Pyrophosphate analogues as inhibitors of DNA polymerases of cytomegalovirus, herpes simplex virus and cellular origin

Bioehimica et Biophysica A cta, 696 (1982) 115-123

I 15

Elsevier Biomedical Press BBA91022

PYROPHOSPHATE ANALOGUES AS INHIBITORS OF DNA POLYMERASES OF CYTOMEGALOVIRUS, HERPES SIMPLEX VIRUS AND CELLULAR ORIGIN BERTIL ERIKSSON a, BO OBERG

a

and BRITTA WAHREN b

a Department of Antiviral Chemotherapy, Research and Development Laboratories, Astra LiJkemedel AB, S6dertiilje and b Department of Virology, National Bacteriological Laboratory, Stockholm (Sweden) (Received June 12th, 1981)

Key words: Cytomegalovirus," Herpes simplex virus; DNA polymerase inhibition; Phosphonoforrnate; Pyrophosphate analog,. Antiviral activity

Several pyrophosphate analogues have been compared for their ability to inhibit the activities of isolated cytomegalovirus (CMV) DNA polymerase, herpes simplex virus type 1 (HSV 1) DNA polymerase and calf thymus DNA polymerase a. The most effective inhibitors were phosphonoformate and phosphonoacetate. Although not identical, the structural requirements for compounds inhibitory to CMV and HSV-1 DNA polymerase were specific, with two negatively charged groups in close vicinity. The CMV DNA polymerase was more susceptible to certain phosphonoacetates containing bulky hydrophobic a-substituents than was the HSV-1 DNA polymerase. No example of the converse preference was found. The inhibition of CMV DNA polymerase by phosphonoformate, hypophosphate, a-hydroxyphosphonoacetate and a-nonylphosphonoacetate was linear non-competitive with the deoxyribonucleoside triphosphates as variable substrates. Phosphonoformate, phosphonoacetate, and to a lesser extent a-hydroxyphosphonoacetate, carbonyldiphosphonate and a-nonylphosphonoacetate also inhibited the focus formation by CMV in ceil-culture. Introduction All human herpesviruses induce D N A dependent DNA polymerases obligatory for the subsequent viral DNA replication. These enzymes differ in their characteristics from the cellular DNA polymerases and are consequently possible targets for selective antiviral compounds [1]. Both pyrophosphate analogues [1-6] and triphosphates of nucleoside analogues [7-9] have been shown to affect herpesvirus DNA polymerases at concentrations not inhibitory to cellular DNA polymerases. Several studies have analysed the structureactivity relation for pyrophosphate analogues on DNA polymerases of certain viruses of the

Abbreviations: CMV, cytomegalovirus; HSV-1, herpes simplex virus type 1 0167-4781/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

herpesvirus group [ 1,4-6,10-12,15]. A few of these compounds have also been shown to inhibit multiplication of herpesvirus in cell cultures [36,11,13,14] and animals [15-17]. Phosphonoacetate was previously found to inhibit cytomegalovirus DNA polymerase [18,19] and CMV infections in mice [20]. Both phosphonoacetate and foscarnet (phosphonoformate) inhibit CMV multiplication [14,18,19,21]. However, no study on the structure-activity relation for pyrophosphate analogues on isolated CMV DNA polymerase or CMV multiplication in cell culture has been reported. We have investigated the effects of 26 pyrophosphate analogues on CMV DNA polymerase, HSV-1 DNA polymerase and cellular DNA polymerase a in order to define the structural requirements for inhibitors. Active compounds were further studied in kinetic experiments to elucidate their mode of inhibition.

