DIAPUCIIICTDV uni 1‘ run 1 101‘ as inhibitors of the herpesvirus of turkey-induced DNA polymerase. At the concentrations tested, only one, 2-phos- phonopropionate, was an inhibitor. The apparent inhibition constant for it was about 50 times greater than the corre- sponding apparent inhibition constant for phosphonoace- tate. DNA polymerase a of duck embryo fibroblasts, the host cell for the herpesviruses, was inhibited by phospho- noacetate. The apparent inhibition constants for the a poly- merase were about 10—20 times greater than the corre- sponding inhibition constants for the herpesvirus-induced DNA polymerase. Duck DNA polymerase B, Escherichia coli DNA polymerase I, and avian myeloblastosis virus re- verse transcriptase were not inhibited by phosphonoacetate. et al., 1968). HVT is used as a vaccine against Marek’s dis- ease (Purchase et al., 1971). In this report, we demonstrate that phosphonoacetate is an effective inhibitor of the DNA polymerase induced by these avian herpesviruses. We report the results of a study of the inhibition patterns produced by phosphonoacetate on in vitro DNA synthesis by the partially purified HVT-in- duced DNA polymerase and propose a mechanism by which the inhibitor works. Finally, we report the results of a study of the effect of some structural analogues of phospho- noacetate on in vitro DNA synthesis by HVT-induced DNA polymerase and examine the effect of phosphonoace- tate on four other DNA polymerases. Materials and Methods Reagents. Phosphonoacetate, disodium salt, was a gift from Abbott Laboratories. 32P-Iabeled sodium pyrophos- phate was purchased from New England Nuclear. Phospho- nopropionate, the trimethyl ester of phosphonoacetate, oz- phenylphosphonoacetate, 2-aminophosphonoacetate, 2- methyl-2-phosphonopropionate, and 2-phosphonopropion- ate were synthesized using published procedures (Chambers and Isbell, 1964; Berry et al., 1972; Isbell et al., 1972). Other reagents were from sources previously described (Boezi et al., 1974), or were from the usual commercial sources. Purification of HVT-Induced DNA Polymerase. The preparation and growth of duck embryo fibroblasts and in- fection with HVT was as previously described (Boezi et al., 1974). The purification of the HVT-induced DNA poly- merase and description of its catalytic and structural prop- erties will be presented in detail elsewhere (manuscript in preparation). In short, however, HVT-induced DNA poly- merase was purified from the nuclear fraction of infected cells by chromatography on phosphocellulose as described by Boezi et a1. (1974). The peak fractions of HVT-induced DNA polymerase activity which eluted from the phospho- HERPESVIRUS-INDUCED DNA POLYMERASE cellulose column at about 0.30 M KC1 were pooled, made 50% (v/v) in glycerol, and stored at -20°C. Only small amounts of HVT-infected duck embryo fibroblasts (about I g wet weight of cells) were used in a single purification pro- cedure and throughout the procedure, bovine serum albu- min was used to stabilize polymerase activity (Boezi et al., 1974). For these reasons, the specific enzymatic activity of HVT-induced DNA polymerase purified through phospho- cellulose chromatography can only be estimated to be about 200 nmol of dNMP incorporated per 30 min per mg of pro- tein and to amount to about a 25-fold purification over the crude nuclear fraction. Further purification was achieved by chromatography on DEAE-cellulose as described by Weissbach et al. (1971). For the experiments reported here, HVT-induced DNA polymerase purified through phospho- cellulose was used. This enzyme fraction, when tested using the standard assay conditions for DNA polymerization con- tained no detectable DNase activity, deoxyribonucleoside triphosphatase activity, or inorganic pyrophosphatase activ- ity. Many of the experiments reported here were also per- formed using the more highly purified HVT-induced DNA polymerase purified through DEAE-cellulose. No differ— ences in the results were seen. Other Polymerases. Escherichia coli DNA polymerase I was purchased from Boehringer Mannheim. AMV reverse transcriptase was a gift of Dr. J. Beard, Life Science Re- search Laboratories. Duck embryo fibroblast DNA poly- merase a from the cytoplasmic fraction and DNA polymer- ase B from the nuclear fraction were purified through DEAE-cellulose as described by Weissbach et al. (1971). MDHV-induced DNA polymerase was purified from the nuclear fraction of infected duck embryo fibroblasts by phosphocellulose chromatography. Assay of the DNA Polymerization Reaction. The stan- dard reaction mixture employed for the HVT- or the MDHV-induced DNA polymerase contained in 200 pl: 50 mM Tris-HCI (pH 8), 1 mM dithiothreitol, 200 mM KCI, 2 mM MgC12, 500 ug/ml of bovine serum albumin, 200 pg/ml of activated calf thymus DNA (DNase I treated, Boezi et al., 1974), 20 uM 3H-labeled deoxyribonucleoside triphosphate (specific radioactivity of 200—1000 cpm/ pmol), 100 pM each of the other three deoxyribonucleoside triphosphates, and DNA polymerase. Incubation was at 37°C for 30 min. Assay of the conversion of 3H-labeled deoxyribonucleoside triphosphate into a trichloroacetic acid insoluble form was as previously described (Boezi et al., 1974). Assay conditions were used so that the rate of DNA polymerization was linear with time and with the amount of DNA polymerase. For the kinetic studies, changes in con- centrations of assay components are as noted in the legends to the figures. In the experiments in which pyrophosphate was added to the reaction mixture as a product inhibitor, supplementary MgClz, in an amount equimolar to the sodium pyrophos- phate added, was used. This supplementary Mng which was in addition to the 2 mM MgClz routinely added to the standard reaction mixture was used to compensate for the chelation of Mg2+ ions by pyrophosphate. The amount of supplementary Mng to be added to the reaction mixtures was determined using the equations described by Moe and Butler (1972). Assay of the dNTP—Pyrophosphate Exchange Reaction. The reaction mixture contained in 200 pl: 50 mM Tris-HCI (pH 8), 1 mM dithiothreitol, 200 mM KCI, 1 mM MgC12, 500 ug/ml of bovine serum albumin, 200 pg/ml of activat- ed calf thymus DNA, 0.1 mM each of the four deoxyri- bonucleoside triphosphates, HVT-induced DNA polymer- ase, and various concentrations (0.14—1.1 mM) of 32P-la- beled sodium pyrophosphate (specific radioactivity of ap- proximately 100 cpm/pmol). In addition to the 1 mM MgC12 routinely added to the reaction mixture, supplemen- tary MgClz, in an amount equimolar to the sodium pyro- phosphate added to the reaction mixture, was used. Incuba- tion was at 37°C for 30 min. The assay measuring the con- version of 32P-labeled pyrophosphate to a Norit-adsorbable form was performed as described by Deutscher and Kornberg (1969). For HVT-induced DNA polymerase, the pyrophosphate exchange reaction was shown to be depen- dent on added enzyme, activated calf thymus DNA, Mg“ ions, and deoxyribonucleoside triphosphates. When assayed using 1.1 mM 32P-labeled sodium pyrophosphate, the reac- tion was found to be linear with time for at least 120 min and was directly proportional to the amount of HVT-in- duced DNA polymerase added to the reaction mixture. The rate of the dNTP-pyrophosphate exchange reaction was about 25% of the rate of the DNA polymerization reaction. Inhibition Patterns. Inhibition patterns and kinetic con- stants were defined according to the nomenclature of Cle- land (l963a,b). Analysis of each reaction mixture was done in duplicate. The data for the double reciprocal plots were evaluated using a computer program based on the method of Wilkinson (1961). For evaluation of the apparent inhibi- tion constants, replots of the intercepts and slopes of the double reciprocal plots were analyzed using a computer pro- gram for least-squares analysis. Results Phosphonoacetate Inhibition of the DNA Polymeriza- tion Reaction Catalyzed by Herpesvirus-Induced DNA Polymerase. Phosphonoacetate was an effective inhibitor of the DNA polymerization reaction catalyzed by MDHV- and by HVT-induced DNA polymerase. The addition of 2-3 pM phosphonoacetate to the standard reaction mixture resulted in a decrease in the rate of the DNA polymeriza- tion reaction by about 50%. Either in the presence or ab- sence of phosphonoacetate, the rate of the reaction was lin- ear for at least 1 hr. Phosphonoacetate Inhibition Patterns for the DNA Po- Iymerization Reaction Catalyzed by H VT-lnduced DNA Polymerase. Phosphonoacetate gave linear noncompetitive inhibition with the four dNTPs as the variable substrate and the activated DNA at a saturating concentration of 200 pg/ml (Figure 1). The apparent inhibition constant (Kn) determined from the replot of the vertical intercepts against phosphonoacetate concentration was 1.5 uM. The apparent inhibition constant (Kis) determined from the replot of the slopes against phosphonoacetate concentration was 1 pM. With activated DNA as the variable substrate, and the four dNTPs at their apparent Michaelis constant concen- trations of 2.5 pM each, phosphonoacetate gave linear non- competitive inhibition (Figure 2). A replot of the vertical intercepts yielded a Ki, of 1.5 pM and a replot of the slopes yielded a K ,5 of 2.5 pM. Phosphonoacetate also gave linear noncompetitive inhibition with activated DNA as the vari- able substrate and with the four dNTPs at 100 uM each. Ki; was determined to be 1.5 pH and Ki, was about 20 pM. The higher Ki, value seen at 100 uM dNTP compared to that seen at 2.5 pM dNTP indicated that the phosphonoa- cetate inhibition pattern was more nearly uncompetitive at —! 