Specificity of the Antiviral Agent Calcium Elenolate

Transcription

1 ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Oct. 1975, p Copyright i 1975 Americn Society for Microbiology Vol. 8, No. 4 Printed in U.S.A. Specificity of the Antivirl Agent Clcium Elenolte JOHN E. HEINZE, ARTHUR H. HALE, AND PHILIP L. CARL* Deprtment of Microbiology, University of Illinois, Urbn, Illinois 6181 Received for publiction 1 June 1975 Clcium elenolte, n ntivirl gent which inhibits reverse trnscriptses, inhibits the growth of chicken embryo fibroblst cells, s well s Echerichi coli nd Bcillus subtilis strins. The drug in vitro inhibits E. coli deoxyribonucleic cid (DNA) polymerse II nd DNA polymerse III holoenzyme, s well s severl unrelted enzymes. The usul DNA polymerse ssy components, with the exception of spermidine, hve no effect on the observed inhibition. Inhibition of DNA polymerse II by the drug ppers to be due to direct nd irreversible effect on the enzyme. However, DNA synthesis in E. coli is no more susceptible to the drug thn is the increse in cell mss. These results suggest tht clcium elenolte is n inhibitor of rther low specificity. Clcium elenolte, monoterpene which is isolted from queous extrcts of the olive plnt (Ole europ) fter mild cid hydrolysis (1, 14), hs been shown to be virucidl in vitro for number of ribonucleic cid (RNA) nd deoxyribonucleic cid (DNA) viruses (15). It lso reduces the yields of virus from hmsters infected with prinfluenz 3 virus (16) nd hs miniml toxicity when dministered to nimls (2). Clcium elenolte inhibits the RNA-dependent DNA polymerses (reverse trnscriptses) of both Moloney nd Ruscher leukemi viruses, 5% inhibition occurring t clcium elenolte concentrtions of 2 ug/ml (75 gm) (5). The drug does not pper to rect with nucleosides or with DNA or RNA (5, 15). It hs been suggested tht the drug cts directly ginst the reverse trnscriptses (5). The drug cn be inctivted by prior incubtion with 2-M equivlents of the mino cids glycine, lysine, cysteine, or histidine, but not with other mino cids (15). Inctivtion requires the presence of free mino group. However, the drug does not inhibit Escherichi coli RNA polymerse or DNA polymerse I t drug concentrtions s high s 2,ug/ml (5). Recently the structure of elenolic cid (1) nd the totl synthesis of DL-methyl elenolte hve been reported (6). We decided to determine if the drug inhibited E. coli DNA polymerse II or Ill since reverse trnscriptses resemble these enzymes in number of properties (7). A specific inhibitor of either E. coli enzyme might prove useful in elucidting the physiologicl roles of these enzymes in the cell. However, we report tht both enzymes re inhibited to similr extent nd, moreover, further studies indicte tht the drug is rther nonspecific. 421 MATERIALS AND METHODS Mterils. Tissue culture dishes were from Flcon Plstics. Powdered Dulbecco modified Egle medium, tryptose phosphte broth, clf serum, nd chicken serum were obtined from Grnd Islnd Biochemicl Co. [3H ]thymine, 7. Ci/mmol, ws purchsed from Schwrz/Mnn. [3H ideoxythymidine 5'-triphosphte, 5 Ci/mmol, ws obtined from New Englnd Nucler. Clf thymus DNA (type V) ws obtined from Sigm nd digested with pncretic deoxyribonuclese (DNse) I (Worthington, EC ) to give mximl DNA polymerse III ctivity (18). Clcium elenolte, lot 9426-JHF-2, ws the generous gift of the Upjohn Compny, Klmzoo, Mich. When dissolved in wter t 1 to 1 mg/ml nd stored frozen, the drug ppered stble for t lest 2 months with repeted freezing nd thwing. Growth of bcteri. The E. coli strins W311 (thya36 nd dr-2) nd D11 (polal, enda-, thya36 nd dr-2) were obtined from the E. coli Genetic Stock Center, New Hven, Conn. The Bcillus subtilis prototrophic strin WB746, isolted by Eugene Nester, ws obtined from Smuel Kpln. Bcteri were grown in M9 miniml medium (4), supplemented with.2% glucose, 1 mm MgSO4, nd 4 ug of thymine per ml. Medium for plting ws solidified with 1.5% gr (Difco). Cell mss ws determined by opticl density t 45 nm in Zeiss PMQII spectrophotometer. Totl cell numbers were determined by counting 2 to 4 cells in Petroff- Huser chmber. The number of vible cells ws determined by plting on M9 gr fter pproprite dilutions. DNA synthesis ws mesured by the incorportion of [3HJthymine into cid-insoluble counts (4) Ġrowth of chicken embryo fibroblsts. Primry chicken embryo fibroblsts were prepred from 1- dy-old chicken embryos s previously described (17). After 4 to 5 dys of growth t 38 C, the cells were removed from the tissue culture pltes with.5% trypsin in tris(hydroxymethyl)minomethne (Tris)- Downloded from on My 14, 218 by guest

