Biochem. J. (1980) 187,419-428 419 Printed in Great Britain

Studies of Inhibition of Rat Synthase and Synthase

Hiroshige HIBASAMI,*§ Ronald T. BORCHARDT,t Shiang Yuan CHEN,t James K. COWARD: II and Anthony E. PEGG*1 *Department ofPhysiology and Specialized Cancer Research Center, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey, PA 17033, U.S.A., tDepartment ofBiochemistry, University of Kansas, Lawrence, KS 66045, U.S.A., and tDepartment ofPharmacology, Yale University School of Medicine, New Haven, CT 06510, U.S.A.

(Received 17 September 1979)

1. S-Adenosyl-L-methionine, S-adenosyl-L-homocysteine, 5'-methylthioadenosine and a number of analogues having changes in the base, sugar or amino acid portions of the molecule were tested as potential inhibitors of and from rat ventral prostate. 2. S-Adenosyl-L-methionine was inhibitory to these reactions, as were other nucleosides containing a sulphonium centre. The most active of these were S-adenosyl-L-ethionine, S-adenosyl-4-methylthiobutyric acid, S-adeno- syl-D-methionine and S-tubercidinylmethionine, which were all comparable in activity with S-adenosylmethionine itself, producing 70-98% inhibition at 1 mm concentrations. Spermine synthase was somewhat more sensitive than spermidine synthase. 3. 5'-Methylthioadenosine, 5'-ethylthioadenosine and 5'-methylthiotubercidin were all powerful inhibitors of both , giving 50% inhibition of spermine synthase at 10-15 ,UM and 50% inhibition of spermidine synthase at 30-45 ,UM. 4. S-Adeno- syl-L-homocysteine was a weak inhibitor of spermine synthase and practically inactive against spermidine synthase. Analogues of S-adenosylhomocysteine lacking either the carboxy or the amino group of the amino acid portion were somewhat more active, as were derivatives in which the ribose ring had been opened by oxidation. The sulphoxide and sulphone derivatives of decarboxylated S-adenosyl-L-homocysteine and the sulphone of S-adenosyl-L-homocysteine were quite potent inhibitors and were particularly active against spermidine synthase (giving 50% inhibition at 380, 50 and 20,UM respectively). 5. These results are discussed in terms of the possible regulation of polyamine synthesis by endogenous nucleosides and the possible value of some of the inhibitory substances in experimental manipulations of polyamine concentrations. It is suggested that 5'-methylthiotubercidin and the sulphone of S-adenosylhomocysteine or of S-adenosyl-3-thiopropylamine may be particularly valuable in this respect.

The correct chemical name for S-adenosyl-L-homo- Polyamine biosynthesis has been studied inten- cysteine 2',3'-acyclic derivative is 2'-{O-l(R)-hydroxy- sively in recent years because of many reports methyl(adenin -9-yl)methyl l -3' -[ S-(R)-homocysteinyll - correlating the production of these amines with cell 3'-deoxy-(S)-glycerol; S-adenosyl-L-homocysteine dialde- growth [reviewed by Tabor & Tabor (1976), Janne hyde derivative is 2'-O-l(R)-formyl(adenin-9-yl)methyll- et al. (1978) and Russell & Durie (1978)1. Con- 3'-S(R)-homocysteinyl-3 '-deoxy-(S)-glyceraldehyde; S- siderable progress in elucidating the function of the adenosyl-L-methionine dialdehyde derivative is 2'-O-K(R)- polyamines has been made by utilizing inhibitors of formyl(adenin - 9 - yl)methyll - 3' - S(R) - methioninyl - 3' - deoxy-(S)-glyceraldehyde; the full structures of Sinefungin their biosynthesis (Williams-Ashman et al., 1976; 5-[5'-deoxy-5'-(C)-adenosyll-L-ornithine } and com- Janne et al., 1978; Pegg, 1978; Mamont et al., 1978; pound A9 1 54C 5-[ 5'-deoxy-5'-(C)-4',5'-didehydro- Seiler et al., 1978; Wifliams-Ashman & Canellakis, adenosylI-L-ornithineF are given in Pugh et al. (1978). 