8 0.20 - / 2 / I , , 0.151- D l : 02 .1020 V 0/3/ 9 a 0'0b/0 g 3 o E 01 «010) — /8 //’: . . o.os- ./‘ ‘ 2 3 /./ [Phosphonoacetate] ,(pM) h b y. 0.1 072 0:3 0.4 0.5 [dNTPI'Jpr' FIGURE 1: Double reciprocal plots with the four dNTPs as the vari- able substrate and phosphonoacetate as inhibitor. Activated DNA was at 200 ug/ ml. The initial velocities were expressed as pmol of 3H-la- beled dCMP incorporated into DNA per 30 min. Phosphonoacetate concentrations were 0 (0), 0.55 all! (0), 1.65 pM (CI), and 2.75 pH (A). Equimolar concentrations of each of the four dNTPs were present in the different reaction mixtures. The replots of the slopes (O) and in- tercepts (0) as a function of phosphonoacetate concentration are shown in the left panel. A 0.20 - / 6 f I A/ 0.15- o .2 ° 40.40 I / B 0. § 0.15- 3 V / /:/ 9/0 O 5 010 /'-02¢7) . . . 0.10 /: O. 0.0 5% ~1- [Phosphonoacetote] , ( uM ) l 1 l l 0.05 0. I0 0.15 0.20 [Activated DNA]",(pg/m1)" FIGURE 2: Double reciprocal plots with activated DNA as the variable substrate and phosphonoacetate as inhibitor. The four dNTPs were at 2.5 uM each. Phosphonoacetate concentrations were 0 (0), 0.55 Mr! (O), 1.1 all! (0), and 2.2 all! (A). the higher concentration of dNTP than it was at the lower concentration. Phosphonoacetate Inhibition Pattern for the dNTP-Py- rophosphate Exchange Reaction Catalyzed by HVT-In- duced DNA Polymerase. Since phosphonoacetate and pyro- phosphate have structural features in common, it was sus- pected that phosphonoacetate might be inhibiting the DNA polymerization reaction by interacting with the polymerase at the pyrophosphate binding site. If so, phosphonoacetate should be a competitive inhibitor of pyrophosphate in the dNTP-pyrophosphate exchange reaction. This was the case (Figure 3). The apparent Ki, value for phosphonoacetate was 1.3 pM. The apparent Km value for pyrophosphate was 0.24 mM. Pyrophosphate Inhibition Patterns for the DNA Poly- merization Reaction Catalyzed by H VT-Induced DNA Polymerase. For comparison to the phosphonoacetate inhi- bition patterns and apparent K: values, inhibition studies using pyrophosphate were performed. With the four dNTPs as the variable substrate, pyrophosphate gave linear non- competitive inhibition. K5; was 1.3 mM and Ki, was 0.7 mM. With activated DNA as the variable substrate and with the four dNTPs at 2.5 uM each, pyrophosphate gave 42x BIOCHEMISTRY v01 15 no 7 1076 LEINBACH ET AL. 1 0.02 '0’ .06 - E 0.10 04 -/ - 0.01 2 n (D .o Intercept. O _O O 0.0 5 [Phosphonoacetate] ,( pH) 2 4 e a [Pyrophosphate lthi' FIGURE 3: Double reciprocal plots of the dNTP-pyrophosphate ex- change reaction with pyrophosphate as the variable substrate and phos- phonoacetate as inhibitor. The initial velocities were expressed as pico- moles of 32P-Iabeled pyrophosphate converted to a Norit-adsorbable form per 30 min. Phosphonoacetate concentrations were 0 (0), 2 pH (0), and 3 pH (0). linear noncompetitive inhibition. K“ was 0.95 mM and Ki, was 1.7 mM. Pyrophosphate gave linear uncompetitive in- hibition with activated DNA as the variable substrate and with the four dNTPs at 20 all! each. K ii was about 0.9 mM. Inhibition by Structural Analogues of Phosphonoace- tate. When tested at a concentration of 200 pM, the fol- lowing analogues of phosphonoacetate produced no signifi- cant inhibition of either the polymerization reaction or of the dNTP—pyrophosphate exchange reaction catalyzed by HVT-induced DNA polymerase: methylene diphosphonate, malonate, phosphoglycolate, sulfoacetate, phosphonopro— pionate, amino methyl phosphonate, a-amino ethyl phos- phonate, trimethyl ester of phosphonoacetate, a-phenyl- phosphonoacetate, 2-aminophosphonoacetate, and 2- methyl-2-phosphonopropionate. 2-Phosphonopropionate was an inhibitor of the polymerization reaction and of the pyrophosphate exchange reaction. As determined in the dNTP-pyrophosphate exchange reaction, the apparent Ki, for 2—phosphonopropionate was about 50 pH. The Effect of Phosphonoacetate on the DNA Polymer- ization Reaction Catalyzed by other DNA Polymerases. DNA polymerase a, but not 6, from uninfected duck em- bryo fibroblasts, was inhibited by phosphonoacetate. The inhibition patterns with the a polymerase for the DNA po- lymerization reaction were similar to those produced with the HVT-induced polymerase, but the apparent K; values were 10-20 times greater. DNA polymerase B, when tested to a phosphonoacetate concentration of 200 pM, was not significantly inhibited. Likewise, neither E. coli DNA poly- merase I nor AMV reverse transcriptase was significantly inhibited. Discussion The results of our study are consistent with the mecha- nism of phosphonoacetate inhibition presented in Figure 4. We propose that in the presence of phosphonoacetate an al- ternate pathway exists in addition to the basic polymeriza- tion pathway. Phosphonoacetate binds to the polymerase at the pyrophosphate binding site and is, thus, a competitive inhibitor of pyrophosphate in the exchange reaction. Phos- phonoacetate may simply dissociate from the EpADNMM” complex or may undergo reaction with the nucleotide at the 3’-end of the DNA primer chain to yield the postulated nu- cleotide, dNMP-PA, and EDNA. Thus, phosphonoacetate inhibition occurs because the EDNAIM” complex is diverted by phosphonoacetate from the main polymerization path- way into an alternate pathway. HERPESVIRUS-INDUCED DNA POLYMERASE For such a reaction scheme, the rate equation describing DNA synthesis in the presence of DNA, dNTP, and phos- phonoacetate is given by eq 1, where KDNA, KiDNA and KDNAI, KiDNAI refer to DNA binding as substrate and product, respectively.2 v/E, = [V1(DNA,dNTP)(DNA)(dNTP) — V2(DNA.PA)(KdNTPK iDNA/ K iDNA’K PA) X (DNA)(PAll/IKiDNAKdNTP + KDNA(dNTP) + KdNTP X (1 + KiDNA/KIDNA’)(DNA) + (1 + KDNA/KiDNA’) X (DNA)(dNTP)]-I- (KdNTPKiDNA/KPAKiDNA’)[KDNA’ + (1 + KDNA’/KIDNA)(DNA)I(PA) + [(l/KiPA) + (KdNTpKiDNA/KidNTPKiDNA'KPA)l X (DNA)(dNTP)(PA)] (1) Under the experimental conditions that we have used, the numerator of eq 1 is approximated by the VHDNAANTP) (DNA)(dNTP) term. The reciprocal of eq 1, arranged with DNA as the variable substrate, takes the form shown in eq 2. At moderate concentrations of dNTP, both the slope and the intercept terms are a function of phosphonoacetate con- centration, and the inhibition pattern is noncompetitive. At high concentrations of dNTP, however, the slope term is in- dependent of phosphonoacetate concentration, and the inhi- bition pattern is uncompetitive. E l K- K ___g = [(KDNA + IDNA dNTP+ v VI(DNA,dNTP) (dNTP) KIDNAKDNA’KdNTP(PA)) 1 + ( KDNA) + 1 + — KiDNA'KPA(dNTP) DNA KiDNA' K i dNTP (1+KDNA)+( 1 + (dNTP) KiDNA' KiPA KiDNAKdNTP K iDNA’K idNTPK PA KiDNAKdNTP < K DNA’ 1+—)) PM] (2) KiDNA’KPA(dNTP) KiDNA ( Our results are consistent with the predictions of eq 2. With activated DNA as the variable substrate and with the dNTP concentration at K m levels, noncompetitive inhibition was observed (Figure 2). With the dNTP concentration at 4OKm, the inhibition pattern was more nearly uncompeti- tive. If eq 2 is rearranged with dNTP as the variable sub- strate, the prediction emerges that the phosphonoacetate in- hibition pattern will be noncompetitive regardless of the concentration of DNA. With dNTP as the variable sub- strate and with activated DNA concentration at about 20km, the inhibition pattern was noncompetitive (Figure 1). Again, our results are consistent with the prediction from the rate equation. Our results are not consistent with phosphonoacetate being a simple dead-end inhibitor in which it could only bind and dissociate from the EDNMM” complex. For the case of a dead-end inhibitor, the rate equation3 predicts that with DNA as the variable substrate the phosphonoacetate 2 The steady-state rate equation was first written in terms of individ- ual rate constants by the method of King and Altman (1956). It was then transformed into a rate equation in terms of kinetic constants as described by Cleland (1963a). Using these procedures, we have also derived and verified the rate equation presented by McClure and Jovin (1975) for the modified ordered bi-bi mechanism. 