Neplanocin A - jbc.org

1 THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 259, No. 7, Issue of April 10, pp. 4353-4358, 1984. 0 1984 by The American Society of Biological Chemists, Inc. Printed in A . Neplanocin A. A POTENT INHIBITOR OF S-ADENOSYLHOMOCYSTEINEHYDROLASE AND OF VACCINIA VIRUS. MULTIPLICATIONINMOUSE L929 CELLS*. (Received for publication, November 14,1983). Ronald T. Borchardt, BradleyT. Keller, and Usha Patel-Thombre From the Departments of Biochemistry and Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66045. Neplanocin A, a novel cyclopentenyl analogof aden- AdoMet-dependent methylation reactions (5, 6). As a conse- osine, is a naturally occurring antibiotic which exhibits quence of inhibiting AdoHcy metabolism cellularmethylation significant antitumor activity against L12 10 leukemia reactions are perturbed, many of which are required formain- in mice (Yaginuma, S.)

2 , Muto, N., Tsujino, M., Sudate, tenance of the normal metabolic integrity of the cell. Y., Hayashi, M., and Otani, M. (1981)J. Antibiot. 34, An example of an essential methylation reaction is found 359-366). In the present study we demonstrate that in the maturation scheme of certain eukaryotic and viral Neplanocin A is also a potent inhibitor of S-adenosyl- messenger RNA molecules. It is known that, in many in- homocysteine (AdoHcy) hydrolase (EC ) having stances, these mRNA molecules must be both capped and a K i of nM for the purified bovine liver enzyme. Analysis of the apparent irreversible inactivation of methylated on their 5' terminus ( m7 Gpppm6A pApm ..) . to promote active translation of the corresponding proteins AdoHcy hydrolase by Neplanocin A indicates that the Downloaded from by guest on August 2, 2018.

3 (7). Methylation of the 5'-cap structure has beendemon- drug is a tight binding inhibitor, exhibiting a stoichi- strated to enhance the efficiency of initiation of translation ometry of one molecule of inhibitor to one molecule (tetramer) of enzyme. at the 5'-endof the mRNA (8). Moreover, it has been shown In addition, we show that Neplanocin A is a potent that thevaccinia virus-specific enzymes which catalyze these inhibitor of vaccinia virus(WR) multiplication inmon- reactions for viral mRNAs ( i e . guanine 7-methyltransferase;. olayer culturesof mouse L-cells. Concentrations of the 2'-0-nucleoside methyltransferase) are susceptible to inhibi- drug as low as and WM in the culture medium tion by AdoHcy (9, 10). It is not surprising, therefore, that produce 84 and 95% inhibition of plaque formation, potent inhibitors of AdoHcy hydrolase such as 3-deazaaden- respectively, while exhibitinglittle toxicity to the host osine (ll),3-deazaaristeromycin (12, 13),and adenosine cells.

4 The inhibitionof virus multiplication by neplan- &aldehyde2 elicit significantantiviral activity against viruses ocin A coincides with a rapid inhibition of AdoHcy requiring a methylated 5'-cap structure on theirmRNAs. hydrolase activity in the infected cells and a subse- Recently, the isolation and characterization of Neplanocin quent 10-fold increase in the intracellular AdoHcyIS- A ((-)-9-[truns-2,truns-3-dihydroxy-4-(hyd roxymethyl)cyclo- adenosylmethionine ratio. These findings suggestthat pent-4-enylladenine) has been reported (14, 15). This com- the antiviral actionsof this compound may be related pound, a novel carbocyclic analog of adenosine in which the to an inhibition of S-adenosylmethionine-dependent ribose moiety is replaced by a cyclopentene ring (Fig.)]

5 L),has macromolecular methylation reactions which are es- been shown to possess antitumor properties with relatively sential to the production of new virus particles ( low cytotoxicity. Considering its structuralsimilarity to aden- viral messenger RNA). osine, it is conceivable that the pharmacological activity of Neplanocin A may be mediated through interaction with an enzyme involved in adenosine metabolism, such as AdoHcy hydrolase. In this paper we report that Neplanocin A is a In recent years, S-adenosyl-L-homocysteine hydrolase (EC potent inhibitor of AdoHcy hydrolase both i n vitro and i n ) has emerged as a specific target for the design of uiuo, and that it elicits potent antiviralactivity against vac- potential chemotherapeutic agents (1-3).