1979). All of the inhibitors presently in use act on § Present address: Department of , Mie either ornithine decarboxylase or S-adenosylmeth- University School of Medicine, Tsu, Mie-ken S 14, Japan. ionine decarboxylase. These decarboxylases have 1I Present address: Department of Chemistry, Rensselaer been regarded as particularly appropriate targets for Polytechnic Institute, Troy, NY 12180, U.S.A. the design of inhibitors because, when assayed in S To whom reprint requests should be addressed. vitro under optimal conditions with saturating Vol. 187 0306-3275/80/050419-10$01.50/1 (c) 1980 The Biochemical Society 420 H. HIBASAMI AND OTHERS concentrations of substrates, their activity in tissue synthesis of analogues of S-adenosylhomocysteine extracts is much less than that of the corresponding have been reported earlier (Borchardt & Wu, 1974, aminopropyltransferases responsible for spermidine 1975, 1976; Borchardt et al., 1974, 1976a, and spermine synthesis (Jiinne et al., 1978). How- 1978a,b). Analogues of S-adenosylmethionine were ever, the fast turnover of the decarboxylases permits prepared as described by Borchardt et al. (1976b). rapid increases in their concentration that can 5'-Methylthiotubercidin was synthesized by the overcome even irreversible inhibitors (Mamont et procedure of Coward et al. (1977). S-Adenosyl- al., 1978; Seiler et al., 1978; Pegg, 1978, 1979), and 3-thiopropylamine sulphoxide was prepared by all of the inhibitors presently available have dis- oxidation of S-adenosyl-3-thiopropylamine with advantages. In particular, they are not very effective 30% (v/v) H202 by using a procedure similar to that at producing substantial depletion of intracellular for the preparation of S-adenosyl-L-homocysteine spermine. We have pointed out that the limiting sulphoxide (Borchardt & Wu, 1974). S-Adenosyl- factor in spermidine and spermine synthesis is the 3-thiopropylamine sulphone was prepared by supply of decarboxylated S-adenosylmethionine oxidation of S-adenosyl-3-thiopropylamine with (Pegg & Hibasami, 1979). The concentration of this ammonium molybdate and perchloric acid by using substrate for the aminopropyltransferases is very a procedure similar to that for the preparation of low in the cell and these enzymes turn over relatively S-adenosyl-L-homocysteine sulfone (Borchardt & slowly. Therefore they may well prove to be Wu, 1974). important sites at which polyamine biosynthesis can be inhibited. Furthermore, the possibility that poly- Preparation ofenzymes amine production in the cell might be subject to Ventral prostate glands were removed from large regulation by endogenous sulphur-containing nu- male Sprague-Dawley-strain rats (300-500g body cleosides has not been considered in any detail. For wt.) purchased from Charles River Breeding Labora- these reasons, the present studies were undertaken to tories, Wilmington, MA, U.S.A. Extracts containing evaluate the inhibitory potency of S-adenosylmeth- both aminopropyltransferase activities were pre- ionine, S-adenosylhomocysteine, 5'-methylthio- pared and fractionated into separate preparations of adenosine and synthetic analogues of these nu- spermidine synthase and spermine synthase by cleosides against rat spermidine synthase and sper- treatment with (NH4)2S04 followed by DEAE- mine synthase. A third rationale for these studies cellulose and Sephadex G-200 chromatography was that several of these analogues have been shown (Hannonen et al., 1972; Pajula et al., 1978). The to be potent inhibitors of various methyltransferase preparations used in the present study reactions. It is of obvious importance to determine to represented purifications of about 110-fold (sper- what extent these inhibitors interfere with poly- midine synthase) and 80-fold (spermine synthase) amine synthesis, another important cellular reaction over the specific activity present in crude ultra- utilizing S-adenosylmethionine. centrifugal prostate extracts. The spermidine syn- thase preparation catalysed the formation of 55 nmol Materials and Methods of spermidine/min per mg of protein and the Chemicals spermine synthase preparation produced 6.2 nmol of S-Adenosyl-L-methionine, 5'-methylthioadeno- spermine/min per mg of protein. These preparations sine, S-adenosyl-L-homocysteine and S-adenosyl- were not homogeneous, but the synthases were free L-ethionine were purchased from Sigma Chemical from each other and neither preparation contained Co., St. Louis, MO, U.S.A., and purified as significant amounts of S-adenosylmethionine de- described by Pegg & Williams-Ashman (1969a,b). carboxylase [less than 1 pmol of '4CO2 pro- S-Adenosyl-L-[Me-'4Clmethionine (50-60 mCi/ duced/min per mg of protein under assay conditions mmol), [1,4-'4Clputrescine (52mCi/mmol) and of Pegg (1979)] or of 5'-methylthioadenosine phos- [tetramethylene- 1,4-'4Clspermidine (62 mCi/mmol) phorylase [(less than 100pmol of methylthioribose were purchased from New England Nuclear Corp., 1-phosphate produced/min per mg of protein under Boston, MA, U.S.A. Decarboxylated S-adenosyl- assay conditions of Pegg & Williams-Ashman methionine, both unlabelled and labelled in the (1969b) with the method of Zappia et al. (1978)1. methyl group, was prepared by the action of S- adenosylmethionine decarboxylase from Escherichia Assay ofaminopropyltransferase activities coli and purified by chromatography on Dowex-50 Aminopropyltransferase activity was determined (H+ form) and high-voltage paper electrophoresis by measuring the production of 5'-[Me-14Clmethyl- (Poso et al., 1976). All other biochemicals thioadenosine from decarboxylated S-adenosyllMe- were products of the Sigma Chemical Co. '4CImethionine in the presence of putrescine (sper- midine synthase) or of spermidine (spermine syn- Preparation ofinhibitors thase) as described by Hibasami & Pegg (1978a). Except for the compounds described below, The assay medium contained 100mM-sodium phos- 1980 INHIBITION OF SPERMIDINE SYNTHASE AND SPERMINE SYNTHASE 421

phate buffer, pH 7.5, 42pM decarboxylated S-aden- 100 osyl[Me-14C]methionine (2,uCi/,umol), 5 mM-dithio- threitol, 0.5 mM-putrescine or 0.5 mM-spermidine, 0 and the enzyme extract in a total volume of 0.2 ml. o 80 The results were expressed as percentages of the control (without addition of inhibitor) activity. -60 -; Sufficient enzyme was added to achieve 5000- 60 10000c.p.m. incorporation into 5'-methylthio- adenosine in 30min incubation at 370C. 40 In some experiments the labelled decarboxylated S-adenosylmethionine was replaced by unlabelled material and radioactive putrescine or spermidine 20 was utilized to follow the reaction. The substrate and product were then separated by high-voltage paper electrophoresis (Hibasami & Pegg, 1978a). < 0 0.1 0.2 0.3 0.4 0.5 1.0 [S-Adenosyl-L-methioninel (mM) Results Fig. 1. Inhibition of spermidine synthase and spermine svnthase by S-adenosvlmethionine Nucleosides were tested for their ability to inhibit The results are expressed as percentages of the spermidine synthase and spermine synthase by control activity measured in the absence of S- addition at concentrations of 0.1 and 1 mm to a adenosylmethionine. Experimental details are given standard assay medium containing 42pM decar- in the text. Results are shown for spermidine boxylated as the synthase (black symbols) assayed by production of S-adenosylmethionine propyl- ['4Clmethylthioadenosine in the presence (0) or amine donor and 0.5 mM-putrescine or -spermidine absence (U) of methylglyoxal bis(guanylhydrazone) as acceptor. Assays were incubated at 370C for or by the incorporation of 1'4Clputrescine into 30min and the amount of 5'-methylthioadenosine spermidine (A). Values for spermine synthase acti- produced was measured. Production of 5'-methyl- vity (open symbols) are shown for assays of the thioadenosine was entirely dependent on the addi- production of l'4Clmethylthioadenosine in the pres- tion of the putrescine acceptor when spermidine ence (0) or absence (O) of methylglyoxal synthase was used and on the presence of the bis(guanylhydrazone) or of the incorporation of spermidine acceptor when spermine synthase was [I4Clspermidine into spermine (A). employed. The results are shown in Table 1. Part 1 of Table 1 shows the results obtained when nucleosides containing a sulphonium centre were tested. S-Adenosyl-L-methionine itself was quite strongly inhibitory, giving about 90% inhibition of the L-isomer. As previously reported with less both enzymes at 1 mM concentrations. Spermine purified enzyme extracts (Hibasami & Pegg, 1978b), synthase was more sensitive than was spermidine S-adenosyl-L-ethionine in which the methyl group synthase (Table 1 and Fig. 1). This inhibition was