116

Materials and Methods

Reagents. The nucleoside triphosphates were from Sigma Chemical Co., St. Louis, MO [3H]dTTP and [3H]dATP were purchased from New England Nuclear, Boston, MA. Activated calf thymus DNA was prepared as described by Schlabach et al. [22]. Calf thymus DNA polymerase a was from Worthington Biochemical Corporation, Freehold, NJ. The synthetic templateprimers used were from Collaborative Research Inc., Waltham, MA. Pyrophosphate analogues. Most of the compounds have been described previously [5]. aEthyl-, a-propyl-, a-nonyl-, a-hydroxy-phosphonoacetates and phosphonomethanesulfonate were synthesized at this laboratory as described by Kovacs et al. (unpublished data), a-Hydroxy-amethylphosphonoacetate was synthesized by G. Stening at this laboratory according to Blum and Worms [23]. N-Phosphonoacetyl-L-aspartic acid was a gift from Dr. L.H. Kedda, Drug Synthesis and Chemistry Branch, division of Cancer Treatment, National Cancer Institute, MD. Virus strains and cell cultures.. The C42 strain of herpes simplex virus type 1 has been described previously [24]. Human cytomegalovirus strain Ad169 was grown in human lung fibroblasts as described previously [ 14]. CMV focus formation. The method used to determine CMV focus formation was as previously described [14]. For inhibition experiments, virus was inoculated on confluent coverslip cultures with human lung fibroblasts. Indirect or anticomplemerit immunofluorescence was performed with human antisera directed against late CMV antigens. Preparation and purification of herpesvirus DNA polymerases. Four 800 cm2 roller bottles with confluent monolayers of human lung fibroblasts were infected with CMV Ad169 at a multiplicity of 0.01. The incubation was carried out for about 10 days until the cells showed a 50-70% cytopatic effect. The infected cells were washed twice with phosphate-buffered saline (PBS) and scraped off with a rubber policeman. The CMV DNA polymerase was isolated by separation on DEAE- and phosphocellulose columns according to published methods [25] with the modifications described previously [5]. 20 #1 enzyme solution from the phos-

phocellulose top fraction incorporated about 50 pmol dTMP in a volume of 100 ttl reaction mixture in 30 rain. The isolated virus enzyme could use poly(dA) • oligo(dT)12_18 but neither poly(dT) • oligo(rA)12_18 nor poly(rC), oligo(dG)12_18 as template-primers. The DNA polymerase activity was also stimulated by more than 200% by the addition of 100 mM KCI. Furthermore, rabbit antiserum against the purified CMV DNA polymerase was used to show the presence of nuclear antigen after CMV infection of fibroblasts. This antiserum did not react with uninfected fibroblasts. These characteristics indicate that the enzyme was the CMV DNA polymerase [25]. HSV-1 DNA polymerase was isolated as previously described [5]. DNA polymerase assays. The CMV DNA polymerase activity was assayed in 100 /tl reaction mixtures containing 100 mM Tris-HC1 (pH 8.0)/20 mM MgC12/2.5 mM dithiothreitol/45 #g bovine serum albumin (fraction V)/100 mM KC1/20/~g activated calf thymus DNA/0.1 mM each of dATP, dCTP and dGTP. Unless otherwise indicated about 0.005 mM [3H]dTTP (1000-5000 cpm/pmol) was used. 20 /tl CMV DNA polymerase solution from the phosphocellulose top fraction was added to the assay and the mixture was incubated and processed as described previously [5]. The assays for HSV-1 DNA polymerase and cellular DNA polymerase a were performed as previously reported [5]. Kinetic studies.. The nomenclature of Cleland [27,28] was used. Data for the different plots were evaluated using a computer programme based on linear regression analysis. Each point in the double -reciprocal plots is given as the mean value of two determinations. The individual values differed by less than 4% from the means. The incorporations were linear under the conditions of incubation time, substrate, and enzyme concentrations used. The inhibition constants presented are mean values from at least two experiments. Results

Effects of diphosphonates Table I shows the effects of pyrophosphate and four diphosphonates on the activities of CMV DNA polymerase, HSV-1 DNA polymerase and

117 TABLE I EFFECTS OF PYROPHOSPHATE AND DIPHOSPHONATES ON ISOLATED DNA POLYMERASES Concentration (gM) giving 50% inhibition

Compound

I

O O tl II HO--P--O--P--OH

I

a DNA polymerase

>500

>500

>500

>500

>500

>500

6

10

100

105

120

>500

>500

>500

>500

OH

O O II II HO--P--CH2--P--OH

I

I

OH

III

HSV-1 DNA polymerase

I

OH

II

CMV DNA polymerase

OH

O O O II II II HO--P--C--P--OH

I

I

OH

OH

o OH O

tl IV

I

~t

HO--P--CH--P--OH

t

I

OH

OH

o OHO

tt V

L

H

HO--P--C-- P--OH

I

I

L

OHCH3OH

cellular D N A polymerase a. Although not shown, 500 # M pyrophosphate (I) caused an inhibition of the viral polymerase activities of about 40%. Replacing the oxygen bridge by a methylene group (II) completely eliminated this slight inhibitory effect. Carbonyldiphosphonate (III, CDP) and methanehydroxydiphosphonate (IV, MHDP), two examples of single substitutions on the methylene bridge, preferentially inhibited the two herpesvirus D N A polymerases. C D P (III) gave a 50% reduction of t h e viral DNA polymerase activities at a concentration of about 10 gM, whereas a 10-fold higher concentration was needed to inhibit the cellular D N A polymerase activity to the same

extent. With two substituents introduced on the methylene carbon, as exempfified by ethane-1hydroxy-l,l-diphosphonate (V, EDHP), the inhibitory potential was lost.