6 Such an approach cinia virus (WR) inmouse L929 cells. has been prompted by the important role that this enzyme is known to play in regulating biological methylation reactions MATERIALSANDMETHODS. ( i e . modulatingtheintracellular AdoHcy'/AdoMet ratio). Neplanocin A was kindly donated by the Toyo Jozo Co., Ltd., AdoHcy hydrolasecatalyzes the reversiblehydrolysis of Japan. AdoHcy to adenosine and homocysteine. Although the equi- Purification of Bovine Liver AdoHcy Hydrohe-AdoHcy hydrolase librium of the reactionfavors synthesis, AdoHcy is efficiently was purified according to the procedure of Palmer and Abeles (16). with a final specific activity of IU (1 IU is defined as 1 pmol of hydrolyzed under physiological conditions because Ado and product formed per min/mg of protein).

7 The protein concentration Hcy are simultaneously removed by several metabolic routes ( mg/ml) was determined from the absorbance at 280 nm, (4). Inhibitionof AdoHcy hydrolase in intact cellular systems = , and by the procedure of Lowry et al. (17). The enzyme results inthe accumulation of AdoHcy, a product inhibitor of preparation was 50% pure as judged by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (18). * This work was supported by United States Public Health Service I n Vitro Assay of Purified AdoHcy Hydrohe-The assay of Research Grant GM-29332. The costs of publication of this article AdoHcy hydrolase activity in thehydrolytic direction was determined were defrayed in part by the payment of page charges. This article by a modified procedure of Richards et al.

8 (19). In a total volume of must therefore be hereby marked advertisement in accordance with 500 pl, the incubation mixture contained 150 mM potassium phos- 18 Section 1734 solely to indicate this fact. phate buffer (pH ), mM EDTA, 100 pM [2,8-3H]AdoHcy,and 4. '. The abbreviations used are: AdoHcy, S-adenosylhomocysteine; . AdoMet, S-adenosylmethionine. * B. T. Keller, and R. T. Borchardt, unpublished observations. 4353. 4354 Antiviral Action of Neplanocin A. EDTA, 40 p~ [2,8-3H]AdoHcy,and 4 units of intestinal adenosine deaminase. The reaction was started by the addition of 320 pl of cell supernatant and incubated for 60 min at 37 "C. The product [2,8-3H]. inosine was then separated from [2,8-3H]AdoHcyas described above. Determination of IntraceUuEar AdoHcy/AdoMet Ratios-Cells (3 X.)

9 Lo6) were removed from 60-mm culture dishes by trypsin-treatment and lysed in 100 pl of IU perchloric acid by rapid freezing on dry HOCHL, yy ice. The samples were stored at -70 'C prior to analysis. In prepara- tion for high pressure liquid chromatography analysis, the samples were slowly thawed and thecell debris removed by centrifugation in Pl OH OH. an Eppendorf microfuge. The supernatant (100 pl) was injected into a Perkin-ElmerSeries 3 high pressure liquid chromatograph equipped FIG. 1. Structure of Neplanocin A. with a Zorbax C-8 reverse phase column (25 cm X mm) (DuPont Co., Wilmington, DE). The AdoHcy and AdoMet were separated by a three-step gradient program at a flow rate of ml/min. Solvent units of intestinal adenosine deaminase. The reaction was started by A contained acetonitrile; Solvent B contained 50 mM sodium phos- the addition of AdoHcy hydrolase and incubated for 5 min at 37 "C.

10 Phate (pH ), 10 mM heptane sulfonic acid; Program: 5-20% A was The reaction was terminated by the addition of 100 pl of 5 N formic for 15 min; 20-40% A was for 10 min; 40-60% A was for 10 min. acid. The reaction mixture and a 500-pl wash of N formic acid Peaks were quantitated by absorption at 254 nm using a Perkin- were layered ontoaSP-Sephadex C-25 column ( X cm), Elmer Sigma Series 10-B Console Data Station. previously equilibrated in N formic acid. The column was eluted with ml of N formic acid and the eluant containing [2,8-3H]. inosine (the product of the hydrolysis of [2,8-3H]AdoHcyand subse- RESULTS. quent deamination of [2,8-3H]adenosine)was 1-ml aliquot I n Vitro Studies of Neplanocin A with Bovine LiverAdoHcy was added to 10 ml of 3a70 scintillation mixture (Research Products International, Mount Prospect, IL) and the amount of radioactivity Hydrohe-It has been demonstrated that Neplanocin A is a determined by liquid scintillation spectrometry.