3 The rate equation for the dead-end inhibitor was derived by the method described by Cleland (1963b). [IONA Ev “z EDNA A ‘tI‘NM""A//‘i:l DNA I7 I'DNA E‘N'c'.PA I4 I’dNTP DNAt-m PA t bum..." “o"i . Earle E ‘ I, DNAt-m "i FIGURE 4: Proposed mechanism of phosphonoacetate inhibition of herpesvirus-induced DNA polymerase. The basic reaction mechanism in the absence of phosphonoacetate (PA) is a modified ordered bi-bi mechanism (Kornberg, I969; McClure and Jovin, 1975). Initial veloci- ty studies (S. S. Leinbach et al., unpublished data) and the pyrophos- phate product inhibition studies presented here are consistent with this mechanism for the HVT-induced DNA polymerase. The postulated compound, dNMP-PA, is a deoxyribonucleoside S’-monophosphate co- valently linked to phosphonoacetate by a phosphodiester bond. inhibition pattern will be uncompetitive regardless of the concentration of dNTP. However, as shown in Figure 2, at Km levels of dNTP, the inhibition pattern which we ob- served was noncompetitive. To verify our proposed scheme, the postulated nucleotide, dNMP-PA, must be identified in the reaction mixtures. Our first attempts to identify the nucleotide have not proved successful. When unlabeled phosphonoacetate and labeled DNA were used as substrates for the reverse reac- tion to polymerization, no labeled nucleotide was detected. The reverse reaction to polymerization, however, apparently goes very poorly since labeled nucleotide (dNTP) was not detected in reaction mixtures which contained either unla- beled pyrophosphate and labeled DNA or labeled pyrophos- phate and unlabeled DNA as the substrates. Success in identifying the nucleotide is more likely to come from stud- ies using radioactive phosphonoacetate of high specific ac- tivity as a substrate in an exchange-type reaction with unla- beled dNTP. Such studies are now being pursued. In our proposed scheme, both phosphonoacetate and py- rophosphate inhibit DNA synthesis in an analogous man- ner. Indeed, the inhibition patterns for these two com- pounds are similar. The apparent inhibition constants for pyrophosphate, however, are two to three orders of magni- tude greater than those for phosphonoacetate. The studies with the analogues of phosphonoacetate give us some information about the structural requirements for binding at the HVT-induced DNA polymerase pyrophos- phate binding site. The results demonstrate that the carbon chain of the phosphono compound must be of specific chain length, that a carboxyl or sulfo group cannot substitute for the phosphono group, that the methylene carbon cannot have bulky or charged substituents, and that an amino, a methyl amino, or a phosphono group cannot substitute for the carboxyl group. Since phosphonoacetate seems to be a general inhibitor of the DNA polymerases induced by the herpesviruses, the pyrophosphate binding site for this group of DNA polymer- ases must be quite similar. The pyrophosphate binding site of DNA polymerase a of ducks appears to be somewhat similar to this site. The pyrophosphate binding sites of DNA polymerase 13 of ducks, E. coli DNA polymerase I, AMV reverse transcriptase, and the a and B polymerases of human Wi—38 cells (Mao et al., 1975), however, appear to be different from this site on the herpesvirus-induced DNA polymerase. References Berry, J. P., Isbell, A. F., and Hunt, G. E. (1972), J. Org. Chem. 37, 4395. Boezi, J. A., Lee, L. F., Blakesley, R. W., Koenig, M., and Towle, H. C. (1974), J. Virol. 14, 1209. Chambers, .1. R., and Isbell, A. F. (1964), J. Org. Chem. 29, 832. Churchill, A. E., and Biggs, P. M. (1967), Nature (lon- don) 215, 528. Cleland, W. W. (l963a), Biochim. Biophys. Acta 67, 104. Cleland, W. W. (I963b), Biochim. Biophys. Acta 67, 173. Deutscher, M. P., and Kornberg, A. (1969), J. Biol. Chem. 244, 3019. Isbell, A. F., Berry, J. P., and Tansey, L. W. (1972), J. Org. Chem. 37, 4399. King, E. L., and Altman, C. (1956), J. Phys. Chem. 60, DOURLENT AND HOGREL 1375. Kornberg, A. (1969), Science 163, 1410. Mao, .I. C.-H., Robishaw, E. E., and Overby, L. R. (1975), J. Virol. I5, 1281. Marek, J. (1907), Dtsch. Tieraerztl. Wochenschr. I 5, 417. McClure, W. R., and Jovin, T. M. (1975), J. Biol. Chem. 250, 4073. Moe, O. A., and Butler, L. G. (1972), J. Biol. Chem. 247, 7308. Nazerian, K., Solomon, J. J., Witter, R. L., and Burmester, B. R. (1968), Proc. Soc. Exp. Biol. 127, 177. Overby, L. R., Robishaw, W. E., Schleicher, J. B., Rueter, A., Shipkowitz, N. L., and Mao, J. C.-H. (1974), Anti- microb. Agents C hemother. 6, 260. Purchase, H. G., Witter, R. L., Okazaki, W., and Burmest- er, B. R. (1971), Perspect. Virol. 7, 91. Shipkowitz, N. L., Bower, R. R., Appell, R. N., Nordeen, C. W., Overby, L. R., Roderick, W. R., Schleicher, .l. B., and VonEsch, A. M. (1973), Appl. Microbiol. 27, 264. Solomon, J. J., Witter, R. L., Nazerian, K., and Burmester, B. R. (1968), Proc. Soc. Exp. Biol. 127, 173. Weissbach, A., Schlabach, A., Fridlender, B., and Bolden, A. (1971), Nature (London), New Biol. 231, 167. Wilkinson, G. N. (1961), Biochem. J. 80, 324. APPENDIX B TREATMENT OF EXPERIMENTAL HERPESVIRUS INFECTIONS WITH PHOSPHONOFORMATE AND SOME COMPARISONS WITH PHOSPHONOACETATE By Earl R. Kern L. A. Glasgow J. C. Overall John M. Reno and John A. Boezi Reprinted from Antimicrobial Agents and Chemotherapy 15:817 (1978) 76 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Dec. 1978, p.817-823 0066-4804/78/0014-081730200/0 Copyright © 1978 American Society for Microbiology Vol. 14, No.6 Printed in U. S.A. Treatment of Experimental Herpesvirus Infections with Phosphonoformate and Some Comparisons with PhosphonoacetateT EARL R. KERN," LOWELL A. GLASGOW,l JAMES C. OVERALL, JR.,' JOHN M. RENO,2 AND JOHN A. BOEZI2 Department of Pediatrics, University of Utah College of Medicine, Salt Lake City, Utah 84132,l and Department of Biochemistry, Michigan State University, East Lansing, Michigan 488232 Received for publication 18 September 1978 Phosphonoformate (PF) at a concentration of 5 to 10 ug/ ml inhibited the growth of type 1 strains of herpes simplex virus (HSV) in tissue culture, whereas 20 to 30 ug/ml was required for inhibition of type 2 strains and about 50 ug/ml was required for murine cytomegalovirus. In mice inoculated intraperitoneally or intracerebrally with HSV or intraperitoneally with murine cytomegalovirus, treatment with 250 to 400 mg of PF per kg twice daily for 5 days had only minimal effectiveness. When mice were inoculated intravaginally (i.vg.) with HSV type 2 and treated i.vg. with 10% PF beginning 3 h after viral inoculation, treatment was effective in completely inhibiting viral replication in the genital tract. If i.vg. therapy was initiated 24 h after infection, when the mice had a mean virus titer of 10° plaque-forming units in vaginal secretions, a significant reduction in the mean virus titer was observed on days 3, 5, and 7 after infection as compared with control animals. In guinea pigs treated i.vg. with 10% PF beginning 6 h after i.vg. inoculation with HSV type 2 there was also complete inhibition of viral replication in the genital tract, and no extenal lesions developed. When therapy was initiated 24 h after infection there was a 4 to 5—Iog decrease in viral titers on days 3, 5, and 7 of the infection and a slight delay in the development of external lesions. Phosphonoacetate (PA) has been reported to be active against herpes simplex virus (HSV) types 1 and 2 and both human and murine cytomegalovirus (MCMV) replication in tissue culture (7, ll, 13) and to be effective when applied topically to treat skin or mucous mem- brane HSV infections of animals (2, 3, 7, 10, 17). The compound has been less effective, however, when administered systemically to HSV-or MCMV-infected animals (1, 2, 7—9, 11). Because PA accumulates in the bone of a variety of experimental animals it has not been considered for use in humans (B. A. Bopp, C. B. Estep, and D. J. Anderson, Fed. Proc. 36: 939, 1977). The efficacy of this drug in herpesvirus infections of animals has generated sufficient interest that a number of analogs have been synthesized in the hope that modification of the parent compound might result in maintenance of antiviral activity with a reduction in toxicity. One of these ana- logs, phosphonoformate (PF), has been reported to be as active as PA in inhibiting the replication of HSV, Marek’s disease herpesvirus, and her- 1’ Publication no. 37 from the Cooperative Antiviral Testing Group, Development and Applications Branch, National In- stitute of Allergy and Infectious Diseases, Bethesda, MD 20014. 