attached to the sulphonium centre is replaced by an not due to the conversion of S-adenosyl-L-meth- ethyl group was also an inhibitor. In the present ionine into the decarboxylated derivative (which if experiments the inhibition by this compound was formed could dilute the specific radioactivity of the somewhat greater than that found before. This may labelled substrate) because the enzyme preparations be partly due to impurities present in the former used were free of S-adenosylmethionine decarboxy- preparation and partly to the more highly purified lase activity. This was confirmed by addition to the enzymes used in the present work. The replacement assays of 10M-methylglyoxal bis(guanylhydra- of methionine by S-methylcysteine, which has one zone), which did not decrease the inhibition by carbon atom less, substantially diminished the S-adenosyl-L-methionine (Fig. 1). Methylglyoxal inhibitory potency. However, S-adenosyl-4-methyl- bis(guanylhydrazone) is a potent inhibitor of S- thiobutyric acid, the deaminated derivative of the adenosylmethionine decarboxylase (Williams- amino acid, was strongly inhibitory to both enzy- Ashman & Schenone, 1972). Furthermore a similar mes, showing that the amino group is not required inhibition of spermidine and spermine synthesis was for binding at the inhibitory site. The intact ribose observed when S-adenosyl-L-methionine was added moiety did appear to be needed, because oxidation of to assay mixtures in which the reaction was the cis-hydroxy groups with periodic acid, yielding measured by following the conversion of labelled S-adenosyl-L-methionine dialdehyde derivative, putrescine or spermidine into the product (Fig. 1). diminished inhibition. S-Adenosyl'D-methionine was also inhibitory to A number of compounds related to S-adeno- the aminopropyltransferases but less active than was syl-L-methionine but having different bases were also Vol. 187 422 H. HIBASAMI AND OTHERS

Table 1. Inhibition of aminopropvltransferases by sub- Table I.-(continued) strate and product analogues The compounds were tested by addition to the Inhibition (%) standard assay medium at the concentrations shown. Experimental details are given in the text. The Concn. Spermidine Spermine percentage inhibition due to the addition is shown. Compound (mM) synthase synthase Sinefungin 1 4 10 Inhibition (%) 0.1 0 2 Compound A9154C 1 53 93 Compound Concn. Spermidine Spermine 0.1 22 70 (mM) synthase synthase S-Adenosyl-L-homo- 1 31 40 cysteine dialdehyde 0.1 3 12 Part 1. Analogues of S-adenosylmethionine derivative S-Adenosyl-L-methionine 1 88 95 0.1 30 75 S-Adenosyl-L-homo- 1 41 58 S-Adenosyl-L-ethionine 1 91 93 cysteine 2',3'-acyclic 0.1 6 10 0.1 36 51 derivative S-Adenosyl-S-methyl- 1 49 74 S-Adenosyl-L-homo- 1 11 24 L-cysteine 0.1 31 cysteine sulphoxide 0.1 3 5 S-Adenosyl-3-thiopropyl- 1 79 61 S-Adenosyl-D-methionine 1 74 91 amine sulphoxide 0.1 28 14 0.1 19 37 (decarboxylated S- S-Adenosyl-4-methylthio- 1 98 97 adenosylhomocysteine butyric acid 0.1 85 84 sulphoxide) S-Adenosyl-L-methionine 1 57 47 S-Adenosyl-L-homo- 99 84 dialdehyde derivative 0.1 16 8 cysteine sulphone 0.1 S-3-Deaza-adenosyl-L- 1 48 61 82 33 methionine S-Adenosyl-3-thiopropyl- 95 80 0.1 10 14 amine sulphone (decarb- 0.1 63 25 S-N6-Methyl-3-deaza- 1 31 1 oxylated S-adenosyl-L- adenosyl-L-methionine 0.1 7 0 homocysteine sulphone) S-7-Deaza-adenosyl- 1 70 83 S-3-Deaza-adenosyl- 1 6 L-methionine (S-tuber- 0.1 23 37 27 cidinylmethionine) L-homocysteine S-N6-Methyl-3-deaza- 1 2 S-Inosyl-L-methionine 1 20 6 7 0.1 5 0 adenosyl-L-homocysteine 1 S-7-Deaza-adenosyl- 1 19 1 7 S-Uridyl-L-methionine 5 2 L-homocysteine S-Cytidyl-L-methionine 1 8 0 S-8-Aza-adenosyl- 1 1 0 L-homocysteine Part 2. Analogues of 5'-methylthioadenosine S-2-Aza-adenosyl- 1 0 4 5'-Methylthioadenosine 1 95 99 L-homocysteine 0.1 82 98 S-N6-Methyladenosyl- 1 6 13 5'-Ethylthioadenosine 1 95 97 L-homocysteine 0.1 82 90 S-Guanyl-L-homocysteine 1 2 9 5'-Methylthio-7-deaza- 1 95 99 S-Inosyl-L-homocysteine 1 4 4 adenosine (5'-methyl- 0.1 64 91 S-Uridyl-L-homocysteine 1 2 4 thiotubercidin) S-Cytosyl-L-homocysteine 1 3 9

