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).

2 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., 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.

3 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. (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.

4 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.

5 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).]

6 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. 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.

7 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). 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).

8 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).

9 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.

10 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. (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.