2 422 HEINZE, HALE, AND CARL hydrochloride plus sline (25 mm Tris-hydrochloride [ph 7.3], 14 mm NCl, 5 mm KCl, nd.7 mm N2HPO4) nd plted t 15 cells/35-mm tissue culture plte. Cells were grown t 41 C in Dulbecco medium contining 1% tryptose phosphte broth, 4% clf serum, nd 1% het-inctivted (56 C for 1 h) chicken serum. All experiments were begun 24 h fter plting. Growth rtes were determined by mesuring cell number with Coulter prticle counter fter removing cells from the pltes with.25% trypsin in Tris-hydrochloride plus sline plus.1% glucose. Uptke of trypn blue,.4% in.9% sline, ws used to estimte cell vibility. In severl experiments the cells were wshed with phosphte-buffered sline, which contined 14 mm NCl, 5.4 mm N2HPO4, 5 mm KCl, 1.8 mm CCl2, 1 mm NH2PO4,.8 mm MgSO4, nd.1% glucose, ph 7.3. After wshing, cells were grown in conditioned medium, which is tht removed from untreted pltes fter 24 h of cell growth. Enzyme inhibition studies. Egg white lysozyme (A grde, EC ) ws obtined from Sigm. Micrococcl nuclese (NFCP, EC ) nd pncretic DNse I (DPFF, EC ) were obtined from Worthington. These enzymes t 4 to 5,g/ml were exposed to clcium elenolte for 3 min t 37 C in 5 mm Tris-hydrochloride, ph 8., t 37 C, nd then ssyed s described by the supplier. E. coli DNA polymerse II ws purified by the method of Kornberg nd Gefter (9), followed by dilysis, phosphocellulose chromtogrphy, nd Sephdex G-15 gel filtrtion (8). The enzyme hd finl specific ctivity of 125 U/mg nd ws stored s recommended by Otto et l. (13). The stndrd ssy for DNA polymerse II contins (.3-ml volume): 33 mm Tris-cette buffer (ph 7.2 t 3 C), 5 mm KCI, 16.7 mm dithiothreitol, 13.3 mm MgCl2, 28 MM (s nucleotide) DNsedigested clf thymus DNA, 33 AM (ech) deoxydenosine 5'-triphosphte, deoxycytosine 5-triphosphte, deoxygunosine 5'-triphosphte, nd [3H Ideoxythymidine 5'-triphosphte (15 counts/min per pmol), bout.7 Mg of enzyme (estimted by ssuming preprtion of specific ctivity 27 U/mg is substntilly pure [8]) nd indicted concentrtions of clcium elenolte. Incubtions were for 3 min t 37 C; nucleotide incorportion into cid-insoluble product ws mesured s previously described (11). Crude extrcts of DNA polymerse III holoenzyme (frction 1) were prepred s described by Wickner nd Kornberg (19) from D11 grown in M9, supplemented with 1% glucose nd.2% Csmino Acids (Difco), to 3.5 x 18/ml. The stndrd ssy for DNA polymerse III holoenzyme contins (.3-ml volume): 33 mm morpholinopropne sulfonte-koh buffer (ph 7.), 16.7 mm dithiothreitol, 6.7 mm MgCl2, 5 mm NCl, 1.4 mm (s nucleotide) DNsedigested clf thymus DNA, 1.3 mm denosine 5'-triphosphte,.15 mg of bulk E. coli phospholipids per ml (19), 133 MM (ech) deoxydenosine 5'-triphosphte, deoxycytosine 5'-triphosphte, deoxygunosine 5'-triphosphte, nd [3H ideoxythymidine 5'-triphosphte (15 counts/min per pmol), bout.1 Mg of enzyme (estimted by ssuming preprtion of specific ctivity 55 U/mg is substntilly pure [191), nd indicted concentrtions of clcium elenolte. Incubtions were for 5 min t 3 C; nucleotide incorportion into cid-soluble product ws mesured s previously described (11). DNA polymerse II ctivity is not detectble in crude extrcts in this ssy (9). RESULTS Spectrum of enzyme inhibition. As shown in Fig. 1, clcium elenolte inhibited both E. coli DNA polymerse II nd DNA polymerse III holoenzyme. Consistent with the previously published report (5), control experiments (dt not shown) hve