Archives of Virology 61, 245--250 (1979) Archives of Virology © by Springer-Verlag 1979~

The Effeet of Pyrophosphate Analofues on Influenza Virus RNA Polymerase and Influenza Virus Multiplication

Brief Report

By

S. STI~IDI{, E. HELOSTRAND, B. L A ~ 5 , A. MlSmR~Y, G. ST~I~'G, and B. 0B]~na

Research and Development Laboratories, Astra Lakemedel AB, SSdert~lje, Sweden

Accepted March 20, 1979

Summary

Analogues of pyrophosphate have been tested as inhibitors of influenza virus~ RNA polymerase activity in cell-free assays. The most active compound, phos- phonoformic acid (PFA), reduced the polymerase act ivi ty to 50 per cent at a concentration of 20 ~M. The inhibition was dependent on the type of d ivalent cation present in the assay. PFA at a concentration of 400 tim also inhibited t h e influenza virus plaque formation by 90 per cent.

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The use of cell-free virM enzymes as test systems has been suggested as a rational approach to select antiviral compounds (4, 7). We have used this method to select inhibitors of influenza virus RNA polymerase. The antiviral properties- of one inhibitor, phosphonoformic acid (PFA), selected in this way has recently- been reported (6).

Several different types of structures have been suggested as inhibitors of- influenza virus multiplication (12). One type, which does not seem to have been systematically investigated, is analogues of pyrophosphate. The cleavage of pyrophosphate from the nucleoside triphosphates during I~]NA polymerization makes it possible tha t the polymerase has a binding site for pyrophosphate. This~ compound can thus be used as a natural starting point for structural variations with the aim of obtaining selective inhibitors.

Analogues of pyrophosphate were tested for inhibition of the virion associated influenza virus RNA polymerase under assay conditions essentially as described by Bishop et al. (2). The nucleotide concentrations used were 1 m~l ATP, 0.02 to. 0.2 mM [3H]-GTP (13 Ci /mmole-- I Ci/mmole), 0.1 mM CTP and 0.1 mM UTP. Glycerol was added to the assay to a final concentration of 8 per cent (v/v). The virus used for the polymerase assay was influenza A Victoria 3/75 ](-47 and

0304-8608/79/0061/0245/$ 01.20

246 S. S~'RIDg et al. :

influenza, B H K 8/73 purchased from Orion OY, Helsinki , F in land , and influenza A W S N ob ta ined from Dr. 1%. Krug. The influenza viruses were grown in eggs and pur i f ied b y differentiM cent r i fugat ion (5). To de te rmine if the inhib i t ion of influenza vi rus R N A polymerase ac t i v i t y was specific, al l py rophospha t e analogues were also tes ted for inhib i t ion of cell-free calf t h y m u s R N A potymerase ac t iv i ty . The calf t h y m u s R N A polymerase A and B were p repa red and assayed according tO KEDINGER st al. (8). Other detai ls of the assay condit ions have been publ i shed .earlier (5). The py rophospha t e analogues were a, lso t e s ted for reduc t ion of inf luenza vi rus p laque fo rma t ion according to BENTLEY and WICKHA~ (1) using the W S N .strain of influenza virus. The compounds were a d d e d to the over lay af ter v i rus adsorp t ion .

Table 1. Inhibition o/ influenza virus R N A polymerase activity and influenza virus plaque ]ormation

Compound

Cone. giving 50% in- hibition of RNA poly- merase from influenza

virus A Victoria

10 mM Mg e+ 10 m~ Mg ~+ 1 mM Mn

Cone. giving > 90% reduction in plaque formation of influ- enza virus A WSN

I HOOC--COOH I I HOOC--CH~--COOIt I I I HOOC--(CHe)2--COOI-I

0 0 it It

~V H O - - P O - - P - -OH 1 t OIt OII

0 0 Ff Ff

¥ I-IO P--CH2- --P--OIl

I I OH OH

0

II ¥I HO~P--O--CII~--COOI-I

I OI-I

0

V I I I-IO--~P--COOH (FFA) I

OH

O

V I I I HO- - :P - -CI I2 - -COOt t (PAA) I

OH

500 > 500 > 500 500 >500 >500

>500 >500 > 500

65 450 >500

450 > 500 > 500

300 >500 > 500

0.3 30 400

0.9 200 >500

Inhibition of Influenza Virus by Pyrophosphate Analogues

Table 1 {continued)

