Acta path. rnicrobiol. s a n d . Sect. B, 87: 75-76, 1979.
BRIEF REPORT TRISODIUM PHOSPHONOFORMATE INHIBITS HEPATITIS B DANE PARTICLE DNA POLYMERASE Erik N~rdenfelt~. E. Helgstrand2 and B. dbergZ
hstitute for Medical Microbiology, University of Lund, Lund and *Research and Development Laboratories. Astra Lakemedel AB, Sodertiilje, Sweden
Nordenfelt. E., Helgstrand. E. & Oberg, B. Trisodium phosphonoformate inhibits hepatitis B Dane particle DNA polymerase. Acta path. rnicrobiol. s a n d . Sect. B. 87: 75-76, 1979. Evidence available indicates that the so-called Dane particles are the hepatitis virus. A DNA polymerase is associated with the core of these particles. The probability that this is the viral DNA polymerase offers the possibility of preventing hepatitis B multiplication by selective inhibition of this enzyme. This investigation reports that trisodium phosphonoformate (PFA) at low concentrations but not phosphonoacetate acid (PAA) inhibits Dane particle associated DNA polymerase. Key words: Hepatitis B; DNA polymerase inhibitor; phosphonoformic acid (PFA). Erik Nordenfelt, Institute of Medical Microbiology, Solvegatan 23, S-223 62 Lund, Sweden.
Accepted as submitted 7.xii.78 Hepatitis B virus (HBV) has yet not been isolated and cultured in vilro. However, all evidence available indicates that the so-called Dane particles are the HBV (Dane e/ a/. 1970). They are about 45 nm in diameter. and have hepatitis B antigen (HB,Ag) on the surface and a core with a separate antigen (HBAg) (Almeida et a/. 197 I ) from which DNA has been isolated (Robinson el a/. 1974). Furthermore, a DNA polymerase has been found to be associated with the core (Hirschman rt a/. I97 I , Kaplan et a/. 1973). The probability that this is the viral DNA polymerase (Krugman et a/. 1974) offers the possibility of preventing hepatitis B multiplication by selective inhibition of the Dane particle DNA polymerase, and this enzyme has recently been utilized to select potential therapeutic agents for hepatitis B infection (Hirschman & Garfinkel 1978). W e now report that trisodium phosphonoformate (PFA) at low concentrations inhibits Dane particle DNA polymerase. In a screening programme utilizing viral enzymes, PFA was found recently to be a good inhibitor of herpes virus DNA polymerase (Helgstrand et al. 1978. Helgstrand & dberg 1978). The structure of PFA is similar to that of phosphonoacetate (PAA), which has the same activity as PFA on herpes virus DNA polymerase (Helgstrand et a/. 1978, Helgstrand & dberg 1978, Reno et a/. 1978). Both structures are similar to pyrophos-
phate and may interact with a pyrophosphate binding site on the polymerase. Serum specimens for DNA polymerase assay were prepared as described previously (Nordenfelt & Kjellkn 1975). The serum was taken from three patients undergoing dialysis and known by regular control to be chronic carriers of HB&. This group of patients has been shown to have high numbers of Dane particles in serum (Nordenjelt & Kjellkn 1975. Couleru ef al. 1973). Electron microscopy studies have been repeated on serum preparations from two of the patients (Ass and EsG) which showed a high number of Dane particles in the pellets with polymerase activity. Assay conditions for the polymerase were the same as described earlier (Kaplan et a / . 1973) with slight modifications (Nordenfelt & Kjellen 1975). Tritiated dTTP (18 Ci/mmole, New England Nuclear) was used as the labelled nucleotide in the reaction. PFA and PAA were synthesized according to the method of Nden 1924. The inhibitors were added to the reaction mixtures at 4' C before incubation was started. The incubation time was 3 h at 37O C and assays were carried out in duplicate. Zero time incorporation was substracted from the 3 h incorporation. The average zero time incorporation was about 5 0 counts/min, and in the absence of inhibitor the 3 h incorporation was 200-300 counts/min. The DNA polymerase activity in the presence of inhibitor is presented as percentage remaining incorporation of [3HbTTP into acid precipi-
75
TABLE I . Inhibition of Dane Particle DNA Polymerase at Different Concentrations of PFA and PAA
Conc
PM
10
20 50
I00 200 500
Percentage remaining activity, mean
83.7 45.6 30.1 11.2 13.2 5.1
PFA No. of determinations
S.D.
activity, mean
S.D.