817 pesvirus of turkeys (16). The mechanism of ac- tion also appears to be similar to PA, that is, inhibition of viral DNA polymerase. The purpose of our experiments was to deter- mine the antiviral activity of PF in tissue culture and in experimental herpesvirus infections of animals that are models of human disease and to compare its activity with that of PA. The experimental viral infections utilized were: (i) intraperitoneal (i.p.) or intracerebral (i.c.) inoc- ulation of mice with HSV type 2, models of herpes encephalitis; (ii) i.p. inoculation of mice with MCMV, a model for disseminated cyto- megalovirus infection; and (iii) intravaginal (i.vg.) inoculation of mice or guinea pigs with HSV type 2, models for genital herpes. With the exception of the guinea pig infection, the path- ogenesis of these experimental infections has been reported in detail in previous publications (8, ll, 12). Due to the lack of availability of PA, the antiviral activities of PA and PF were com- pared directly only in tissue culture and in the genital infections of mice and guinea pigs. MATERIALS AND METHODS Animals and virus inoculation. Three-week-old female Swiss Webster mice (Simonsen Laboratories, 818 Gilroy, Calif.) were inoculated by the i.p. route with 2 x 10‘ plaque forming units (PFU) of HSV or 10‘ PFU of MCMV, which usually resulted in 80 to 100% mor- tality. Two-week-old mice were inoculated i.c. with 2 to 5 PFU of HSV, which resulted in 95 to 100% mortality. Seven-week-old female mice or 200-g female guinea pigs (Charles River Breeding Laboratories, Inc., Wilmington, Mass.) were inoculated i.vg. with 10‘5 or 10‘ PFU of HSV type 2, respectively, using a plastic catheter attached to a syringe. These concentrations of virus usually resulted in a 70 to 90% genital infection rate. Viruses, media, cell cultures, and virus assay. The McIntyre, E-377, MS, E-326, and X-79 strains of HSV were obtained from Andre Nahmias, Emory Uni- versity, Atlanta, Ga. Strain E-196 was from Harold Haines, University of Miami, Miami, Fla. The strains designated HL-3, HL-34, Wilson, and Heeter were isolated in our laboratory from patients with oral or genital lesions. These strains were typed using a mod- ification of the chick embryo microtest as described by Yang et al. ( 19). The origin and preparation of pools of the Smith strain of MCMV and vaccinia virus have been previously described (4, 5). The media utilized, preparation of cell cultures, and assay for HSV have been described in a previous publication (6). Antiviral agents. The disodium salt of PA (mo- lecular weight 184) was provided by Abbott Labora- tories, North Chicago, Ill. The trisodium PF hexahy- drate (molecular weight 300) was synthesized in the Department of Biochemistry laboratories, Michigan State University, East Lansing, Mich. (16). Both PA and PF were dissolved in phosphate-buffered saline. In addition, a 5% PA cream and a 10% suspension of PF in 0.4% agarose were utilized. All drug solutions were prepared just before use and administered i.p. or i.vg. in a volume of 0.1 ml. Susceptibility of viral strains to PF and PA. The susceptibility of a number of HSV strains and of MCMV and vaccinia virus to the two drugs was deter- mined by a 50% plaque-reduction assay as described previously (6). Serial twofold dilutions of each drug in twice-concentrated minimal essential medium were mixed with an equal volume of 1.0% agarose. The overlay mixture containing drug was added to mono- layer cultures of mouse embryo fibroblast (MEF) cells 1 h after inoculation with about 50 PFU of each of the virus strains. Assay for virus in vaginal secretions. Vaginal swabs for isolation of HSV were obtained on days 1, 3, 5, 7, and 10 after inoculation. The swabs were placed in 1.0 ml of tissue culture medium, and frozen at -70°C until assayed for virus on fetal lamb kidney cells. Viral titers are expressed as PFU per milliliter of tissue culture medium. To rule out carry-over of drug from the genital tract to the viral assay system, all swabs were collected 12 h after treatment. Samples from drug-treated animals were also tested in vitro for antiviral activity. No evidence of drug in sufficient concentration to inhibit HSV replication was detected in the vaginal swabs tested. Statistical evaluation. For comparison of final mortality rates in untreated and drug-treated mice, the data were evaluated by the Fisher exact test. The KERN ET AL. AN'runcnos. AGENTS Gunmen. Mann-Whitney U test was used for comparing the mean day of death between untreated and drug- treated animals. The virus titer-day area under the curve and the lesion score-day area under the curve were generated through the use of a computer pro- gram. These data from untreated and drug-treated animals were compared also by the Mann-Whitney U test. For all statistical analyses, a P value of (0.05 was considered to be significant. RESULTS Susceptibility of viral strains to PA and PF in vitro. The susceptibility of five strains of HSV type 1, five strains of HSV type 2, MCMV, and vaccinia virus to the two drugs was deter- mined in MEF cells. The mean values from two separate experiments are listed in Table 1. Since the two compounds have different molecular weights, the 50% inhibitory levels are expressed both in micrograms per milliliter and millimolar concentration. Most of the type 1 strains of HSV were inhibited by 4 to 10 pg of PA per ml and 5 to 15 pg of PF per ml. The type 2 strains required lOtoZOpgofPApermland20t030pgofPF per ml for 50% inhibition. When the inhibitory levels of the two drugs are compared on an equimolar basis, however, there is little difier- ence between the susceptibility of either virus type to PA or PF. The type 1 strains were about twofold more sensitive than type 2 strains to both compounds. Another member of the her- pesvirus group, MCMV, was inhibited by about 15 pg of PA per ml and about 50 pg of PF per ml. Vaccinia virus, another DNA virus, was not inhibited by concentrations of 50 to 100 pg of either drug per ml. Effect of treatment with PF on mortality of mice inoculated i.c. or i.p. with HSV type 2. After i.c. inoculation, untreated mice became paralyzed on days 5 to 7 and died on days 6 to 8. The effect of treatment with 400 mg of PF per kg given i.p. twice daily for 5 days is summarized in Table 2. In experiment 1 the virus control group treated with phosphate-buffered saline had a final mortality of 91% and a mean day of death of 6.6 days. Treatment with PF initiated 2, 24, or 48 h after virus inoculation had no effect on final mortality, but did significantly increase the mean day of death. Similar data were ob- tained in experiment 2. After i.p. inoculation, untreated mice devel- oped ruffled fur and hunching on days 5 to 7, and most of the animals died between 7 and 12 days after viral challenge. The results of treat- ment in this model infection with 250 to 300 mg of PF per kg i.p. twice daily for 5 days are summarized in Table 3. In experiment I treat- ment initiated 2 or 24 h after infection was effective in reducing final mortality. In the sec- VOL. 14, 1978 ANTI-HERPESVIRUS ACTIVITY OF PHOSPHONOFORMATE 819 TABLE 1. Susceptibility of type 1 and 2 strains of HSV, vaccinia virus and MCM V to PA and PF in MEF cells 50% inhibitory levels“ Virus strains ug/ml 1 SD mM PA PF PA PF HSV type 1 McIntyre 30.8 :t 8.2 78.1 i: 31.0 0.167 0.260 E-377 4.6 :t 2.2 8.4 :1: 2.1 0.025 0.028 HL-3 3.6 i 0.9 4.9 :t 2.7 0.020 0.016 HL34 6.1 :t 4.4 13.6 a: 5.1 0.033 0.045 Wilson 4.1 :t: 1.6 8.0 :L- 0.1 0.022 0.027 HSV type 2 MS 15.8 t 10.5 32.8 :t 7.5 0.086 0.110 E-326 12.9 :t 4.2 26.9 :r 4.4 0.070 0.090 X-79 15.6 :t 8.3 31.0 :t 7.0 0.085 0.103 E-196 13.1 i 6.3 20.5 :t 0.7 0.071 0.068 Heeter 13.5 :t 5.0 22.5 :t 0.07 0.073 0.075 MCMV 15.1 57.0 0.082 0.190 Vaccinia >50.0 >50.0 “ Determined by plaque reduction assay. SD, Standard deviation. TABLE 2. Effect of treatment with PF on the mortality of 2-week-old mice inoculated i.c. with HS V type 2 Mortality Treatment‘l MDD‘ No.‘ % Experiment 1 PBS 20/22 91 6.6 PF +2 h 17/20 85 8.9" +24 h 16/ 19 84 7.4‘ +48 h 18/19 95 7.6' Experiment 2 PBS 20/ 20 100 5.4 PF +2 h 18/ 18 100 9.3" +24 h 20/20 100 6.8’ +48 h 19/20 95 7.1" Drug control 1/ 10 10 ° Treatment was 400 mg of PF per kg i.p. twice daily for 5 days. b MDD, Mean day of death. ‘ Number of mice dead/number treated. d P < 0.001. ‘ P < 0.01. and experiment, where the dosage of drug was slightly increased, only therapy begun 2 h after viral inoculation reduced mortality. Effect of treatment with PF on mortality of mice inoculated i.p. with MCMV. To de- termine the effectiveness of treatment with PF in another disseminated viral infection, mice were inoculated i.p. with MCMV. Control ani- mals exhibited diminished activity, ruffled fur, and hunching by day 3, and most animals died between days 5 and 7. The results of treatment with 250 mg of PF per kg i.p. twice daily for 5 days are listed in Table 4. In experiment 1 only treatment begun 2 h after infection effectively reduced mortality; no reduction at any of the time periods was observed in the second exper- iment. Effect of i.vg. treatment with PF on a genital HSV type 2 infection of mice. Genital TABLE 3. Effect of treatment with PF on the mortality of weanling mice inoculated i.p. with HSV type 2 Mortality Treatment MDD“ No.“ % Experiment 1" PBS 13/ 15 87 11.0 PF +2 h 1/13 8" 12.0 +24 h 6/ 15 40' 10.8 +48 h 8/ 15 53 11.5 Experiment 2’ PBS 14/ 14 100 8.0 PF +2 h 1/12 8" 5.0 +24 h 12/15 80 7.9 +48 h 9/ 12 75 10.0‘I Drug control 1/ 13 8 ° MDD, Mean day of death. b Number of mice dead/number treated. ‘ 250 mg of PF per kg i.p. twice daily for 5 days. 0' P < 0.001. ' P < 0.05. ’ 300 mg of PF per kg i.p. twice daily for 5 days. ‘ P < 0.01. 820 KERN ET AL. TABLE 4. Effect of treatment with PF on the mortality of weanling mice inoculated i.p. with MCM V Mortality Treatment“ MDD" No.