indicted tht the inhibition of DNA polymerse II by clcium elenolte is not influenced by ny of the stndrd DNA polymerse II or DNA polymerse III holoenzyme ssy components except spermidine, which therefore ws not used in the ssy of DNA polymerse III holoenzyme ctivity in extrcts. Thus the pprent differences in the susceptibility of the two enzymes cnnot be explined by the differences in the enzyme ssys. Inhibition of DNA polymerse II ctivity by clcium elenolte could not be overcome by incresing the concentrtion of DNA, deoxyribonucleoside triphosphtes, or mgnesium, but could be overcome by incresing the concentrtion of enzyme (Tble 1). Thus inhibition ppers to be directed ginst the enzyme itself. Furthermore, the inhibition of DNA polymerse II by clcium elenolte ppered to be irreversible (Tble 1). DNA polymerse II ws exposed to 5 Ag of clcium elenolte per ml for 5 min t 37 C nd then diluted 3-fold into the stndrd ssy mixture, which contined no cl- N EC w ' 5' ANTIMICROB. AGENTS CHEMOTHER. u A V 2 5L I S S Colcium Elenolle, pg/ml. FIG. 1. Inhibition of severl enzymes by clcium elenolte. The enzymtic ctivity observed in the bsence of the drug is tken s 1%. Symbols: A, micrococcl nuclese;, egg white lysozyme;, E. coli DNA polymerse HI;, E. coli DNA polymerse III holoenzyme; A, pncretic DNse L Downloded from on My 14, 218 by guest

3 VOL. 8, 1975 TABLE 1. Fctors which influence the inhibition of DNA polymerse II by clcium elenolte Exp conditions Observed inhibition Stndrd ssy conditions.42 Incresing DNA concentrtion fivefold to 1.4 mm.4 Incresing deoxyribonucleoside triphosphte concentrtions fourfold to 133 MM ech.42 Decresing mgnesium concentrtion fourfold to 3.3 mm Incresing enzyme concentrtion fourfold.27 Incresing enzyme concentrtion 1-fold 18 Exposure of enzyme to 5 Asg of clcium elenolte per ml for 5 min t 37 C before diluting 3-fold into stndrd ssy contining no clcium elenolte.. 66 The stndrd ssy of DNA polymerse II ws modified s indicted nd the inhibition of ctivity by 2 Mg of clcium elenolte per ml ws determined. cium elenolte. The finl concentrtion of clcium elenolte in the ssy mix ws thus 17 Mg/ml, concentrtion tht gve negligible inhibition of DNA polymerse II (Fig. 1). However, in this experiment, DNA polymerse II ws strongly inhibited compred to control exposed to wter, indicting tht the effects of exposure to 5,Mg of clcium elenolte per ml were irreversible. The effect of clcium elenolte on the ctivity of severl other enzymes is lso shown in Fig. 1. Although pncretic DNse I ws only slightly inhibited t high drug concentrtions, lysozyme nd micrococcl nuclese both were more susceptible to inhibition by clcium elenolte thn DNA polymerse II. Indeed micrococcl nuclese, which ws 5% inhibited t 2,Mg/ml (7.5,MM), ws 1-fold more susceptible to the drug thn re virl reverse trnscriptses (5). In vivo trget of inhibition in E. coli. Since DNA polymerse III, which is essentil for DNA repliction in E. coli (3, 12), ws inhibited by clcium elenolte, we wnted to determine if DNA synthesis in E. coli ws preferentilly inhibited by clcium elenolte in vivo. Preliminry experiments (dt not shown) hd estblished tht the growth of B. subtilis WB746, s well s E. coli W311, ws inhibited by clcium elenolte. Thus we exmined the effects of clcium elenolte on n exponentilly growing culture of E. coli (Fig. 2). Figure 2A is the control nd indictes tht the cells were in blnced exponentil growth s indicted by the prllel increse in ll prmeters. SPECIFICITY OF CALCIUM ELENOLATE 423 Figure 2B shows the effects of 2 Mg (.75 mm) of clcium elenolte per ml. The drug ppered bcteriosttic t this concentrtion since ll prmeters chnged in prllel from 6-min doubling time to 75-min doubling time fter 2 min of exposure to the drug. Note tht even t drug concentrtion which hd only smll effect on the growth of this strin, DNA synthesis did not pper to be more susceptible to inhibition thn ws the increse in cell mss. Figure 2C shows the effects of 5 ug (1.88 mm) of clcium elenolte per ml. DNA