Selective inhibition of poliovirus growth by d -penicillamine in vitro

VIROLOGY

30, 618-622 (1966)

Selective

Inhibition

of

Poliovirus

D-Penicillamine

Growth

By

in Vitro’

G. L. GESSA, B. LODDO, G. BROTZU, M. L. SCHIVO, A. TAGLIAMONTE, A. SPANEDDA, G. BO, AND W. FERRARI Institutes

of Pharmacology, Hygiene,

Microbiology,

Pharmacology,

and Virology, University of Cagliari, of Modena, Italy

and Institute

of

University

Accepted

July

19, 1966

n-Penicillamine inhibits poliovirus growth in vitro. This effect is related to a parallel inhibition of viral RNA synthesis. n-Penicillamine possesses a very narrow antiviral spectrum. In fact it exerts a weak inhibition against Coxsackie and ECHO viruses and shows no inhibitory effect against herpes simplex, vaccinia, and vesicular stomatitis viruses. The inhibitory action of n-penicillamine is suppressed by phenylalanine, tyrosine, tryptophan, and leucine and by Cu and Fe salts. L-penicillamine is ineffective as an antiviral agent. INTRODUCTION

The influence of guanidine on poliovirus replication in vitro can be suppressed by a number of amino acids, methionine being the most active one (Lwoff and Lwoff, 1964; Loddo and Schivo, 1965). In an attempt to define the structure-activity relationships for antiguanidine action among amino acids, we have demonstrated that the antiguanidine action of methionine does not stem from its methylating properties; in fact equally active antiguanidines are its ethyl analog, ethionine (Loddo et al., 1966), and its demethylated oc-aminoacid analogshomocysteine and a-aminobutyric acid; p- and y-ammobutyric acid are ineffective (Gessa et al., 1966). As a working hypothesis we have proposed that guanidine may act, at least in part, by competing with someamino acids, essential for virus rephcation (perhaps during replicase synthesis), and that a potential antipolio agent might be found among some analogs of those amino acids that most actively antagonize guanidine (Gessaet al., 1966). 1 This work was supported No. 227.

by a NATO

Grant 618

Consequently, we have studied the influence of n-penicillamine, a valine analog, on poliovirus replication in vitro. MATERIALS

AND METHODS

Chemicals. n-Penicillamine was kindly given by Dista Product,s Ltd. Liverpool, U.K.; n-penicillamine was furnished by Aldrich Chemicals Co. Virus strains. All the enterovirus strains used (polio 1 Brunhenders and Mahoney; polio 2 MEF 1; polio Sabin types 1, 2, and 3; ECHO 1, 2, 4, 7; Coxsackie B2 and B6) were kindly supplied by Istituto Superiore di Sanita, Roma. Vaccinia virus was isolated from vaccinia lymph of Istituto Sieroterapico Milanese. Herpes simplex virus and VSV (vesicular stomatitis virus) were provided by Dr. R. W. March of the National Cancer Institute, Bethesda, Maryland. KB cells and Detroit 6 cells (clone 12) were routinely grown in Eagle’s MEM with 5 % calf serum. For experiments about 5 X lo5 cells were seededon small petri dishes in the same medium buffered with Tris and kept at 37” for 16 hours. Thereafter, the confluent cell monolayers were infected with 10’ plaque-forming units

ANTIPOLIO

ACTIVITY

(PFU) of the virus to be tested. The infected cultures, unless otherwise specified, after incubation at room temperature for 1 hour, were washed four times with Hanks’ BSS buffered with Tris at pH 7.3; and finally 3 ml of BSS, containing the compounds to be tested, was added. After 12 hours’ incubation at 37”, l-ml samples of the overlay medium were withdrawn and titrated by the plaque technique of Dulbecco and Vogt (1954). RESULTS

Effect of D-Penicillanaine

on Poliovirus

Growth

n-Penicillamine inhibited very effectively poliovirus 1 growth at a concentration far below the maximum tolerated by cell cultures both in KB and in Detroit 6 cells (Table 1). Table 2 shows the effect of TABLE

1

IKHIBITION BY D-PESICILLAMINE OF POLIOVIRUS 1 (BRUNHENDERS), REPLICATION IN KB CELLS AND DETROIT 6 (CLONE 12) CELLS D-Penicihamine concentration in the medium &g/ml)

3008 100 33 11 a Maximum concentration tures for 48 hours at 37”.