‘ % Experiment 1 PBS 10/ 15 67 5.9 PF +2 11 3/15 20" 5.0 +24 h 6/ 15 40 7.0 +48 h 6/15 40 6.5 Experiment 2 PBS 12/15 80 5.5 PF +2 h 9/15 60 5.6 +24 h 9/15 60 6.9 +48 h 7/ 15 47 7.4“ Drug control 1/ 15 7 " 250 mg of PF per kg i.p. twice daily for 5 days. " MDD, Mean day of death. ‘ Number of mice dead/number treated. d P < 0.05. Auriuicaos. AGENTS Casual-HER. HSV infections of humans are one of the major areas of emphasis for viral chemotherapy. Since PA has been one of the most effective com- pounds we have tested in our mouse model of genital HSV infection, we next evaluated PF in this infection. The effect of treatment with 10% PF suspended in 0.4% agarose administered i.vg. twice daily for 7 days on viral replication in the genital tract is illustrated in Fig. 1. Treatment was begun either 3 or 24 h after viral inoculation. A group of mice that received a 5% PA cream preparation beginning 3 h after infection was also included. In the untreated control group the geometric mean titer of virus in vaginal secre- tions reached levels of about 10‘ PFU/ml on days 1, 3, and 5 and dropped to about 10" PFU/ml on day 7. Similar titers of virus were detected in animals treated with the agarose placebo. Treatment i.vg. with PA or PF initiated 3 h after viral inoculation was highly successful in preventing viral replication in that virus was TREATMENT OF GENITAL HERPES SINPLEX VIRUS (HSVI INFECTION OF MICE WITH 5". PA m IOVoPF 1 'I' I ‘ o o 1 - o . d ’ a 8 .. soon HSV Titer Loon pin/ml H O 3 3 I O as; 7.; 3.0< - 1'3. '0 «o _. O O I.o-______ - ____________ ”FIT -uasas III ITfiI [I357 57 Days Post Infection I 3 19F+24h1vg FIG. 1. Treatment of genital HS V type 2 infection of mice with PA or PF. Treatment by the i.vg. route with 5% PA or 10% PF, twice daily for 5 days, was initiated at 3 or 24 h after infection. Symbols: (C) virus titer for each animal; (A—Ngeometric mean HS V titer of all animals for each day sampled. VOL. 14, 1978 isolated from none of the PA-treated mice and from only one of those that received PF. Treat- ment with PF initiated 24 h after infection, when 14 of 15 animals were infected with a mean titer of virus of 10"5 PFU / ml, reduced the number of infected mice to 10 of 15 with a mean viral titer of 102 PFU/ ml. To compare statistically the differences in mean viral titers between untreated and drug- treated animals, we utilized a computer program to calculate the mean virus titer-day area under the curve (18). The mean areas for each group were then compared by the Mann-Whitney U test (Table 5). Treatment either with PA at 3 h or with PF at 3 or 24 h after viral inoculation significantly reduced viral replication in the gen- ital tract. The placebo had no effect. The final mortality rate of the infected ani- mals in the previous experiment was reduced from 64 and 93% in the control and placebo- treated groups, respectively, to 0% in the groups treated with PA or PF at 3 h after viral inocula- tion. Therapy with PF was not effective in re- ducing mortality (64%) when initiated 24 h after infection. Development of a genital HSV type 2 in- fection of guinea pigs and treatment with PF. The use of a genital infection of mice as a model for human herpes genitalis has two major drawbacks: (i) most infected mice develop an acute encephalitis and die within 10 days, so long-term follow-up is not possible, and (ii) with the virus strains we have utilized no external lesions develop, so we are unable to assess the efficacy of therapy on the course of lesion devel- ANTI-HERPESVIRUS ACTIVITY OF PHOSPHONOFORMATE 821 opment. To develop a model which more closely resembles HSV genitalis in humans, we inocu- lated 200-g guinea pigs i.vg. with the MS strain of HSV type 2. After inoculation with this strain, there is a pattern of viral replication in the genital tract similar to that observed in mice and, more importantly, lesions appear on the external genitalia beginning 3 to 4 days after i.vg. inoculation. Lesions were scored by a single investigator throughout the experiment accord- ing to the scale: 1+, redness or swelling; 2+, a few small vesicles; 3+, several large vesicles; 4+, several large ulcers and maceration. A declining score was used during the healing stage. The mean lesion score for all the animals was calcu- lated for each day of observation. The effect of i.vg. treatment with 10% PF suspended in 0.4% agarose is shown in Fig. 2. Treatment was initiated 6 or 24 h after viral inoculation and continued twice daily for 7 days. In the first experiment (Fig. 2, upper row) the untreated HSV infected animals had peak mean vaginal viral titers of 105 PFU/ ml on day 1; these gradually declined through day 10. External le- sions first appeared on day 4, and peak mean lesion scores were observed on days 8 to 10. Similar results were obtained in the agarose placebo-treated group. When treatment with PF was begun 6 h after viral inoculation, there was complete inhibition of viral replication in the genital tract of most animals, and no external lesions developed. In the second experiment (Fig. 2, bottom row), the untreated virus control and the placebo-treated animals followed the same course as described above. If i.vg. treat- TABLE 5. Area under the curve analysis“ Experimental groups Mean area under the curve Animals Group Vaginal virus titers Lesion scores Mice Control 26.0 Placebo +3 h 27 .5" PA +3 h 0‘ PF +3 h 0.1” PF +24 h 18.9c Guinea pigs Experiment 1 Control 34.0 25.3 Placebo +6 h 333" 21.9” PF +6 h 3.6” 0‘ Experiment 2 Control 30.0 25.3 Placebo +24 h 29.3” 20.3” PF +24 h 13 1‘ 18.2“ ‘ Area under the curve analysis for comparison of mean vaginal virus titers and mean external lesion scores between untreated and drug-treated mice and guinea pigs inoculated intravaginally with HSV type 2. ’ Placebo not significantly different from untreated control. ‘ P < 0.001 when compared with placebo. “' Not significantly different from placebo; P - 0.05 when compared with untreated control. 822 KERN ET AL Ammrcaoa. Acaurs CHEMOTHER- TREATMENT OF GENITAL HERPES SIMPLEX VIRUS (HSV) INFECTION OF GUINEA PIGS WITH IO'lo PHOSPHONOFORMATE (PH 9‘ O ,u o 'o Moon HSV “for Log” ptu/Inl H " a (4+ -‘ : . o L3§ c- . b2+ +_ - --' -- _—I+ .5 + ‘0. Moon Lesion Score H ‘t‘ 3 5 7 I2 3 5 I HSV Control I PIaccbo+24II PF+24II Days Post Infection FIG. 2. Treatment of genital HS V type 2 infection of guinea pigs with PF: Treatment by the i. vg. route was initiated at 6 or 24 h after infection with 10% PF twice daily for 7 days. Symbols: I.) virus titer for each animal; (A—A) geometric mean HSV titer of all animals for each day sampled; H mean score of external lesions. ment with PF was initiated at 24 h after infec- tion, when 9 of 10 animals were infected with a mean viral titer of 10”, there was a reduction in the number of infected animals to four, greater than a 4-log decrease in mean titers of virus on day 3, and a slight delay in the development of lesions. In other experiments, treatment with PA produced similar results (data not pre- sented). Values for the mean virus titer-day area under the curve and mean lesion score-day area under the curve for each group were calculated and analyzed for statistical significance (Table 5). Treatment with PF, initiated either 6 or 24 h after infection, significantly altered viral repli- cation in the genital tract, but only early treat- ment had an effect on lesion development. DISCUSSION The minimal inhibitory levels of PF deter- mined in our plaque reduction assay for several type 1 and type 2 strains of HSV and for MCMV are similar to those reported for PA by us and other investigators (7, 11, 13). When compared on a micrograms-per-milliliter basis, the viruses tested were generally about twofold more sus- ceptible to PA than PF; however, when com- pared on a millimolar basis, little difference was noted. In mice inoculated i.c. or i.p. with HSV or i.p. with MCMV, treatment with PF was successful only if begun 2 to 24 h after viral inoculation. In these same animal model infections, treatment with PA has also been effective only with early treatment (1, 2, 8, 11). Therefore, with systemic administration for HSV and MCMV infections of mice, PF appeared to be about as effective as PA. We have reported previously that in mice inoculated with HSV by the i.c. or i.p. route treatment with either adenine arabinoside or adenine arabinoside 5’-monophosphate was ef- fective in preventing mortality when therapy was initiated 48 to 96 h after viral inoculation (8). From these data it appears that both PA and PF were less effective than adenine arabi- noside or adenine arabinoside 5’-monophos- phate in these experimental infections. In a gen- ital HSV infection of mice, i.vg. treatment with PA initiated 3 or 24 h after viral inoculation was effective in preventing or reducing HSV repli- cation in the genital tract (7). Treatment i.vg. with PF was also highly effective in altering HSV replication in the genital tract of both mice VOL. 14, 1978 and guinea pigs when administered as late as 24 h after viral inoculation. Additionally, early treatment (6 h after challenge) was effective in preventing the development of external lesions in guinea pigs. In contrast, treatment initiated later in the course of infection in guinea pigs, i.e., 24 h after infection, failed to retard the development of the lesions. In both genital HSV model infections PF appeared to be as effective as PA. PA has also been reported to be effica- cious against a