I I
I44
60.0
-
-
I53 I42
24.0 30.0 -
2 2
-
72. I
17.1
2
Patient
-
5 7 5 8
-
3 2 2 3 I
1 -
11.2
2
1
1
22.2 17.8 17.9 15.3
table radioactivity, as compared to the incorporation without inhibitor. A control serum from H B a g negative persons was always included but did not show any DNA polymerase activity. A summary of the results is given in Table I . The concentration dependence of the inhibition by PFA is presented and also compared with the effect of PAA. The Dane particle DNA polymerase was inhibited to 50 9;by 20 pM PFA, while 20 pM PAA seemed to have a slight stimulatory effect. At 100 pM, almost all DNA polymerase activity was inhibited by PFA. but PAA was still stimulatory. A slight inhibition was caused by 500 pM PAA. The lack of inhibition of the DNA polymerase by PAA correlates with earlier results where no inhibition was seen at concentrations up to 1 mM (Ma0 & Robishaw 1975). PFA and PAA have similar activities on herpes virus DNA polymerase, which is inhibited to 50% at 3 and 7 pM, respectively (Helgstrand et a / . 1978, Helgsirand & dberg 1978). A difference in inhibition was observed with influenza virus RNA polymerase where PFA was more inhibitory, and for vaccinia virus multiplication where PAA was the more active compound (Helgstrand et a / . 1978). Despite the slight difference in structure between PFA and PAA, their activities on viral enzymes could evidently be quite different. No differences between PFA and PAA in inhibition of cellular DNA polymerases a, B and y have been observed (Helgstrand et a/. 1978, Reno et al. 1978). At concentrations up to 500 pM, PFA and PAA do not inhibit DNA, RNA or protein synthesis in cell cultures (Helgstrand et a / . 1978, Stenberg & Larsson 1978). A concentration of 1 mM PFA is necessary to reduce DNA synthesis'and cell proliferation to 50% in cell cultures, and this inhibition is reversible (Sienberg & Larsson 1978). A very low toxicity in rats and dogs and no metabolism in mice have been observed for PFA (Flodh. H . & Lundstrom. J . personal communication I978), and it does not have the dermal toxicity observed for PAA (Alenius ei al. 1978). Control of viral hepatitis B is an important medical problem. Prevention by passive immunization has met with some success and a vaccine is forseeable in the near future (For review see Cossarr 1977). A specific chemotherapy against hepatitis B would, however, be a
76
PAA No. of determinations
Patient AA
Patient Ass
EsG
Percentage remaining-
-
-
-
Patient Ass
2
Patient EsG
Patient AA
I
-
1 1 1
-
I
-
I
well needed complement, and attempts in that direction have recently been described (Hirschman & Garfinkel 1978). The lack of cell toxicity of PFA (Helgstrand et a / . 1978, Stenberg & Larsson 1978) and its selective inhibition of Dane particle DNA polymerase suggest that an evaluation of its effect on hepatitis B in animals should be undertaken. W e thank Mrs Eva Miller for excellent technical assistance. The investigation was supported by the Swedish Medical Council (project No B77-16X-286501).