Effects of a-substituted phosphonoacetates The inhibition of CMV D N A polymerase by a-substituted phosphonoacetates is shown in Table II and is compared to the effect on HSV-1 D N A polymerase and D N A polymerase a. Drastic changes in activity occurred with increasing lengths of the substituted alkyl chain. Both the a-methyl (VII) and a-ethyl (VIII) derivatives were 20-50times less effective than phosphonoacetate (VI) and affected the viral enzymes almost equally.

118 T A B L E II E F F E C T S O F P H O S P H O N O A C E T A T E A N D a - S U B S T I T U T E D A N A L O G U E S ON ISOLATED D N A POLYMERASES Concentration (/~M) giving 50% inhibition

Compound

C M V D N A polymerase 0

a D N A polymerase

H

II I VI

HSV- I D N A polymerase

..~o

HO--P--C--C"

I

0.4

0.4

35

\OH

I

OHH O

H

ii I VII

,p

HO--P--C--

C

[ I

18

15

>500

~'OH

OHCH 3 O

H

II I VIII

.~o

HO--P--C--C

~

I I

>500

a

13

8a

30

200 a

>500 a

10 a

160 a

~OH

OHCH: --CH 3 O IX

H

)1 HO--P f

CI c ~ o I- ~OH

OH O X

CH 2--CH 2--CH 3 H

H O - - P~- - / -O "- C "

1.5

I I

~OH OH(CH2)s-CH 3 O

H

I

II XI

HO--P

o

C--C ~ I \OH

I

18

100

3

2

500

>500

>500

0 0

H

i) I XII

.,,o

HO--P--C--C."

I I

a

100 a

"OH

OHOH O

CH 3

H I

XIII

~o

HO--P--C--C

I I

\OH

OH OH a Kovacs et al., personal communication.

>500

119

TABLE III EFFECTS OF PHOSPHONOFORMATE AND RELATED COMPOUNDS ON ISOLATED DNA POLYMERASE Compound

Concentration (ttM) giving 50% inhibition CMV DNA polymerase

O II ~O HO--P--C ~

XIV

I

HSV-1 DNA polymerase

0.3

a DNA polymerase

0.3

40

\OH

OH O O II II HO--P--P--OH

XV

150

150

250

I I OHOH 0

0

\/

/ -.\

xvI

HO

Slope

13o

>500

OH

Interc~ ~ (v)

(• )

J_ V

0.2.

- 0,2

0.1.

-0.1

.

012

1i0

0~5 [ Phosphono for mate~

1.E

1.0 •

IIM

0.,5"

P 02

05

1

2

1 PM'I

Fig. I. Inhibition of CMV DNA polymerase by phosphonoformate with the four deoxyribonucleoside triphosphates as substrates. Phosphonoformate concentrations were • • , 0 gM; -~ - 4-, 0.2 pM; • • , 0.5 gM and • I , 1 gM. Conditions and assay procedures were as described. The velocity (V) is expressed as pmol dTMP incorporated/30 min and 40 gl reaction mixture. Inserted are the secondary plots of slopes and intercepts vs. increased concentrations of phosphonoformate. The calculated values of Kis and Kii were 0.82 and 0.28 gM, respectively.

120

With longer or larger substituents introduced, as for a-propyl- (IX) or a-phenyl-phosphonoacetates (XI), a 6-7-times preferential inhibition of CMV DNA polymerase was observed. This was also evident for a-nonylphosphonoacetate (X). Next to phosphonoacetate (VI) the latter compound was the most potent inhibitor of isolated CMV DNA polymerase activity, a-Hydroxyphosphonoacetate (XII), which was also a relatively potent inhibitor affected the viral DNA polymerases almost equally. However, a double substitution introduced into the a-position, as examplified by a-hydroxy-amethylphosphonoacetate, led to a considerable decrease in inhibitory effect as compared to the effects of the corresponding monosubstituted compounds (VII, XII). Generally, the a-substituted phosphonoacetates were considerably less inhibitory to cellular DNA polymerase a than to CMV or HSV-1 DNA polymerase.