genital HSV infection of hamsters (15), skin lesions induced by HSV and vaccinia virus in mice and rabbits (2, 17), and herpes keratitis in rabbits (3, 17). The data reported in this paper suggest that PF would also be effec- tive in these other model infections. Although PA is a very effective antiviral agent when applied topically against a number of ex- perimental HSV infections, it has not been tested in humans because of potential toxicity. Several investigators have noted skin irritation associated with the drug (14), and there is one report in which the drug accumulated in bone of a number of animal species (Bopp et al., Fed. Proc. 36:939, 1977). In our studies using topical treatment with PA or PF on mouse and guinea pig genitalia, no adverse effects were noted. Fur- ther toxicology studies are needed, however, to determine if alteration of the molecule has elim- inated the toxicity associated with PA. The ef- fectiveness of PF in the treatment of genital HSV infections of mice and guinea pigs indicates that additional studies are needed to further define the potential for this drug in the treat- ment of mucocutaneous HSV infections in hu- mans. ACKNOWLEDGMENTS This work was supported by Public Health Service contract NOI-AI-42524 from the Antiviral Substances Program, De- velopment and Application Branch, and by Public Health Service grant AI-102l7, both from the National Institute of Allergy and Infectious Diseases, and by Public Health Service grant no. CA-l7554 from the National Cancer Institute. J. C. O. is an investigator of the Howard Hughes Medical Institute. We thank James T. Richards and Sally Miramon for their excellent technical assistance. LITERATURE CITED 1. Fitzwilliam, J. F., and J. F. Griffith. 1976. Experimen- tal encephalitis caused by herpes simplex virus: com- parison of treatment with Tilorone hydrochloride and phosphonoacetic acid. J. Infect. Dis. 133(Suppl.): A221-A225. 2. Friedman-Rica, A. E., A. A. Fondak, and R. J. Klein. 1976. Phosphonoacetic acid treatment of shape fibroma and vaccinia virus skin infections in rabbits. J. Invest. Derrnatol. 66:953-102. 3. Ger-stein, D. D., C. R. Dawson, and J. 0. 0h. 1975. Phosphonoacetic acid in the treatment of experimental herpes simplex keratitis. Antimicrob. Agents Chemo- ANTI-HERPESVIRUS ACTIVITY OF PHOSPHONOFORMATE 4. 10. ll. 12. 13. 14. 15. 16. 17. 18. 19. 823 ther. 7 :285-288. Kelsey, D. K., E. R. Kern, J. C. Overall, Jr., and L. A. Glasgow. 1976. Effect of cytosine arabinoside and 5- iodo-2'-deoxyuridine on a cytomegalovirus infection in newborn mice. Antimicrob. Agents Chemother. 9:458-464. . Kern, E. R., J. C. Overall, Jr., and L. A. Glasgow. 1975. Herpesvirus hominis infection in newborn mice: treatment with interferon inducer polyinosinic-polycy- tidylic acid. Antimicrob. Agents Chemother. 7 :793-800. . Kern, E. R., J. C. Overall, Jr., and L. A. Glasgow. 1975. Herpesvirus hominis infection in newborn mice: comparison of the therapeutic efficacy of l-B-D-arabi- nofuranosylcytosine and 9-fl-D—arabinofuranosyladen- ine. Antimicrob. Agents Chemother. 7:587-595. . Kern, E. R., J. T. Richards, J. C. Overall, Jr., and L. A. Glasgow. 1977. Genital Herpesvirus hominis infec- tion in mice. II. Treatment with phosphonoacetic acid, adenine arabinoside, and adenine arabinoside 5' mono- phosphate. J. Infect. Dis. 135:557-567. . Kern, E. R., J. T. Richards. J. C. Overall, Jr., and L. A. Glasgow. 1978. Alteration of mortality and patho- genesis of three experimental Herpesvirus hominis in- fections of mice with adenine arabinoside 5'omonophos- phate, adenine arabinoside, and phosphonoacetic acid. Antimicrob. Agents Chemother 13:53-60. . Lefltowitz, E., M. Worthington, M. A. Conliffe, and S. Baron. 1976. Comparative effectiveness of six anti- viral agents in herpes simplex type 1 infection of mice. Proc. Soc. Exp. Biol. Med. 152:337-342. Meyer, R. F ., E. D. Varnell, and H. E. Kaufman. 1976. Phosphonoacetic acid in the treatment of experimental ocular herpes simplex infections. Antimicrob. Agents Chemother. 9:308-311. Overall, J. C., Jr., E. R. Kern, and L. A. Glasgow. 1976. Effective antiviral chemotherapy in cytomegalo- virus infection of mice. J. Infect. Dis. l33(Suppl.):A237-A244. Overall, J. C., Jr., E. R. Kern, R. L. Schlitzer, S. B. Friedman, and L. A. Glasgow. 1975. Genital Herpes- virus hominis infection in mice. I. Development of an experimental model. Infect. Immun. 11:476-480. Overby, L. R., E. E. Robishaw, J. B. Schleicher, A. Rueter, N. L. Shipkowitz, and J. C.-H. Mao. 1974. Inhibition of herpes simplex virus by phosphonoacetic acid. Antimicrob. Agents Chemother. 6:360-365. Palmer, A. E., W. T. London, and J. L. Sever. 1977. Disodium phosphonoacetate in cream base as a possible topical treatment for skin lesions of herpes simplex virus in cebus monkeys. Antimicrob. Agents Chemo- ther. 12:510-512. Renis, H. E. 1977. Chemotherapy of genital herpes sim- plex virus type 2 infections of female hamsters. Anti- microb. Agents Chemother. 11:701-707. Reno, J. M., L. F. Lee, and J. A. Boezi. 1978. Inhibition of herpesvirus replication and herpesvirus-induced de- oxyribonucleic acid polymerase by phosphonoformate. Antimicrob. Agents Chemother. 13:188—192. Shipkowitz, N. L., R. R. Bower, R. N. APPOH. C. W. Nordeen, L. R. Overby, W. R. Roderick, J. B. Schleicher, and A. M. Von Esch. 1973. Suppression of herpes simplex virus infection by phosphonoacetic acid. Appl. Microbiol. 26:264-267. Spruance, S. L., J. C. Overall, Jr., E. R. Kern, G. G. Krueger, V. Pliam, and W. Miller. 1977. The natural history of recurrent Herpes simplex labialis. N. Engl. J. Med. 297:69-75. Yang, J. P. 8., W. Chiang, J. L. Gale, and N. S. T. Chen. 1975. A chick-embryo cell microtest for typing of Herpesvirus hominis. Proc. Soc. Exp. Biol. Med. 148:324-328. APPENDIX C INHIBITION OF EUKARYOTIC DNA POLYMERASES BY PHOSPHONOACETATE AND PHOSPHONOFORMATE By Carol L. K. Sabourin John M. Reno and John A. Boezi Reprinted from Archives of Biochemistry and Biophysics ‘lQZ: 96 (1978) 84 ARCHIVES or BIOCHEMISTRY AND BIOPHYSICS Vol. 187, No. 1, April 15, pp. 96-101, 1978 Inhibition Of Eucaryotic DNA Polymerases by Phosphonoacetate and Phosphonoformate‘ CAROL L. K. SABOURIN, JOHN M. RENO, AND JOHN A. BOEZI Department ofBiochemistry. Michigan State University. East Lansing. Michigan 48824 Received September 6, 1977; revised November 17, 1977 Phosphonoacetate was found to be an inhibitor of the DNA polymerase a from three human cells. HeLa. Wi-38. and phytohemagglutinin-stimulated lymphocytes. The inhibition patterns were determined. The apparent inhibition constants (K.,) were about 30 pM. Thus the DNA polymerase a is 15 to 30 times less sensitive to phosphonoacetate than the herpesvirus-induced DNA polymerase. The DNA polymerase a from Chinese hamster ovary cells and calf thymus was also inhibited. The DNA polymerases )3 and y from the eucaryotic cells were relatively insensitive to phosphon- oacetate. The sensitivity of the DNA polymerase (I: and the relative insensitivity of the DNA polymerase B and y appeared to be general characteristics of the vertebrate polymerases. DNA polymerases from two other eucaryotic cells, yeast DNA polymerase A and B and tobacco cell DNA polymerase, were inhibited by phosphonoacetate. and to about the same extent as the a-polymerases. Fourteen phosphonate analogs were examined for inhibition Of the HeLa DNA polymerase a. Only one, phosphonoformate. was an inhibitor. The mechanism of inhibition for phosphonoformate was analogous to that for phosphonoacetate. Phosphonoacetate is an effective inhibi- tor Of the replication of herpesviruses (1- 4). The inhibition of viral replication is through an effect on the viral-induced DNA polymerase (5—9). At the concentra- tions at which phosphonoacetate is an ef- fective antiherpesvirus agent, it has no Obvious cytotoxic effects. At higher con- centrations, however, phosphonoacetate is cytotoxic (2, 5, 10) and cellular DNA syn- thesis is inhibited. Cell growth is impaired and the cells are apparently arrested at interphase. Mao et al. (6) and Mao and Robishaw (7) reported that the inhibitory effect of phos- phonoacetate 0n the herpesvirus-induced DNA polymerase was specific. They re- ported that the herpesvirus-induced DNA polymerase was highly sensitive to phos- phonoacetate, but that the cellular DNA ' This work was supported in part by Grant CA- 17554 awarded by the National Cancer Institute, DHEW. and by Grant AI-14357 awarded by the National Institute of Allergy and Infectious Dis- eases, DHEW. Michigan Agricultural Station No. 8243. 