synthesis, cell division, nd cell growth cesed within 2 min of exposure to the drug. The drug ppered to be highly bctericidl t this concentrtion since the vible cell count dropped drmticlly fter the ddition of the drug. Effects of clcium elenolte on chicken embryo fibroblsts. It hd been reported tht clcium elenolte t concentrtion of 2 Mg/ml is nontoxic to mouse embryo fibroblst cells in culture (5). We hve shown tht the drug t this concentrtion slows the growth rte of E. coli nd inhibits number of enzymes in vitro. Since clcium elenolte cn be inctivted by mino cids (15), which re present in tissue culture medium, we hve exmined the effects of the drug on chicken embryo fibroblst cells in culture under severl experimentl conditions (Tble 2). When the cells were wshed with phosphte-buffered sline to remove mino cids in the medium nd then exposed to clcium elenolte in phosphte-buffered sline E 6 o T,me (minutes) FIG. 2. Effects of clcium elenolte on n exponentilly growing culture of E. coli W311 t 37 C in M9 medium. At the time indicted by the rrow, [3Hlthymine ws dded to 1 MCi/ml, nd the culture ws divided into three portions: (A) control; (B) 2,ug of clcium elenolte dded per ml; nd (C) 5 Mg of clcium elenolte dded per ml. I 2 S 612, E -!i,.x Downloded from on My 14, 218 by guest

4 424 HEINZE, HALE, AND CARL for 3 min, drug concentrtion of 5 Ag/ml ppered to be toxic wheres 2 Mg/ml drmticlly slowed the rte of growth. A drug concentrtion of 5,ug/ml hd no effect on the growth rte. However, s shown in Tble 2, the effects of clcium elenolte on chicken embryo fibroblst cells depended strongly on the medium nd the length of exposure to the drug. Even under the mildest conditions employed (3-min exposure in growth medium), the growth rte ws still slightly inhibited t 2,g of clcium elenolte per ml. DISCUSSION These studies with clcium elenolte hve shown tht the drug inhibits the growth of E. coli nd B. subtilis strins. The drug inhibits E. coli DNA polymerse II nd DNA polymerse III holoenzyme in vitro. The smll difference in pprent susceptibility of the enzymes cnnot be explined by differences in the ssy conditions of the enzyme, but my reflect differences in the purity of the enzyme preprtions since the drug ppers to rect directly nd irreversibly with DNA polymerse II nd inhibits severl unrelted enzymes. Clcium elenolte does not pper to function s sulfhydryl-blocking gent (5, 15), but is inctivted by incubtion with mino cids (15) nd spermidine. The drug ppers to rect with free mino groups (15). Although DNA polymerses II nd III re inhibited by clcium elenolte in vitro, DNA synthe- TABLE 2. Effects of clcium elenolte on the growth of chicken embryo fibroblst cells Clcium Rtio of doubling times Exp elenolte (experimentl culture/ (g/ml) control culture) l _b 2c d Cells wshed twice with phosphte-buffered sline (PBS), exposed to the drug in PBS for 3 min, then wshed twice with PBS before conditioned growth medium dded. b The cell number decresed slightly over 5-dy period. Twelve hours fter exposure to the drug, greter thn 9% of the cells were permeble to trypn blue. c Cells exposed to the drug in growth medium for 3 min, then wshed once with PBS before conditioned growth medium dded. d Cells continuously exposed to the drug in growth medium. ANTIMICROB. AGENTS CHEMOTHER. sis in E. coli is no more susceptible to the drug thn is the increse in cell mss. These observtions suggest tht the primry effect of clcium elenolte in E. coli is inhibition of growth, probbly by covlent rection with free mino cids nd proteins. Clcium elenolte lso ffects the rte of growth of chicken embryo fibroblst cells in culture, the extent of the effect depending on the drug concentrtion nd the experimentl conditions. Our results suggest tht cre must be exercised when the ntivirl effects (5, 15) of clcium elenolte re exmined, since not only re number of enzymes likely to be inhibited, but lso the tissue culture cells themselves my be ffected by the drug, depending on its concentrtion nd the