Part 3. Analogues of S-adenosyl-L-homocysteine S-Adenosyl-L-homo- 1 7 47 cysteine 0.1 0 12 S-Adenosyl-3-thiopropyl- 1 55 65 tested as inhibitors, but none was as active as the amine (decarboxylated 0.1 4 12 parent compound. Replacing the adenine by ino- S-adenosylhomo- sine, uracil or cytidine resulted in the complete loss cysteine) of inhibitory activity, indicating the importance of S-Adenosyl-3-N-acetyl- 1 12 34 the adenine base for binding to the enzyme. 0.1 0 8 thiopropylamine Analogues containing derivatives of adenine were S-Adenosyl-4-thiobutyric 1 41 72 inhibitory, the 3-deaza and 7-deaza acid derivatives S-Adenosyl-4-thio- 1 59 85 showing activity approching that of S-adenosyl- butyrate methyl ester 0.1 17 52 methionine itself. Addition of a methyl group at N-6 S-Adenosyl-L-cysteine 1 21 62 substantially diminished the inhibitory potency. 0.1 0 15 A product of both aminopropyltransferase reac- S-Adenosyl-D-homo- 1 0 5 tions is 5'-methylthioadenosine. This product has cysteine 0.1 2 0 previously been reported to be inhibitory to sper-

1980 INHIBITION OF SPERMIDINE SYNTHASE AND SPERMINE SYNTHASE 423 mine synthase (Hibasami & Pegg, 1978b; Pajula & active. Surprisingly, the sulphone and its decar- Raina, 1979), and as shown in Table 1 it was a boxylated derivative were quite strongly inhibitory to powerful inhibitor, producing virtually complete both enzymes, but particularly active against sper- inhibition at 0.1 mm concentrations. This inhibition midine synthesis. was greater than found in our earlier experiments in Modifications to the base moiety of S-adeno- which a crude enzyme preparation was used sylhomocysteine were deleterious to the inhibitory (Hibasami & Pegg, 1978b). It is probable that, as action towards spermine synthase. Only the 3-deaza- suggested by Pajula & Raina (1979), this dis- and 7-deaza-adenosine derivatives had any sig- crepancy was due to the presence of 5'-methyl- nificant activity. Substitutions of methyl or ethyl thioadenosine phosphorylase in the crude enzyme groups at N-6 of adenosine, modifications forming fractions, which could have lowered the concen- 2-aza or 8-aza derivatives or replacement with other trations of the added nucleoside. Spermidine syn- purines or pyrimidines led to the loss of all activity thase was also inhibited by 5'-methylthioadenosine, (Table 1). although not quite so potently as was spermine Detailed kinetic investigations of the mode of synthase. Previous studies did not show inhibition action of the various compounds found to be strong of spermidine synthesis by 5'-methylthioadenosine inhibitors were not attempted because of the (Williams-Ashman et al., 1972; Hibasami & Pegg, complex kinetics of the aminopropyltransferases. In 1978b), but, as mentioned above, the enzyme confirmation of a previous report (Coward et al., preparations used contained 5'-methylthioadeno- 1977), it was found that spermidine synthase was sine phosphorylase. 5'-Ethylthioadenosine was prac- strongly inhibited at high substrate concentrations tically as active as the methyl derivative in inhibiting (Fig. 2). It was also found that the apparent Km both enzymes, and 5'-methylthiotubercidin, in which the adenine was replaced by 7-deaza-adenine, was only slightly less potent. The remainder of Table 1 (part 3) details the effects on these reactions of S-adenosylhomocy- 80r steine and related compounds. Spermidine synthase was not significantly affected by S-adenosyl-L- homocysteine, but, in agreement with our previous 7070 report (Hibasami & Pegg, 1978b), spermine syn- x thase was inhibited by 1 mm concentrations. Modi- E 60 fication of the amino acid portion of the molecule by ciC) removal of either the carboxy or the amino group increased the inhibitory activity somewhat for both £ 50 enzymes. Decarboxylated S-adenosylhomocysteine and the methyl ester of S-adenosyl-4-thiobutyric acid inhibited both enzymes by more than 50% when present at 1 mm concentrations. The removal 1.