0003-9861/78/1871-0096S02.00/0 Copyright © 1978 by Academic Press, Inc. All rights of reproduction in any form reserved. polymerases a and [3 of human Wi-38 cells, the host cells for their herpesvirus infec- tions, were not inhibited by phosphonoac- etate. Huang (2) and Hirai and Watanabe (11) also reported that the DNA polymer- ases a and B of Wi-38 cells were not inhibited. In contrast, Bolden et al. (12) showed that, although the B—polymerase of human HeLa cells was not inhibited by phosphonoacetate, the a-polymerase was inhibited. Moreover, Bolden et al. (12) reported that the HeLa a-polymerase was as sensitive to phosphonoacetate as the herpesvirus-induced DNA polymerase. For duck embryo fibroblasts, Leinbach et al. (9) demonstrated that the a-polymer- ase was sensitive to phosphonoacetate, but that it was 15 to 30 times less sensitive to the inhibitor than the herpesvirus-induced DNA polymerase. With regard to the human cells, Overby et al. (13) suggested that the apparent differences in the sensitivities of the a- polymerases from Wi-38 and HeLa cells might be explained by the fact that the two cell types arose from different sources. PHOSPHONOACETATE AND PHOSPHONOFORMATE INHIBITION 97 Wi-38 cells originated from normal embry- onic lung tissue (14) and HeLa cells arose from cervical cancer tissue (15). They argued that normal and transformed cells might have different a-polymerases. In this report, we have reexamined the effect of phosphonoacetate on the a-polym- erases of Wi-38 and HeLa cells. We have also examined the a-polymerase of phyto- hemagglutinin-stimulated human lym- phocytes. In addition, we have looked at the a—polymerases of Chinese hamster ovary cells and calf thymus as well as the 'DN A polymerases from two nonvertebrate eucaryotic cells, yeast and tobacco cells. Finally we have investigated the effect of several phosphonate analogs on the HeLa a-polymerase. EXPERIMENTAL PROCEDURES Reagents. Phosphonoacetic acid was from Rich- mond Organics. Trisodium phosphonoformate and triethyl phosphonoformate were prepared following the procedure of Nylen (16). Deoxythymidine 5'- phosphophosphonoacetate was a gifl: from Dr. Susan S. Leinbach of this laboratory. Phosphonoacetamide and N -methylphosphonoacetamide were gifts from Dr. George Stark, Stanford University. Acetonyl phosphonate was a gift from Dr. Ronald Kluger, University of Toronto. The a-polymerase from calf thymus and the y-polymerase from fetal calf liver were obtained from Worthington. Other reagents were from sources previously described (9) or from the usual commercial sources. Cell cultures. HeLa (CCL 2) and Wi-38 (CCL 75) cells were purchased from the American Type Cul- ture Collection. Chinese hamster ovary (CHO)2 cells, proline requiring, were obtained from Dr. Louis Siminovitch, University of Toronto. HeLa, Wi-38, and CH0 cell cultures were tested at 37°C aerobically and anaerobically for mycoplasma con- tamination, as described by Hayflick (l7), and were not found to be contaminated. HeLa and CHO cells were grown in suspension culture in F-14 and F-19 media (Gibco), respectively, supplemented with 10% fetal calf serum. Wi-38 cells (passages 16—29) were grown in monolayer culture in G~13 medium (Gibco) supplemented with 10% fetal calf serum. Saccharo- myces cerevisiae, X2180 diploid strain, was obtained from Dr. William L. Smith of this department and was grown in glucose-yeast extract-casamino acids medium. Tobacco XD cells (Nicotiana tabacum L. var. Xanthi) were a gift from Dr. Philip Filner of ’ Abbreviations used: CHO, Chinese hamster ovary; DEAE, diethylaminoethyl; d'I'I‘P, deoxythy— midine 5’-triphosphate; dNTP, deoxyribonucleoside triphosphate. this department and were grown according to the procedure of Filner (18). All cells were harvested in midlog phase and frozen at ~70°C until use. .Lym- phocytes were purified from human blood by centri- fuging over Ficoll—Paque (Pharmacia) (19). The lymphocytes were diluted in G~13 medium contain- ing 10% fetal calf serum and 0.8% (v/v) phytohemag- glutinin-P (Difco) and cultured for 4.5 days. Assay of the DNA polymerization reaction. The standard reaction mixture for the III-polymerase contained in 200 pl: 50 mm Tris—hydrochloride (pH 8.0), 1 mm dithiothreitol, 500 pig/ml of bovine serum albumin, 10 mm MgCl,, 20 mm KCI, 200 pg/ml of activated calf thymus DNA (DNase I treated), 20 an aH-labeled deoxyribonucleoside triphosphate, 100 pm each of the other three deoxyribonucleo- side triphosphates, and a-polymerase. For the B- polymerase and the yeast DNA polymerases, KCl was omitted from the standard reaction mixture. For the tobacco cell DNA polymerase, KCl was omitted, dithiothreitol was 20 mm, and MgCl, was 6 mM. The standard reaction mixture for the fetal calf livery-polymerase contained in 200 pl: 50 mm Tris- hydrochloride (pH 8.0), 2.5 mm dithiothreitol, 500 rig/ml of bovine serum albumin, 4 mm MgCl,, 100 mu KCI, 50 rig/ml of poly(rA) - oligo(dT),,-.,, 100 pm 3H-labeled d'I'l‘P, andy-polymerase. Incubation was at 37°C for 30 min. Assay of the conversion of 3H- labeled deoxyribonucleoside triphosphate into a tri- chloroacetic acid-insoluble form was as previously described (20). Assay conditions were used so that the rate of DNA polymerization was linear with time and with the amount of DNA polymerase. For the kinetic studies, changes in the concentrations of assay components are as noted in the legends to the figures. Purification of the DNA polymerases. HeLa and CHO cells were fractionated into a nuclear and a cytoplasmic fraction according to the procedure of Chang and Bollum (21). The purification of the a- polymerase from the cytoplasmic fraction and the B-polymerase from the nuclear fraction was as de- scribed in the procedure of Weissbach et al. (22). This procedure involved purification by DEAE-cel- lulose and phosphocellulose column chromatogra- phy. The peak fractions of DNA polymerase activity from the phosphocellulose column were pooled, made 50% (v/v) in glycerol, and stored at —20°C. The specific enzymatic activity (nanomoles of deox- yribonucleoside triphosphate incorporated into an acid-insoluble form in 30 min at 37°C per milligram of protein) was 2400 for the HeLa a-polymerase and 1300 for the CH0 a-polymerase. The purified a- polymerases showed a Single peak of enzymatic activity at about 7 .5 S in a linear 20 to 40% glycerol gradient containing 0.45 M KCl. The Wi-38 a-polym- erase was purified as above except that the phospho- cellulose chromatography step was omitted. Glyc- erol gradient centrifugation was used to purify the 98 SABOURIN, RENO, AND BOEZI a—polymerase from phytohemagglutinin-stimulated lymphocytes, as described by Bertazzoni et al. (23). DNA polymerases A and B from S. cerevisiae were isolated through DEAE-cellulose chromatography according to Wintersberger and Wintersberger (24). The DNA polymerase from tobacco cells was pre- pared as described by Srivastava (25) and was fur- ther purified by DEAE-cellulose chromatography. Inhibition patterns. Inhibition patterns and ki- netic constants were defined according to the no- menclature of Cleland (26, 27). Analysis of each reaction mixture was done in duplicate. The data for the double-reciprocal plots were evaluated using a computer program based on the method of Wilk- inson (28). For evaluation of the apparent inhibition constants, replots of the intercepts and slopes of the double-reciprocal plots were analyzed using a com- puter program for least-squares analysis. RESULTS Phosphonoacetate inhibition of the DNA polymerization reaction catalyzed by HeLa DNA polymerase a. Phosphonoacetate was an inhibitor of the DNA polymeriza- tion reaction catalyzed by the HeLa a- polymerase. The addition of 25 to 30 pm phosphonoacetate to the standard reaction mixture resulted in a decrease in the rate of the DNA polymerization reaction by about 50%. In either the presence or the absence of phosphonoacetate, the rate of the reaction was linear for at least 1 h. Phosphonoacetate gave linear noncom- petitive inhibition with the four dNTPs as the variable substrate and activated DNA at a saturating concentration of 200 fig/ml (Fig. l). The apparent inhibition constant (Kn) determined from the replot of the vertical intercepts against the phosphon- oacetate concentration was 29 12151. The apparent inhibition constant (K.,) deter- mined from the replot of the slopes against phosphonoacetate concentration was 53 pM. Phosphonoacetate also gave linear noncompetitive inhibition with activated DNA as the variable substrate, the three dNTPs at 100 MM each, and the 3H-labeled dTTP at 20 pM. The K” for phosphonoace- tate was determined to be 29 pM and the K is 200 pM. The above results therefore demonstrate that the HeLa a-polymerase is indeed sen- sitive to phosphonoacetate. It is, however, less sensitive than the herpesvirus-in- duced DNA polymerase. The apparent in- oowh / Q 2 006 0.0l0'- 0/9 30005 2 . Ag/ 0 004 a — A 8 5. 