References: Alenius. S., Dinter, Z . & &berg, B.: Antimicrob. Agents Chemother. 14: 408-41 3, 1978. Almeida, J. D., Rubenstein, D. & Stort. E. J.: Lancet II: 1224-1228, 1971. - Cossart, Y. E.: Bailliere Tindall, London. 1977. - Coulero. O., Moulias. B., Peter, F.. German, A . & Loeper, J.: Nouv. Press Med. 2: 16331643. 1973. -Dane, D. S., Cameron, C . H . & Briggs. M.: Lancet I : 695-698, 1970. - Helgstrand, E., Eriksson, B., Johansson, N.-G., Lannero. B.. Larsson, A , . Misiorny. A , , Noren, J . O.,Sjoberg. B.. Stenberg, K . , Stening, G.. Stridh. B.. Alenius. S . & Philipson. L.: Science 201: 8 19821. 1978. - Helgstrand. E. & &berg. B.: Current chemotherapy 329-330, 1978. - Hirschman. S. Z . & Garfinkel. E.: Nature 271: 681-683, 1978. - Hirschman, S . Z.. Vernance. S . J . & Schaffner, F.: Lancet I: 1099-1 103, 1971. - Kaplan. R . P.. Greenman, R . L . , Gerin. J . L . . Purcell, R . H . & Robinson. W. S . : Virol. 12: 995-1 105. 1973. - Krugman, S., Hoofnagel, J . M., Gerety, R . J . , Kaplan, P. M. & Gerin, J . R . : New Eng. J. Med. 290: 1331-1335. 1974. - Mao. J . C.-H. & Robishaw. E. E.: Biochem. 14: 5475-5479, 1975. Nordenfelt, E. & Kjellen. L.: lntervirology 5 : 225-232, 1975. - Nylen, P.: Chem. Berichte 57B: 1023-1038. 1924. - Reno, J . M., Lee. L. F. & Boezi, J . A . : Antimicrob Agents Chemother. 13: 188-1 92, 1978. Robinson, W. S.. Clayron, D. A . & Greenman, R . L.: J. Virol. 14: 384-93 I , 1974. - Stenberg. K . & Larsson. A , : Antimicrob Agents Chemother. In press, 1978.
BRIEF REPORT TRISODIUM PHOSPHONOFORMATE INHIBITS HEPATITIS B DANE PARTICLE DNA POLYMERASE Erik N~rdenfelt~. E. Helgstrand2 and B. dbergZ
hstitute for Medical Microbiology, University of Lund, Lund and *Research and Development Laboratories. Astra Lakemedel AB, Sodertiilje, Sweden
Nordenfelt. E., Helgstrand. E. & Oberg, B. Trisodium phosphonoformate inhibits hepatitis B Dane particle DNA polymerase. Acta path. rnicrobiol. s a n d . Sect. B. 87: 75-76, 1979. Evidence available indicates that the so-called Dane particles are the hepatitis virus. A DNA polymerase is associated with the core of these particles. The probability that this is the viral DNA polymerase offers the possibility of preventing hepatitis B multiplication by selective inhibition of this enzyme. This investigation reports that trisodium phosphonoformate (PFA) at low concentrations but not phosphonoacetate acid (PAA) inhibits Dane particle associated DNA polymerase. Key words: Hepatitis B; DNA polymerase inhibitor; phosphonoformic acid (PFA). Erik Nordenfelt, Institute of Medical Microbiology, Solvegatan 23, S-223 62 Lund, Sweden.