- of one carbon atom from the amino acid moiety ° 30 (S-adenosyl-L-cysteine) did not significantly alter the inhibition of spermine synthase. The antifungal 0 t 020 antibiotic Sinefungin, which is related to S-adenosyl- = 3 homocysteine but has the sulphur atom replaced t: 10 by -CH(NH2)- (see footnote on title page), was not inhibitory at the concentrations used, but the related compound A9145C was strongly inhibitory, parti- cularly to spermine synthase. S-Adenosyl-D-homo- cysteine was inactive against either enzyme. Altera- 0 40 80 120 160 200 240 tions to the ribose moiety of the molecule where the 1 /l Decarboxylated S-adenosylmethionineJ (mM- ') C-2'-C-3' bond was cleaved, producing either the Fig. 2. Effect of concentration of decarboxylated S- dialdehyde or the corresponding alcohol, did not adenosvlmethionine on the spermidine synthase reaction affect the inhibition of spermine synthesis sig- A double-reciprocal plot of the formation of 5'- [Me- 4Clmethylthioadenosine at different concen- nificantly, but these compounds were slightly more trations of decarboxylated S-adenosylmethionine is potent inhibitors of spermidine production than was shown. Assays were carried out by incubation for S-adenosylhomocysteine itself. 15min at 370C in the presence of 0.5mM-put- The sulphoxide of S-adenosylhomocysteine was rescine. Experimental details are given in the text. only slightly inhibitory to both enzymes, but the The formation of product was linearly related to the decarboxylated derivative was significantly more time of incubation for at least 30 min. Vol. 187 424 H. HIBASAMI AND OTHERS values for putrescine or spermine varied greatly compounds found to be inhibitory, the effect of according to the concentration of decarboxylated inhibitor concentration was tested under the stan- S-adenosylmethionine used as substrate (A. E. Pegg dard assay conditions. All the inhibitors showed & H. Hibasami, unpublished work). In order to clear dependence on concentration added. Results obtain some estimate of the relative potency of the are shown in Fig. 1 for S-adenosyl-L-methionine, in Fig. 3 for S-adenosylhomocysteine sulphone and in Fig. 4 for 5'-methylthiotubercidin. S-Adenosyl-L- methionine and 5'-methylthioturbercidin were clearly much more inhibitory towards spermine synthase °. 100 (producing 50% inhibition at about 30 and 15,UM respectively) than towards spermidine synthase (210 0 and 45,UM). S-Adenosyl-L-homocysteine sulphone so 80 (Fig. 3) was much more inhibitory to spermidine I-I synthase, producing 50% inhibition at 20,UM, ,) 60 whereas spermine synthase required 2 10pM. Cd Table 2 summarizes the inhibitory potential of those compounds showing activity in the experi- 0 40 ments of Table 1. At least six concentrations over 0. Experimental details are given in the text. Only those compounds effective to inhibit by 25% at 0 0.1 mm were included. S.U 0 Concn. needed for 50% inhibition (UM) 0 - f CU Spermidine Spermine Compound synthase synthase :U 0. S-Adenosyl-L-methionine 210 30 -01cA S-Adenosyl-L-ethionine 185 95 c) S-Tubercidinyl-L-methionine 305 175 0. 0 S-Adenosyl-4-methylthio- 40 40 butyric acid 1- 5'-Methylthioadenosine 30 10 az 0 5'-Ethylthioadenosine 30 15 ._ 5'-Methylthiotubercidin 45 15 220 0 0.1 0.2 0.3 S-Adenosyl-4-thiobutyric acid <500 15'-MethylthiotubericidinJ (mM) S-Adenosyl-4-thiobutyrate 500 75 methyl ester Fig. 4. Inhibition of spermidine synthase and sperrmine Compound A9154C 790 35 svnthase by 5'-methylthiotubercidin S-Adenosyl-3-thiopropylamine 380 715 The results are expressed as percentages of tthe sulphoxide control activity measured in the absence of tthe S-Adenosyl-L-homocysteine 20 210 inhibitor. Experimental details are given in the text. sulphone Results are shown for spermidine synthase (a) a nd S-Adenosyl-3-thiopropylamine 50 310 spermine synthase (0). sulphone 1980 INHIBITION OF SPERMIDINE SYNTHASE AND SPERMINE SYNTHASE 425 compounds for which 50% inhibition was achieved steine, which already contain a propylamine group at less than 100#uM. The only nucleosides that and therefore do not need prior decarboxylation. showed this degree of inhibition towards spermidine Such an effect would appear to be an inhibition, since synthase were S-adenosyl-L-homocysteine sulphone the labelled substrate used in our standard assay