2 V / / 5 002‘" 0/9 0005/8 ./a 20 40 so ./ [Phosphonoacetate] (pM) I I l 005 0:0 0 l5 [dNTP]" (le-I FIG. 1. Double-reciprocal plots with the four dNTPs as the variable substrate and phosphonoace- tate as inhibitor of the purified HeLa car-polymerase. Activated DNA was at 200 pig/ml. The initial veloc- ities were expressed as picomoles of ’H-labeled dTMP incorporated into DNA per 30 min. Phosphonoace- tate concentrations were 0 (O), 20 (O), 40 (D), and 60 pm (A). Equimolar concentrations of each of the four dNTPs were present in the different reaction mixtures. The replots of the slopes (O) and inter- cepts (O) as a function of phosphonoacetate concen- tration are shown in the left panel. hibition constants for the herpesvirus-in- duced DNA polymerase were in the 1 to 2 pM range (7, 9). Phosphonoacetate inhibition of the DNA polymerization reaction catalyzed by Wi- 38 DNA polymerase .a. Contrary to the results published by Mao et al. (6), Mao and Robishaw (7), Huang (2), and Hirai and Watanabe (11), phosphonoacetate was an inhibitor of the Wi-38 a-polymerase. The inhibition constants and the inhibi- tion patterns were similar to those of the HeLa a-polymerase. Phosphonoacetate gave linear noncompetitive inhibition with the four dNTPs as the variable sub- strate and activated DNA at a saturating concentration of 200 rig/ml. The K., was 15 [.LM and the K,s was 25 pM. With activated DNA as the variable substrate, the three dNTPs at 100 pm each, and the 3H-labeled dTTP at 20 pM, phosphonoacetate gave linear noncompetitive inhibition. The K“ was 33 pM and the K, was 150 pM. The effect of phosphonoacetate on the DNA polymerization reaction catalyzed by other a-polymerases. The a-polymerase from a third human cell source was exam- ined for its sensitivity to phosphonoacetate. The a-polymerase from phytohemaggluti- Q 020 PHOSPHONOACETATE AND PHOSPHONOFORMATE INHIBITION 99 nin-stimulated lymphocytes was as sensi- tive as the a—polymerase from HeLa and Wi-38 cells. Indeed, the inhibition by phos- phonoacetate seems to be a general char- acteristic of a-polymerases. For example, the CH0 a-polymerase and the one from calf thymus were inhibited. For the CH0 a-polymerase the inhibition patterns and apparent inhibition constants were exam- ined and found to be the same as those reported above for the HeLa a-polymer- ase. The effect of phosphonoacetate on the DNA polymerization reaction catalyzed by other DNA polymerases. Initial results by Mao et al. (6), Bolden et al. (12), and Leinbach et al. (9) showed that the [3- polymerase of eucaryotic cells was not sig- nificantly inhibited by phosphonoacetate. Our results agree. The addition of 200 uM phosphonoacetate to the standard reaction mixture produced no significant inhibition in the rate of the DNA polymerization reaction catalyzed by the HeLa or CHO B- polymerase. The B-polymerases of Wi-38 cells and human lymphocytes were also insensitive to phosphonoacetate. Like- wise, the y-polymerase of fetal calf liver was not significantly inhibited. The HeLa y-polymerase had previously been shown by Knopf et al. (29) not to be significantly sensitive to phosphonoacetate. The DNA polymerases from two nonver- tebrate eucaryotic cells were examined for their susceptibility to phosphonoacetate. The DNA polymerases A and B from S. cerevisiae were inhibited. The rate of DNA synthesis for both DNA polymerases A and B was decreased 50% by the addition of 15—20 pM phosphonoacetate to the stan- dard reaction mixture. Similarly, the DNA polymerase from tobacco cells was sensitive to phosphonoacetate at about the same concentration. The effect of other phosphonate com- pounds on the DNA polymerization reac- tion catalyzed by HeLa DNA polymerase 02. Recently it was discovered by Reno et al. (submitted for publication) that phos- phonoformate is an effective inhibitor of the herpesvirus-induced DNA polymerase. Phosphonoformate is also an inhibitor of the HeLa a-polymerase. With the four dNTPs as the variable substrate and acti- vated DNA at a saturating concentration of 200 rig/ml, phosphonoformate, like phosphonoacetate, gave linear noncom- petitive inhibition (Fig. 2). A replot of the vertical intercepts yielded a K n of 24 pM and a replot of the flows gave a K ,3 of 59 ptM. Phosphonoformate also gave linear noncompetitive inhibition with activated DNA as the variable substrate. A replot of the vertical intercepts gave a K H of 32 pM and a replot of the slopes yielded a K .8 of 176 pM. Phosphonoformate, therefore, ap- pears to be equally effective as phosphon- oacetate in its inhibition of the HeLa a- polymerase. Reno et al. (submitted for publication) have shown that phosphonoformate in- hibits the herpesvirus-induced DNA po- lymerase in a manner analogous to that of phosphonoacetate. This also seems to be the case for the HeLa a-polymerase. A multiple inhibition analysis (30, 31) of the DNA polymerization reaction indicated that phosphonoformate and phosphonoac- etate are mutually exclusive inhibitors and, therefore, bind at the same site on the a-polymerase. For the multiple inhi- bition analysis the concentration of phos- phonoformate was varied in the presence of fixed concentrations of phosphonoace- tate. AS shown in Fig. 3, a plot of UV against phosphonoformate concentration resulted in a series of parallel lines, indi- cating that the two phosphonate com- 004- InIercepI a 8 002i- 8 0 8/0/ a / OOI / / . 20 40 so /:/s/ [Phosphonotomiore] (pM) 1 1 1 1 o 05 0. l0 out o 20 [dNTP]" (pur' FIG. 2. Double-reciprocal plots with the four dNTPs as the variable substrate and phosphonofor- mate as inhibitor of the purified HeLa a-polymer- ase. Activated DNA was at 200 rig/ml. Phosphono- formate concentrations were 0 (O), 20 (O), 40 (Cl), and 60 an (A). 100 .0 9 UI I [Phosphonoformate] (pM) FIG. 3. Multiple inhibition of the HeLa a-polym- erase by phosphonoacetate and phosphonoformate. Activated DNA was at 200 rig/ml and the four dNTPs were at 7 psi each. Phosphonoacetate con- centrations were 0 (O), 10 (O), 20 (D), and 30 [.LM (A). pounds are mutually exclusive inhibitors. Other phosphonate compounds produced no significant inhibition of the rate of the polymerization reaction catalyzed by the HeLa a-polymerase when tested to a con- centration of 200 pM. The compounds which were tested were imidodiphosphate, methylene diphosphate, sulfoacetate, 2- phosphonopropionate, 3 - phosphonopro— pionate, 2-phenylphosphonoacetate, de- oxythymidine 5’ - phosphophosphonoace- tate, trimethyl phosphonoacetate, triethyl phosphonoacetate, triethyl phosphonofor- mate, phosphonoacetamide, N -methyl- phosphonoacetamide, and acetonyl phos- phonate. DISCUSSION This report demonstrates that the a-po- lymerases from three human cells, HeLa, Wi-38, and phytohemagglutinin-stimu- lated lymphocytes, and the enzymes from calf thymus and CHO cells are inhibited by phosphonoacetate. The apparent inhi- bition constants (K n) are about 30 pM. The HeLa a-polymerase has previously been shown by Bolden et al. (12) to be inhibited by phosphonoacetate. This report confirms their result. In addition, we report the apparent inhibition constants and inhibi- tion patterns for the a-polymerase. In con- trast to the results Of Bolden et al. (12), SABOURIN, RENO, AND BOEZI who reported that the a-polymerase and the herpesvirus-induced DNA polymerase were equally sensitive to phosphonoace- tate, our results demonstrate that the a- polymerase is, in fact, 15 to 30 times less sensitive. Our results showing that the Wi-38 a-polymerase is sensitive to phos- phonoacetate contradict the results re- ported by other authors (2, 6, 7, 11). We found that the B-polymerases of three human cells, HeLa, Wi-38, and lym- phocytes, are relatively insensitive to phosphonoacetate. Our result for HeLa is in agreement with that of Bolden et al. (12) and our result for Wi-38 agrees with the results reported by others (2, 6, 7, 11). Moreover, the B-polymerase of CHO cells, as reported here, and the B-polymerase of duck embryo fibroblasts, as previously re- ported by Leinbach et al. (9). are not inhibited by phosphonoacetate. As ob- served by Knopf et al. (29) with HeLa y- polymerase, the y-polymerase of fetal calf liver was also relatively insensitive to phosphonoacetate. Therefore, the sensitiv- ity of the a-polymerase and the relative insensitivity of the B- and y-polymerases appear to be general characteristics of ver- tebrate polymerases. Of the many phosphonate compounds which were examined for inhibition of the HeLa a-polymerase, only one, phosphono- formate, was shown to be an inhibitor. Phosphonoformate was also an inhibitor of the yeast DNA polymerases A and B and the tobacco cell DNA polymerase. Re- cently, in this laboratory, Reno at al. (sub- mitted for publication) discovered that phosphonoformate is an effective inhibitor of herpesvirus-induced DNA polymerase and that the mechanism of inhibition by phosphonoformate is analogous to that of phosphonoacetate. Leinbach et al. (9) re- ported that phosphonoacetate inhibits the herpesvirus-induced DNA polymerase by interacting at the pyrophosphate-binding site. For the experiments reported here with HeLa a-polymerase, phosphonoforo mate and phosphonoacetate were shown to be mutually exclusive inhibitors which bind at the same site on the a-polymerase. Presumably, this site is the pyrophos- phate-binding site. PHOSPHONOACETATE AND PHOSPHONOFORMATE INHIBITION At high concentrations, about 10 to 20 times greater than the concentration which effectively blocks herpesvirus repli- cation, phosphonoacetate is cytotoxic (2, 5, 10) and cellular DNA synthesis is in- hibited. Although not proven, it is not unreasonable to assume that at high con- centrations, phosphonoacetate inhibits cell growth as a result of the inhibition of cellular DNA synthesis through a specific inhibition of the a-polymerase. 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