247

Conc. giving 50% in- hibition of RNA poly- merase from influenza Conc.giving > 90%

virus A Victoria reduction in plaque 10 m~I Mg s+ 10 mM Mg ~+ formation of influ- 1 mM Mn enza virus A WS2q

Compound ~ ~ ~M

O J~

IX I-IO--P--(CH~) e--COOH > 500 > 500 > 500 I

OH

O CIIa

X HO--P- -CH--COOH 500 > 500 > 500

OH

O IJ

XI HO t J C H e ~ H ~ 110 >500 >500 I

OH

O

XI I H~CeO--P--CH2o-COOH >500 >500 >500 ] OH

Compounds Nos. : 1, II, IIl, IV, Vl, XI are commercially available Compounds :No. : IX was synthesized according to NYL~ (i I) Colnpounds iXTos. : VII, VIII, X, XII were synthesized according to NYL~ (i0) Compounds No. : V was synthesized according to SCECWA~ZE:~BACH and ZIJRC (16)

Table 1 shows the s tructure-act ivi ty relationship for inhibition of influenza virus RNA polymerase and reduction in plaque formation caused by the pyro- phosphate analogues. The concentrations giving 50 per cent inhibition of influenza virus RNA polymerase ac t iv i ty were quite different with and without manganese in the assay, but the same pat te rn of inhibition was observed. In the presence of manganese, P F A (VII), phosphonoacetic acid (VIII , PAA) and pyrophosphate (IV) were most inhibitory. Some inhibition was a]so observed for 2-phosphono- propionic acid (X), methylenediphosphonic acid (V), phosphoglycolic acid (VI) and aminomethylphosphonic acid (XI). In the absence of manganese P F A was the only strong inhibitor of influenza RIgA polymerase. P F A seems to have the optimal distance between the two acid groups and a decrease in ac t iv i ty was observed as the distance is increased in compound V I I I and IX. A substi tuent on the methylene-group in PAA also diminished the inhibition. None of the compounds showed any inhibition of calf thymus RNA polymerase A and B at 500 ~ concentration either with or without manganese in the assay.

17 Arch. Virol. 6113

248 S. STRIDII et al. :

The inhibi t ion by P F A at different concentrat ions of magnesium and manganese in the cell-free assay is shown in Table 2. Optimal sensi t ivi ty to P F A was obta ined a t a magnes ium concentra t ion of 8 mM or a manganese concentra t ion of 2 m~. However, there was a large difference in the concentrat ion of P F A necessary for 50 per cent inhib i t ion in the presence of magnes ium as compared to manganese. A combinat ion of manganese and magnesium in the assay resulted in the same sensi t iv i ty to P F A as manganese alone.

Table 2. Inhibition o/ influenza virus A Victoria R N A synthesis by P F A at dif/erent concentrations o / M n ~+ and~or Mg 2+

~xM PFA ~x:g PFA ln~I Mn'-' + ~ PFA giving 50°/0 giving 50(}/o and giving 50%

mM Mg 2+ inhibition m~ Mn~ ~ inhibition 8 mM Mg 2+ inhibition

1 >500 0.25 10 2 190 0.50 0.8 4 40 t.0 0.4 8 20 2.0 0.2

t0 30 4.0 0.2 12 30 6.0 0.5

0.1 3 0.5 0.4 1 0.2 2 0.2

Table 3. Inhibition by P.FA o] influenza virus R:VA polymerase /rein influenza A strains W S N and Victoria, and influenza B HI!~