Slope (e)

Effects of phosphonoformate and related compounds The structure-activity relation for phosphonoformate and a few other compounds with two closely situated and negatively-charged groups are shown in Table III. Phosphonoformate (XIV) was the most potent inhibitor, whereas hypophosphate (XV) and oxalate (XVI) affected the viral polymerases to a lesser extent. Esterification of either the phosphono or carboxyl group of phosphonoformate yielded inactive compounds (data not shown). In this series there was no significant difference in inhibition of CMV and HSV-1 DNA polymerase activities.

Effects of otherpyrophosphate analogues N- Phosphonoacetyl- L- aspartate (PALA) reduced both CMV and HSV-1 DNA polymerase activities to 50% at a concentration of about 300 #M. The following compounds were inactive at

Interce ~ (~) 1

-0.5

0 50

150

500

EHypophosphate

IJM

J

1

Fig. 2. Inhibition of CMV D N A polymerase by hypophosphate with the four deoxyribonucleoside triphosphates as substrates. The concentrations used were • 0 , 0 ,aM; -4 - 4-, 50 ,aM; • • , 150 ,aM and • II, 500 `aM. Conditions and assay procedures were as described in Fig. 1. Calculated Kis and Kii values are 528 and 260 `aM, respectively.

121 500 /~M: glycolate, glycerate, malonate, carbamylphosphate, c a r b a m o y l m e t h a n e p h o s p h o n a t e , benzoylmethanephosphonate, a - aminoethylphosphonate, aminomethylphosphonate, 3-phosphonopropionate, sulfonoacetate and phosphonomethanesulfonate.

Mechanism of inhibition The mechanisms of inhibition by some of the active compounds on C M V D N A polymerase were studied in kinetic experiments. Figs. 1 and 2 show the Lineweaver-Burk plots for phosphonoformate and hypophosphate, with the deoxyribonucleoside

triphosphates as variable substrates. The lines intersect at one point in the lower left-handed quadrant. According to Cleland [27,28] this type is referred to as linear non-competitive. However, this must be distinguished from the simple or classic linear non-competitive inhibition which gives an intersection of the lines on the horizontal axis. The same inhibition pattern was also obtained by phosphonacetate, and a-hydroxyphosphonoace -tate and a-nonylphosphonoacetate, (data not shown). With activated D N A as variable substrate and the four deoxyribonucleoside triphosphates at

TABLE IV INHIBITION OF CMV FOCUS FORMATION BY PYROPHOSPHATE ANALOGUES. THE COMPOUNDS WERE TESTED AT A CONCENTRATION OF 500 ttM Compound

Per cent inhibition of focus formation Multiplicity of infection---0.1

III

O O O II II II HO--P--C--P--OH

I

Multiplicity of infection-- 0.01

0

85

I00

100

I

OH

OH

O H

LI I VI

.,O

HO--P--C--C"

I

I

\OH

OHH O H X

IL I __O HO--P--C--C~ I I --OH OH(CH2)sCH3

0

35 a

O H

II XIl

~o

I

HO--P--C--C~" I ] \OH OHOH

30

70

100

100

O II

XIV

~O

HO--P--C ~"

I OH

a Visible cell toxicity.

\OH

122

a saturating level, uncompetitive inhibition patterns were observed for phosphonformate and anonylphosphonoacetate (data not shown).

Inhibition of CMV focus formation by CMV DNA polymerase inhibitors As shown in Table IV, several of the active DNA polymerase inhibitors at 500 /~M also inhibited focus formation by CMV in cell culture. The inhibitory effect was determined at two different multiplicities of infection. The most active compounds were phosphonoac'etate and phosphonoformate, whereas carbonyldiphosphonate and ahydroxyphosphonoacetate affected the focus formation to a lesser extent. The effect of anonylphosphonoacetate at a concentration of 500 /~M caused visible cell toxicity. Decreased multiplicity of infection resulted in an increased inhibitory effect by the compounds studied. Discussion

Several nucleoside analogues [29-31] have an antiherpes activity based on their selective phosphorylation by herpesvirus-induced thymidine kinase. The absence of a CMV-induced thymidine kinase [32] makes it necessary to find inhibitors of this virus based on other enzyme activities. This study was based on the assumption that pyrophosphate analogues may interfere with a pyrophosphate binding site on the CMV DNA polymerase and thereby inhibit the viral DNA synthesis. Several of the analogues investigated were found to inhibit CMV DNA polymerase at low concentrations e.g., carbonyldiphosphonate, phosphonoacetate, a-nonylphosphonoacetate, a-hydroxyphosphonoacetate and phosphonoformate showing that both a diphosphonate, a-substituted phosphonoacetates, and a simple combination of a phosphono and a carboxyl group could be inhibitory. With a few exceptions the structural requirement for active inhibitors seems specific with two acidic functions at close proximity. A sulfonyl group could neither replace the phosphono nor the carboxylic group. Furthermore, a double substitution on the methylene bridge between the negatively charged groups rendered the compound inactive. As for HSV-1 DNA polymerase [5] esterification of either acid group in phosphonoformate