experimentl protocol. Clcium elenolte seems to be drug of rther low specificity. Since it pprently rects only with the mino group of certin mino cids (15), it seems resonble to postulte tht the drug inhibits the vrious enzymes by recting with the e-mino group of exposed lysine residues nd the exposed N-terminl mino group of polypeptide chins. The vrition in the susceptibility of enzymes (nd cells) my reflect the number of such residues tht re exposed to the drug nd the effect of their modifiction on enzymtic ctivity. Consistent with this model is the observtion tht bovine serum lbumin is required t bovine serum lbumin to drug molr rtios of t lest 2 to 1 to prevent inhibition of micrococcl nuclese by 2,ug of clcium elenolte per ml (dt not shown). Thus the drug ppers to rect with bovine serum lbumin, contrry to published results (5), but only t smll number of residues per protein molecule. Although these results suggest tht clcium elenolte hs rther low specificity, the drug my still prove useful s probe of cellulr nd virl surfces nd of protein structure. ACKNOWLEDGMENTS We thnk Pt Chen for ble technicl ssistnce, nd The Upjohn Compny, Klmzoo, Mich., for providing generous smple of clcium elenolte. This work ws supported by Public Helth Service grnt GM from the Ntionl Institute of Generl Medicl Sciences. LITERATURE CITED 1. Beyermn, H. C., L. A. vndijck, J. Levislles, A. Meler, nd W. L. C. Veer The structure of elenolide. Bull. Sci. Chem. Frnce 1: Elliott, G. A., D. A. Buthul, nd E. N. DeYoung Preliminry sfety studies with clcium elenolte, n ntivirl gent, p Antimicrob. Agents Chemother Gefter, M. L., Y. Hirot, T. Kornberg, J. A. Wechsler, Downloded from on My 14, 218 by guest

5 VOL. 8, 1975 nd C. Brnoux Anlysis of DNA polymerses II nd Im in mutnts of Escherichi coli thermosensitive for DNA synthesis. Proc. Ntl. Acd. Sci. U.S.A. 68: Hnn, M. H., nd P. L. Crl Reinitition of deoxyribonucleic cid synthesis by deoxyribonucleic cid initition mutnts of Escherichi coli: role of ribonucleic cid synthesis, protein synthesis, nd cell division. J. Bcteriol. 121:219-2, Hirschmn, S. Z Inctivtion of DNA polymerses of murine leukemi viruses by clcium elenolte. Nture (London) New Biol. 238: Kelly, R. C., nd I. Schletter Totl synthesis of dl-methyl elenolte. J. Am. Chem. Soc. 95: Kornberg, A Eukryotic cell DNA polymerses, p In DNA synthesis. W. B. Freemn nd Compny, Sn Frncisco. 8. Kornberg, T., nd M. L. Gefter Purifiction nd DNA synthesis in cell-free extrcts: properties of DNA polymerse II. Proc. Ntl. Acd. Sci. U.S.A. 68: Komberg, T., nd M. L. Gefter Deoxyribonucleic cid synthesis in cell-free extrcts. IV. Purifiction nd ctlytic properties of deoxyribonucleic cid polymerse III. J. Biol. Chem. 247: McKellr, F. A., R. C. Kelly, E. E. vntmelen, nd C. Dorschel Structure nd stereochemistry of elenolic cid. J. Am. Chem. Soc. 95: Moses, R. E., nd C. C. Richrdson Repliction SPECIFICITY OF CALCIUM ELENOLATE 425 nd repir of DNA in cells of Escherichi coli treted with toluene. Proc. Ntl. Acd. Sci. U.S.A. 67: Nusslein, V., B. Otto, F. Bonhoeffer, nd H. Schller Function of DNA polymerse III in DNA repliction. Nture (London) New Biol. 234: Otto, B., F. Bonhoeffer, nd H. Schller Purifiction nd properties of DNA polymerse III. Eur. J. Biochem. 34: Pnizzi, L., M. L. Scrpti, nd G. Orient The constitution of Oleuropein, bitter glycoside hving hypotensive ction, from olives. Gzz. Chim. Itl. 9: Renis, H. E In vitro ntivirl ctivity of clcium elenolte, p Antimicrob. Agents Chemother Soret, M. G Antivirl ctivity of clcium elenolte on prinfluenz infection of hmsters, p Antimicrob. Agents Chemother Weber, M. J Hexose trnsport in norml nd Rous srcom virus-trnsformed cells. J. Biol. Chem. 248: Wickner, R. B., B. Ginsberg, I. Berkower, nd J. Hurwitz DNA polymerse II of Escherichi coli. I. The purifiction nd chrcteriztion of the enzyme. J. Biol. Chem. 247: Wickner, W., nd A. Kornberg A holoenzyme form of DNA polymerse III: isoltion nd properties. J. Biol. Chem. 249: Downloded from on My 14, 218 by guest