Virus Mn~+ 5Ig~+~ Mg~+ M:n2+ Mg~+ ApG b polymerase 1 m~ 8 m~ 8 m~I 1 m~ 8 m~ 0.1 m~

A WSN 0.3 100 0.2 31 A Victoria 0.4 27 0.2 29 B H K 0.14 29 0.9 61

The values shown are the concentrations of PFA in ~zs~ giving 50 per cent inhibition of enzyme activity

, [3It]-GTP exchanged to [att]-UTP b With ApG in the assay the incubation time was 15 minutes

Inh ib i t ion by P F A of polymerases from influenza A viruses, WSN and Vic- toria, and influenza B virus H K was the same in the presence of Mn 2+ and in the presence of ApG together with Mg ~+ as shown in Table 3. The use of ApG and Mg ~+ in the assay s t imulates the enzyme act iv i ty especially for the WSN polymer- use and p robab ly leads to a funct ional R N A product (3, t3). Wi th on ly Mg 2+ in the WSN polymerase assay the low polymerase ac t iv i ty prevented an exact de te rmina t ion of the 50 per cent inhibi t ion by P F A bu t a value a round 100 [xM was obtained. The same inhibi t ion b y P F A was observed when [aH]-GTP was subs t i tu ted with [aH]-UTP in the assay using polymerase from influenza s t rain A Victoria 3/75 X-47.

As shown in Table 1 P F A was the only substance in this series tha t caused a plaque reduct ion of influenza virus. The results obta ined from the plaque reduc- t ion assay corresponds more closely to the cell-free assay with magnes ium than

Inhibi t ion of Influenza Virus by Pyrophosphate Analogues 249

wi th manganese (Table 3). This is in ag reemen t wi th the resul ts of PLOTCH and KRUG (13, 14) suggest ing t h a t Mn 2~ could cause wrong ini t ia t ions.

Phosphonoformic ac id has r ecen t ly been found to be an efficient inh ib i tor of herpesvi rus D N A po lymerase and also of herpesvi rus infect ion in an ima l s (6, 15). I t is in te res t ing to no te t h a t P F A and P A A inhib i t herpcsvi rus D N A po lymerase and v i rus mul t ip l i ca t ion a t abou t the same concen t ra t ion while the i r ac t iv i t ies aga ins t inf luenza vi rus po lymerase and mul t ip l i ca t ion are qui te different . The same difference in inh ib i t ion p a t t e r n for P F A and P A A has r ecen t ly been found for reverse t r ansc r ip t a se (17) and hepa t i t i s B I ) N A po lymerase (9). The inh ib i t ion of the herpesvi rus D N A po lymerase b y P F A is noncompe t i t i ve wi th respect to nucleoside t r iphospha tes (].5) and p r e l im ina ry resul ts indica te t h a t this is also the case for inf lnenza v i rus I~NA polymerase .

Aeknowledflments The excellent, technical assistance of Mrs. M. Eriksson is gratefully acknowledged.

Re~erenees 1. BENTLEY, J. , ~¥ICKHAM, E. A. : A simple plaque assay for influenza A~ viruses and

its application to antiviral screening. Arch. ges. Virusforsch. 33, 234~ -241 (1971). 2. BIs~o]~, D. H. L., OBIJESKI, J . F. , SIM~'SO~, R. W. : Transcription of the influenza

ribonucleic acid genome by a virion polymerase. I. Optimal conditions for i n vitro act iv i ty of the ribonucteic a c i d - - d e p e n d e n t ribonucleic acid polymerase. J . Virol. 8, 66--~73 (1971).

3. BouLoY, M., P]~OTCH, S. J., K~UG, R. 1Vi. : Globin mRNAs are primers for the t ranscript ion of influenza viral R N A i n vitro. Proc. Natl . Acad. Sei. (U.S.A.) 75, 4886--4890 (t978).