led to inactive compounds. Apart from phosphonoacetate [ 12,18,19], no other pyrophosphate analogue has previously been reported to inhibit CMV DNA polymerase activity. Certain a-substituted phosphonoacetates preferentially inhibited the CMV DNA polymerase activity. This was evident with large hydrophobic a-substituents as in compounds IX, X and XI. No example was found of an inhibitor that was significantly more inhibitory to HSV-1 DNA polymerase than to CMV DNA polymerase. The active compounds were considerably less inhibitory to cellular DNA polymerase a than to HSV-1 or CMV DNA polymerase (Tables I-III). The only exception was hypophosphate, which also has been shown to irreversibly inhibit cell division [33]. The kinetic study with CMV DNA polymerase revealed that the mechanism of inhibition was linear non-competitive for phosphonoformate, hypophosphate, a-hydroxyphosphonoacetate and anonylphosphonoacetate. The same mechanism of inhibition has previously been found for HSV-1 DNA polymerase [4,5] with phosphonoformate and phosphonoacetate. Hypophosphate was shown to inhibit HSV-1 DNA polymerase activity in a competitive manner [5]. It seems unlikely that only hypophosphate among the pyrophosphate analogues should compete with the nucleoside triphosphates for the substrate binding site. However, the reason for this discrepancy between the two herpesvirus DNA polymerases might be explained if the interaction of hypophosphate with a presumed pyrophosphate binding site on the enzyme also reduced the affinity for the deoxyribonucleoside triphosphates in the case of HSV-1 DNA polymerase but not for CMV DNA polymerase. The inhibition of CMV multiplication in cell culture (Table IV) by pyrophosphate analogues seems to correspond to the inhibitory effect of these compounds on the cell-free CMV DNA polymerase. Phosphonoformate and phosphonoacetate were previously reported to inhibit CMV multiplication [14,18,19], whereas carbonyldiphosphonate, a-nonylphosphonoacetate and ahydroxyphosphonoacetate are new inhibitors. The present investigation has shown that compounds with structural similarities to pyrophosphate can be effective inhibitors of CMV DNA polymerase activity and CMV multiplication. The

123

results also indicate an increasing specificity in structural requirements on inhibitors of the pyrophosphate binding site when CMV, HSV-1 and cellular DNA polymerase a are compared, with CMV having the least stringent requirements. Acknowledgements We thank Mrs. G. Br~innstr6m for excellent technical assistance. Mrs. B. Andersson, M. Kropp and G. Norlin for patient typing, and Dr. Desmond M. Lake-Bakaar for linguistic correction of this manuscript. References 1 Helgstrand, E. and Oberg, B. (1980) Antibiot. Chemother. 27, 22-69 2 Mao, J.C.-H., Robishaw, E.E. and Overby, L.R. (1975) J. Virol. 15, 1281-1283 3 Helgstrand, E., Eriksson, B., Johansson, N.-G., Lanner6, B., Larsson, A., Misiorny, A., Nor~n, J.O., Sj6berg, B., Stenberg, K., Stening, G., Stridh, S., Oberg, B., Alenius, S. and Philipson, L. (1978) Science 201,819-821 4 Reno, J.m., Lee, L.F. and Boezi, J.A. (1978) Antimicrob. Agents Chemother. 13, 188-192 5 Eriksson, B., Larsson, A., Helgstrand, E., Johansson, N.-G. and Oberg, B. (1980) Biochim. Biophys. Acta 607, 53-64 6 Boezi, J.A. (1979) Pharm. Ther. 4, 231-243 7 MUller, W.E.G., Zahn, R.K., Bittlingmaier, K. and Falke, D. (1977) Ann. N.Y. Acad. Sci. 284, 34-48 8 Furman, P.A., St. Clair, M.H., Fyfe, J.A., Rideout, J.L., Keller, P.M. and Elion, G.B. (1979) J. Virol. 32, 72-77 9 Eriksson, B., ()berg, B. and Gauri, K.K. (1981) Antiviral Chemotherapy: Design of Inhibitors of Viral Functions, Hamburg 1980 (Gauri, K.K., ed.) Academic Press lO Leinbach, S.S., Reno, J,M., Lee, L.F., Isbel, A.F. and Boezi, J.A. (1976) Biochemistry 15, 426-430

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