PFU

produced

In KB cells 1.2 5 6 4.5

x x X x

1.4

x

8 x 1.5 x 9 x

tolerated

107 103 103 104 106

by cell cul-

TABLE 2 INHIBITION OF POLIOVIRUS 1 (BRUNHENDERS) REPLICATION IN KB CELLS BY D-PENICILLAMINE (44 fig/ML) ADDED AT VARIOUS TIMES AFTER VIRAL INFECTION Interval infection

between cell and treatment (minutes)

No treatment 5 60 90 120 180 240

PFU

produced 12 hours after infection

4 8 2 5 8 8

x x x x x x

10’ 103 103 104 104 104 105

619

D-PENICILLAMINE

n-penicillamine, added to the cultures at various intervals after virus infection. The drug was effective in inhibiting virus growth when added up to 3 hours after infection. Cells pretreated with n-penicillamine (66 pg/ml) for 9 hours, and then washed, maintain their ability to support virus growth (Table 3). E$ect of D-Penicillamine Synthesis

on Poliovirus

RNA

It can be seen from Table 4 that n-penicillamine inhibition of virus growth is related to a parallel inhibition of the synthesis of infectious viral RNA. Antiviral

Spectmm of n-Penicillamine

The effect of n-penicillamine against the growth of different strains of polio 1, 2, and 3, Coxsackie, and ECHO viruses was studied. The results given in Table 5 show that n-penicillamine inhibits polio 2 and 3 to the same extent as polio 1, but is much less

in 12 hours In Detroit 6 ce!ls

107 103 103 104 106

OF

TABLE REPLICATION TREATED CILLAMINE

3

OF POLIOVIRUS IN KB CELLS PREFOR 9 HOURS AT 37” WITH D-PENIAND WASHED BEFORE INFECTION

Treatment of cell monolayers before virus infection

PFU produced 12 hours after virus infection

Hanks’ BSS D-Penicillamine, 66 fig/ml in Hank’s BSS

8 x 9 x

TABLE

4

INHIBITION BY D-PENICILLAMINE RNA SYNTHESIS IN KB CELLS POLIOVIRUS 1 (BRUNHENDERS) n-Penicillamine in the medium Wml)

100 33

106 106

Infectious 12 hours

OF IKFECTIO~S INFECTED WITH

RNA produced after infectiona 6 x 2 x 8 x

104 102 102

a Total RNA was extracted according to the method of Gierer and Schramm (1956) and evaluated according to the method of Ellem and Colter (1960) modified by Loddo et al. (1962).

620

GESSA,

effective in inhibiting Coxsackie and Echo viruses. This unique specificity of the inhibitory effect of D-penicillamine was further invesTABLE INHIBITION REPLICATION

By

VIRUSES

5

~PENICILLAMINE OB DIFFERENT IN

-

KB

0~ THE ENTEROCELLS

concentration

strain

(pg/ml)

44

0

Polio Polio Polio

1 Brunhenders 1 Mahoney 2 MEFr

Polio Polio Polic

1 Sabin 2 Sabin 3 Sabin

Coxsackie Coxsackie ECHO ECHO ECHO ECHO

Bs Bg 1 2 4 7

i

9 2.2 8 7

CATION PENDENT Concentration in the

BY

1073 107

x

X lo78 x lo72 x 10713 X 107,3

TABLE INHIBITION

I

7 x 2 x

132

1078

I

x

106

x

105

X x

lo”4 lo76

X X

lo6 lo*

x

107 3 x lo7 6 X

107

lo7

6

D-PENICILLAMINE

OF THE

IN KB CELLS OF POLIOVIRUS STRAIN

A

REPLI-

GUANIDINE-DE(BRUNHENDERS)”

PFU produced 12 hours after

of the compounds medium (&ml)

HCl

D-Penicillamine Guanidine

a Polio

b Titrated 100 pg/ml.

7 BETWEEN GUANIDINE IN INHIBITING POLIOREPLICATION IN KB

CELLS

6 X X 4 x 3 x 2 x

lo3 lo6

103

No

x 7 x

103 103

Guanidine

Concentration of the compounds in the medium &g/ml)

105

33 + 66 33 + 44 1 Brunhenders serial in

7.5

made

transfers in increasing up to 200 pg/ml.

the

presence

produced 12 hours

drug HCl

D-Penicillamine

guanidine-deguanidine

of guanidine

PFU after

103

HCI

+o-penicillamine

pendent by concentrations

5.6

The inhibitory effect of D-penicillamine strongly decreases in the presence of Eagle’s medium (Table 8). For this reason and for the reasons expressed in the introduction, we have investigated whether this suppressing effect was due to some amino acid contained in Eagle’s medium. Therefore, all amino acids, at various concentrations, were tested against D-penicillamine. Table 9 shows that phenylalanine, tyrosine, tryptophan, and leucine were very TABLE

infection*

33 16 66 44

The similarities between the antiviral spectra of D-penicillamine and guanidine prompted us to test the action of the former against a guanidine-dependent poliovirus strain (Loddo et al., 1962). As shown in Table 6, D-penicillamine is not able to replace guanidine in sustaining the growth of guanidine-dependent polioviruses, but actually inhibits them. Moreover, D-penicillamine and guanidine act additively against a guanidine-sensitive poliovirus (Table 7).