4. COHEN, S. S. : A s trategy for the chemotherapy of infectious diseases. Science 197, 431--432 (1977).

5. EI~IKSSON, B., I~ELGSTRAND, E., JOI~IA~SSON, N.-G., LA~SSON, A., MISlOR~-Y, A., N o g ~ , J. O., ]?mLIPSON, L., STJ~]NBERG, K., STENI2~G, G., ST]~IDtt, S., ~IIERG, B. : Inhibi t ion of influenza virus ribonucleic acid polymerase by ribavirin tr iphosphate. Antimicrob. Ag. Chemother. 11, 946~951 (1977).

6. I~ELGSTI%AaND, E., ElgIli~SSOl% B., JOICIANSSOI% ~T.-G., LANNEt¢6, B., LARSSO~-, A., MISIOI~NY, A., I~Ot¢]~N, J. 0. , SJOBElCG, B., STENBElCG, K., STENING, G., STalDH, S., 0BERG~ B., ALENIUS, S.~ PI4ILIPSOlV, L.: Trisodium phosphonoformate, a new antiviral agent. Science 201, 819- ~821 (1978).

7. HELGSTRA~'D, E., OBE]ac, B. : AntivirM screening based on ceU free polymerase models and a new selective inhibitor. 10th Int . Congr. Chemother., Sept. 6, 1977. Curr. Chemother. 329--330 (1978).

8. I~EDINGEIZ, C., GISSING'EN, F., GNIAZDOWSKI, M., MONDEL, J.-L., Cr~A~BON, P. : Animal-dependent IZNA polymerase. I . A large-scale solubilization and separation of A and B calf thymus R.NA polymerase activities. Europ. J. Biochem. 28, 269 to 276 (1972).

9. NOI~DENFELT, E., I~tELGSTI~AND, E., OBERG, B. : Trisodium phosphonoformate inhibits hepat i t is B Dane particle DNA polymerase. Aeta Path. Mircobiot. Seand. In press 1979.

10. NYLON, P. : Beitrag zur Kenntnis der Organischen Phosphor-Verbindungen. I. Die Reakt ion zwisehen Natr iumphosphi t und Chloressigsaurem Natr ium. Chem. Ber. 57B, 1023--1038 (1924).

11. NYL]~sr, P . : Zur Kenntnis der Organischen Phosphorverbindungen. I I . I_~ber ~-Phosphonpropionsaure und a-Phosphon-n-Butters£ure. Chem. Bet. 59, 1119 --1128 (1926).

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250 S. STgZD~I et al. : Inhibit ion of Influenza Virus by Pyrophosphate Analogues

12. OXFORD, J. S. : Specific inhibitors of influenza virus replication as potential chemo- prophylactic agents. J. Al~timierob. Chcmother. 1, 7--23 (1975).

13. PLOTC~, S. J., K~.r~'G, R. M.: Influenza virion transcriptase: Synthesis in vitro of large polyadenylie acid-cont, aining complementary RNA. J. Virol. 21, 24--34 (1977).

14. i)r~o:rc~r, S. J., KRu¢, R. M. : Segments of influenza virus complementary RNA synthesized in vitro. J. Virol. 25, 579--586 (1978).

15. I~F~NO, J. M., LEE, L. F., Bo~zI, J. A. : Inhibit ion of herpesvirus replication and herpesvirus-induced deoxyribonucleic acid polymerase by phosphonoformate. Antimicrob. Ag. Chemother. 13, 188--192 (1978).

16. SCaWA~ZEag~ACE, G., Z~rRc, J. : Die Pyro- und die Unterphosphors/iure im Ver- gleich mit organischen DiphosphonsAuren. Monatshefte ffir Chemic 81, 202--212 (1950).

17. SUIgDQVlST, B,, 0BERG, B.: Inhibi tory effect of phosphonoformate on reverse transcriptase activity. I I I d CEC International Conference oil bovine Ieukosis, A]fort-Paris (1978).

Authors' address: S. S:r}~ID~I, Research and Development Laboratories, Astra L/i.kemedel AB, S-151 85 S6dertfilje, Sweden.

Received February 26, 1979