was (20,UM), S-adenosyl-3-thiopropylamine sulphone the usual propylamine donor, decarboxylated S- (50,UM), S-adenosyl-4-methylthiobutyric acid adenosylmethionine. All of the inhibitory com- (40,uM), 5'-methylthioadenosine (30uM), 5'-ethyl- pounds shown in Table 2 were therefore tested for thioadenosine (30M) and 5'-methylthiotubercidin their abilities to support the reaction with a different (45,UM). The first two compounds were respectively assay method in which conversion of labelled 10 and 6 times more active against spermidine putrescine into spermidine or of labelled spermidine synthase than against spermine synthase. The into spermine was measured. In the standard assay greatest specificity towards spermine synthase was approx. 9000 c.p.m. was incorporated into the seen for compound A9154C, which was at least product, but when decarboxylated S-adenosylmeth- 20 times more active towards this enzyme than ionine was replaced by the other compounds no towards spermidine synthase. Other compounds significant radioactivity was found (Table 3). that were selectively active against spermine Neither S-adenosylmethionine nor S-adenosylethio- synthesis were S-adenosyl-L-methionine, S-adenosyl- nine was active, emphasizing the absence of S- 4-thiobutyrate methyl ester and 5 '-methylthio- adenosylmethionine decarboxyalse from the pre- tubercidin. These were 3-7 times more potent parations of enzymes used. inhibitors of spermine rather than spermidine Inhibition of spermidine synthase by S-adeno- synthesis. syl-L-homocysteine sulphone and of spermine syn- It was possible that some of the compounds tested thase by 5'-methylthiotubercidin was studied in in these studies were, in fact, substrates able to more detail because of the potential usefulness of donate a propylamine group to the amine acceptor. these compounds as inhibitors of polyamine pro- This was a particularly important problem for the duction in vitro. Both compounds were reversible decarboxylated derivatives of S-adenosylhomocy- inhibitors. After dialysis overnight to remove them from the enzyme, activity was restored to that found in preparations treated similarly except for exposure to the inhibitor. Neither compound inhibited more strongly if preincubated with the enzyme in the absence of one or both substrates. Table 3. Abilities of various nucleosides to act as substratesfor aminopropyltransferases Discussion Assays were carried out in the standard medium but with decarboxylated S-adenosylmethionine being Mammalian aminopropyltransferases have not replaced by 1 mm concentrations of the nucleoside been studied extensively. However, the information shown and with '4C-labelled putrescine or spermidine that is available clearly indicates that, unlike being used to follow the reaction. Experimental Escherichia coli, which contains only one amino- details are given in the text. N.D., Not determined. propyltransferase preferring putrescine as acceptor but able to synthesize spermine under certain Spermidine Spermine synthase activity synthase activity conditions (Bowman et al., 1973), mammalian cells (c.p.m./30min (c.p.m./30 min contain distinct spermidine synthase and spermine Addition incubation) incubation) synthase (Pegg & Williams-Ashman, 1969a, 1970; Decarboxylated 9961 8555 Hannonen et al., 1972; Hibasami & Pegg, 1978a,b; S-adenosyl- Pajula et al., 1978). The present work confirms the methionine existence of separate enzymes and emphasizes their S-Adenosyl-L- 37 62 different properties in that their responses to some of methionine the inhibitors were quite different. This could have S-Adenosyl-L- 45 N.D. important physiological and pharmacological con- ethionine sequences, as discussed below. S-Adenosyl-L- 12 12 The present results may also provide some homocysteine information on the interaction between decarboxyl- sulphone S-Adenosyl-3-thio- 15 18 ated S-adenosylmethionine and the enzymes. The propylamine complete absence of inhibitory action by analogues sulphoxide of S-adenosylmethionine or S-adenosylhomocy- S-Adenosyl-3-thio- 14 23 steine containing bases other than adenine or its 3- propylamine and 