Accepted as submitted 7.xii.78 Hepatitis B virus (HBV) has yet not been isolated and cultured in vilro. However, all evidence available indicates that the so-called Dane particles are the HBV (Dane e/ a/. 1970). They are about 45 nm in diameter. and have hepatitis B antigen (HB,Ag) on the surface and a core with a separate antigen (HBAg) (Almeida et a/. 197 I ) from which DNA has been isolated (Robinson el a/. 1974). Furthermore, a DNA polymerase has been found to be associated with the core (Hirschman rt a/. I97 I , Kaplan et a/. 1973). The probability that this is the viral DNA polymerase (Krugman et a/. 1974) offers the possibility of preventing hepatitis B multiplication by selective inhibition of the Dane particle DNA polymerase, and this enzyme has recently been utilized to select potential therapeutic agents for hepatitis B infection (Hirschman & Garfinkel 1978). W e now report that trisodium phosphonoformate (PFA) at low concentrations inhibits Dane particle DNA polymerase. In a screening programme utilizing viral enzymes, PFA was found recently to be a good inhibitor of herpes virus DNA polymerase (Helgstrand et al. 1978. Helgstrand & dberg 1978). The structure of PFA is similar to that of phosphonoacetate (PAA), which has the same activity as PFA on herpes virus DNA polymerase (Helgstrand et a/. 1978, Helgstrand & dberg 1978, Reno et a/. 1978). Both structures are similar to pyrophos-
phate and may interact with a pyrophosphate binding site on the polymerase. Serum specimens for DNA polymerase assay were prepared as described previously (Nordenfelt & Kjellkn 1975). The serum was taken from three patients undergoing dialysis and known by regular control to be chronic carriers of HB&. This group of patients has been shown to have high numbers of Dane particles in serum (Nordenjelt & Kjellkn 1975. Couleru ef al. 1973). Electron microscopy studies have been repeated on serum preparations from two of the patients (Ass and EsG) which showed a high number of Dane particles in the pellets with polymerase activity. Assay conditions for the polymerase were the same as described earlier (Kaplan et a / . 1973) with slight modifications (Nordenfelt & Kjellen 1975). Tritiated dTTP (18 Ci/mmole, New England Nuclear) was used as the labelled nucleotide in the reaction. PFA and PAA were synthesized according to the method of Nden 1924. The inhibitors were added to the reaction mixtures at 4' C before incubation was started. The incubation time was 3 h at 37O C and assays were carried out in duplicate. Zero time incorporation was substracted from the 3 h incorporation. The average zero time incorporation was about 5 0 counts/min, and in the absence of inhibitor the 3 h incorporation was 200-300 counts/min. The DNA polymerase activity in the presence of inhibitor is presented as percentage remaining incorporation of [3HbTTP into acid precipi-
75
TABLE I . Inhibition of Dane Particle DNA Polymerase at Different Concentrations of PFA and PAA
Conc
PM
10
20 50
I00 200 500
Percentage remaining activity, mean
83.7 45.6 30.1 11.2 13.2 5.1
PFA No. of determinations
S.D.
activity, mean
S.D.
I I
I44
60.0
-
-
I53 I42
24.0 30.0 -
2 2
-
72. I
17.1
2
Patient
-
5 7 5 8
-
3 2 2 3 I
1 -
11.2
2
1
1
22.2 17.8 17.9 15.3
table radioactivity, as compared to the incorporation without inhibitor. A control serum from H B a g negative persons was always included but did not show any DNA polymerase activity. A summary of the results is given in Table I . The concentration dependence of the inhibition by PFA is presented and also compared with the effect of PAA. The Dane particle DNA polymerase was inhibited to 50 9;by 20 pM PFA, while 20 pM PAA seemed to have a slight stimulatory effect. At 100 pM, almost all DNA polymerase activity was inhibited by PFA. but PAA was still stimulatory. A slight inhibition was caused by 500 pM PAA. The lack of inhibition of the DNA polymerase by PAA correlates with earlier results where no inhibition was seen at concentrations up to 1 mM (Ma0 & Robishaw 1975). PFA and PAA have similar activities on herpes virus DNA polymerase, which is inhibited to 50% at 3 and 7 pM, respectively (Helgstrand et a / . 