LACK OF INTERFERENCE AND D-PENI~ILLAMINE VIRUS 1 (BRUNHENDERS)

No drug Guanidine

tigated. The effect of this drug on herpes simplex, vaccinia, vesicular stomatitis (VSV) viruses in the same cell systems was determined. In no cases was activity detected at the highest level of D-penicillamine tested (333 pg/ml).

Compounds Able to Antagonize the Inhibitory Action of D-Penicillamine on Poliovirus

1064

X

AL.

Inhibition by wPenicillamine of GuanidineDependent Poliovirus Strains

PFU produced at 12 hours in :he presence of D-penicillamine Virus

ET

Guanidine HCl

+

33 16 100 44

1.2 8.4 6 4 1.8

X

107

x

104

X x x

lo5 103 10”

x

103 104

HCI

D-penicillamine

16 + 100 16 + 44

1.3

ANTIPOLIO

ACTIVITY

OF

effective in suppressing the inhibitory action of D-penicillamine against poliovirus. Valine, methionine, and L-cysteine were found to have a weak suppressing effect on the inhibition by D-penicillamine. All other amino acids gave negative results. Choline and trimethylamine, two compounds highly active in suppressing guanidine inhibition on poliovirus (Loddo et al., 1966), were inactive against n-penicillamine. It is interesting that 5-hydroxytryptophan, dioxyphenylalanine, and cr-methyltyrosine were found inactive in antagonizing D-penicillamine inhibition of poliovirus. Actually, 5-hydroxytryptophan showed a sigTABLE INEIBITION

BY D-PENICILLAMINE

CATION OF POLIOVIRUS CELLS MAISTAIA-ED

OF THE

REPLI-

1 (BRUNHENDERS) HANKS’ BSS

KB

nificant inhibitory effect against poliovirus replication, in vitro. Moreover, since n-penicillamine is a copper-chelating agent, the influence of Cu and other metal ions on the virus inhibitory activity of D-penicillamine was checked. As shown in Table 10, the action of Dpenicillamine is antagonized by Cu and Fe salts, but not by Mn, Hg, Pb, and Zn salts. Finally, in order to establish stereospecificity of penicillamine for its inhibitory propTABLE

IN

IN OR IN

ON

THE

KB

POLIO

D-Penicillamine concentration (rdml!

-

PFU

in 12 hours

BSS

In Eagle MEM

In Hank’s 1.2 1.4 6 5 9

300 100 33 11

produced

x x X x x

107 103 103 104 105

TABLE SUPPRESSION

BY AMINO

L-Phenylalanine L-Leucine L-Tryptophan L-Tyrosine L-Isoleucine L-Valine L-Methionine L-Cysteine

acid”

X 107 X 104 X lo5 x 10” X 106

9

ACIDS

ACTION OF D-PENICILLAIMINE PLICATION OF POLIO

Amino

1.6 1.9 7.2 3 6

OF THE

INHIBITORY

ON THE MULTI1 (BRUNHENDERS)

Lowest molar concentration capable of suppressing the inhibition of n-penicillamine (44 &ml) at 12 hours 8 x 8 x 1.6 1.4 1.4 1.4 1.4

10-j 10-b 10-4 X 10-4 x 10-a x 10-a x 10-s x 10-S

a They were added to the cell cultures at the same time as D-penicillamine, i.e., 1 hour after virus infection. The following amino acids were active at concentrations above 3 X 10-a: DLethionine, DL-homocysteine, L-histidine, DL-w aminobutyric acid.