1978, Helgsirand & dberg 1978). A difference in inhibition was observed with influenza virus RNA polymerase where PFA was more inhibitory, and for vaccinia virus multiplication where PAA was the more active compound (Helgstrand et a / . 1978). Despite the slight difference in structure between PFA and PAA, their activities on viral enzymes could evidently be quite different. No differences between PFA and PAA in inhibition of cellular DNA polymerases a, B and y have been observed (Helgstrand et a/. 1978, Reno et al. 1978). At concentrations up to 500 pM, PFA and PAA do not inhibit DNA, RNA or protein synthesis in cell cultures (Helgstrand et a / . 1978, Stenberg & Larsson 1978). A concentration of 1 mM PFA is necessary to reduce DNA synthesis'and cell proliferation to 50% in cell cultures, and this inhibition is reversible (Sienberg & Larsson 1978). A very low toxicity in rats and dogs and no metabolism in mice have been observed for PFA (Flodh. H . & Lundstrom. J . personal communication I978), and it does not have the dermal toxicity observed for PAA (Alenius ei al. 1978). Control of viral hepatitis B is an important medical problem. Prevention by passive immunization has met with some success and a vaccine is forseeable in the near future (For review see Cossarr 1977). A specific chemotherapy against hepatitis B would, however, be a
76
PAA No. of determinations
Patient AA
Patient Ass
EsG
Percentage remaining-
-
-
-
Patient Ass
2
Patient EsG
Patient AA
I
-
1 1 1
-
I
-
I
well needed complement, and attempts in that direction have recently been described (Hirschman & Garfinkel 1978). The lack of cell toxicity of PFA (Helgstrand et a / . 1978, Stenberg & Larsson 1978) and its selective inhibition of Dane particle DNA polymerase suggest that an evaluation of its effect on hepatitis B in animals should be undertaken. W e thank Mrs Eva Miller for excellent technical assistance. The investigation was supported by the Swedish Medical Council (project No B77-16X-286501).
References: Alenius. S., Dinter, Z . & &berg, B.: Antimicrob. Agents Chemother. 14: 408-41 3, 1978. Almeida, J. D., Rubenstein, D. & Stort. E. J.: Lancet II: 1224-1228, 1971. - Cossart, Y. E.: Bailliere Tindall, London. 1977. - Coulero. O., Moulias. B., Peter, F.. German, A . & Loeper, J.: Nouv. Press Med. 2: 16331643. 1973. -Dane, D. S., Cameron, C . H . & Briggs. M.: Lancet I : 695-698, 1970. - Helgstrand, E., Eriksson, B., Johansson, N.-G., Lannero. B.. Larsson, A , . Misiorny. A , , Noren, J . O.,Sjoberg. B.. Stenberg, K . , Stening, G.. Stridh. B.. Alenius. S . & Philipson. L.: Science 201: 8 19821. 1978. - Helgstrand. E. & &berg. B.: Current chemotherapy 329-330, 1978. - Hirschman. S. Z . & Garfinkel. E.: Nature 271: 681-683, 1978. - Hirschman, S . Z.. Vernance. S . J . & Schaffner, F.: Lancet I: 1099-1 103, 1971. - Kaplan. R . P.. Greenman, R . L . , Gerin. J . L . . Purcell, R . H . & Robinson. W. S . : Virol. 12: 995-1 105. 1973. - Krugman, S., Hoofnagel, J . M., Gerety, R . J . , Kaplan, P. M. & Gerin, J . R . : New Eng. J. Med. 290: 1331-1335. 1974. - Mao. J . C.-H. & Robishaw. E. E.: Biochem. 14: 5475-5479, 1975. Nordenfelt, E. & Kjellen. L.: lntervirology 5 : 225-232, 1975. - Nylen, P.: Chem. Berichte 57B: 1023-1038. 1924. - Reno, J . M., Lee. L. F. & Boezi, J . A . : Antimicrob Agents Chemother. 13: 188-1 92, 1978. Robinson, W. S.. Clayron, D. A . & Greenman, R . L.: J. Virol. 14: 384-93 I , 1974. - Stenberg. K . & Larsson. A , : Antimicrob Agents Chemother. In press, 1978.