OF THE

INHIBI-

(40

1 (BRUNHENDERS)

rg/ml)

REPLICATION

CELLS

Concentration of the antagonist” b-dml)

Compounds in the medium

MEM

EAGLE

10

ANTAGONISM BY HEAVY METALS TORY ACTION OF D-PENICILLAMIKE IN

8

621

D-PENICILLAMINE

PFU produced at 12 hours

1.8 x 9 x 3 x

-

D-Penicillamine + CUSO~ + CuC12.2 Ha0 + CuCl + FeS04 + FeClg.H20 + Pb acetate + Pb(NO& + MnS04 + ZnSOe + Zn acet,ate + CaClz

0.2 0.12 0.12 10 100 25 16 50 0.5 0.33 100

2.5 8 1.5 4 9 8 9 8

X x x x x x x x

107 103 106 10” 106 lo6 106 104 104 10” 103 103 103

a The metal salt concentrations used were the maximal tolerated by cell cultures, and they did not influence viral growth. The salts were added at the same time as o-penicillamine. TABLE

11

FAILURE OF L-PENICILLAMINE (BRUNHENDERS) IN KB

CELLS

TO SUPPRESS

D-PENICILLA~\IINE

Compounds added to the infected cells L-Penicillamine L-Penicillamine D-Penicillamine n-Penicillamine + L-penicillamine a Maximum cultures for

TO INHIBIT AND ITS

concentration 12 hours.

POLIO ABILITY

ACTION

iah1

PFU produced at 12 hours

4ooa 40 40 40 +

107 4 x 106 6 X lo6 1.2 x 101 6 X lo6

40

tolerated

by

cell

1

622

GESSA,

erties, the antiviral action of L-penicillamine was studied. As shown in Table 11 this compound was completely inactive as an antiviral agent; actually it was able to suppress the antiviral action of the n-isomer. DISCUSSION

D-penicillamine has a remarkably specific action against poliovirus. Against other viruses it has little or no inhibitory effect. Dpenicillamine possesses an antiviral spectrum narrower than that of guanidine. The mechanism of action of n-penicillamine in inhibiting poliovirus replication is not clear. The lack of inhibitory action of L-penicillamine indicates that a considerable stereospecificity is required and that the antiviral action of n-penicillamine does not stem from other known biological activities which Dpenicillamine shares with L-penicillamine, i.e., Cu-chelating capacity and pyridoxine antagonism. The antagonism exerted by some amino acids reinforces our working hypothesis that n-penicillamine acts by competing with some amino acids essential for poliovirus replication. It is interesting to note that 6 out of S amino acids that antagonize n-penicillamine are among those which antagonize guanidine. Moreover, the fact that 5-hydroxytryptophan, a-methyl-1-tyrosine, and dioxyphenylalanine fail to antagonize n-penicillamine, indicate that the natural amino acids, tryptophan, tyrosine, and phenylalanine cannot be substituted even by very close analogs. Since n-penicillamine has a very low toxicity in animals and in man (Gibbs and Walshe, 1966), it deserves a further investigation as an antipolio agent in viva.

ET

AL. REFERENCES

DULBECCO, R., and VOCT, M. (1954). Plaque formation and isolation of pure lines with poliomyelitis viruses. J. Exptl. Med. 99, 167-182. ELLEM, K. A. O., and COLTER, J. S. (1960). The interaction of infectious ribonucleic acids with a mammalian cell line. I. Relationship between the osmotic pressure of the medium and the production of infectious centers. V&logy 11, 434-443. GESSA, G. L., LODDO, B., and SCHIVO, M. L. (1966). Antagonism0 da parte dell’omocisteina e dell’acido a-amino butirrico dell’attivit8 antipolio, in vitro, della guanidina. Boll. Sot. Ital. Biol. Sper. 42, 813-815. GIBBS, K., and WALSHE, J. M. (1966). Penicillamine and pyridoxine requirements in man. Lancet 175-180. GIERER, A., and SCHRAMM, G. (1956). Die Infektiosit%t der Nucleinsgure aus Tabakmosaikvirus. Z. Naturjorsch. llb, 138-142. LODDO, B., and SCHIVO, M. L. (1965). Attiviti antagonista di alcuni aminoacidi sulla inibizione da guanidina dello sviluppo de1 poliovirus in vitro. Boll. Sot. Ital. Biol. Sper. 41, 960-963. LODDO, B., FERRARI, W., SPANEDDA, A., and BROTZU, G. (1962). In vitro guanidino-resistante and guanidino-dependence of poliovirus. Experientia 18, 518. LODDO, B. GESSA, G. L., SCHIVO, M. L., SPANEDDA, A., BROTZU, G., and FERRARI, W. (1966). Antagonism of the yuanidine interference with poliovirus replication by simple methylated and ethylated compounds. Vi’irology 28, 707-712. LODDO, B., SCARPA, B., and MUNTONI, S. (1963). Infective ‘ribonucleic acid’ extraction from poliovirus by sodium dodecylsulfate. Experientia 19, 246-247. LWOFF, A., and LWOFF, M. (1964). Neutralisation par divers m&abolites de I’effet inhibiteur de la guanidine sur le dBveloppement du poliovirus. Compt. Rend. Acad. Sci. 259, 949-952.