7-deaza derivatives suggest that a purine with a sulphone 6-amino group is essential. The diminished inhibi- Vol. 187 426 H. HIBASAMI AND OTHERS tion by the N6-methyl derivatives suggest that RNA methylases (Borchardt, 1977; Leboy et al., binding involves a free amino group at the 6- 1978; Borchardt & Pugh, 1979). However, their position. The results with the amino acid variants of uptake into cells and biological stability have not yet S-adenosylmethionine suggest that the adenosyl been fully evaluated. Although 5'-methylthioadeno- moiety (preferably with an intact ribose ring) and a sine and 5'-ethylthioadenosine were strong inhibi- sulphonium centre are sufficient to permit inhibitory tors, the ubiquitous distribution and high activity of binding. The (l-amino group of S-adenosyl-L-meth- 5'-methylthioadenosine phosphorylase, which at- ionine was clearly not needed for inhibition, since tacks both of these compounds (Pegg & Williams- S-adenosyl-4-methylthiobutyric acid was a potent Ashman, 1969b; Garbers, 1978; Zappia et al., inhibitor. However, since S-adenosyl-D-methionine 1978), may limit their ability to inhibit polyamine and S-adenosyl-S-methyl-L-cysteine were less active production in vivo. However, 5'-methylthiotuber- than S-adenosyl-L-methionine, the presence of a side cidin is not degraded by this enzyme but is a chain of appropriate length and stereospecificity is of competitive inhibitor (Coward et al., 1977; Zappia some importance. et al., 1978). It could therefore inhibit polyamine It should be pointed out that the synthetic synthesis both by a direct effect and indirectly by sulphonium compounds used in this study (all those preventing the breakdown of 5'-methylthioadeno- except S-adenosyl-L-methionine, S-adenosyl-L-eth- sine. 5'-Methylthiotubercidin and 5'-methylthio- ionine and decarboxylated S-adenosylmethionine) adenosine were found to inhibit proliferation of are epimeric mixtures of the enantiomers at the human lymphocytes stimulated to proliferate by sulphur atom. It is probable that the enzyme mitogens (Ferro, 1979), but it is not known whether reactions use only one of these enantiomers of the effect is mediated or accompanied by changes in decarboxylated S-adenosylmethionine (Poso et al., polyamine concentrations. At present 5'-methyl- 1976; Coward et al., 1977), which is probably the thiotubercidin appears the most likely compound to S-configuration since Cornforth et al. (1977) have be of value in inhibiting spermine synthesis, but there shown that naturally occurring S-adenosylmeth- are many other known analogues of 5'-methyl- ionine exists in this form. Therefore it is quite thioadenosine that appear worthy of consideration in possible that of those compounds existing as this respect (Montgomery et al., 1974). epimeric mixtures one enantiomer is much more The results in Table 1 should also be considered active than the other, and care should be exercised in when analogues of S-adenosylmethionine, S-adeno- comparing results between two compounds one of sylhomocysteine and 5 '-methylthioadenosine are which was a mixture and the other a single used with the aim of inhibiting methyltransferase enantiomer. reactions. The present experiments suggest that a Almost all of the compounds containing a wide variety of such compounds also have the sulphonium centre were more potent inhibitors of potential to interfere with polyamine synthesis. The spermine synthesis than of spermidine production. compounds SIBA (5'-deoxy-5'-S-isobutylthio- This was also the case with the three 5'-alkyl- adenosine), Sinefungin and compound A9145C have thio-5 '-deoxynucleosides tested. Perhaps the most evoked considerable interest because of their anti- surprising finding in the present experiments is the viral and oncostatic properties (Pugh et al., 1978; inhibitory action of certain derivatives of S-adeno- Robert-Gero et al., 1979; Borchardt & Pugh, 1979). sylhomocysteine, although we had previously obser- These may be due to inhibition of methylation, ved some inhibition of crude preparation of sper- particularly in the case of Sinefungin and compound mine synthase by 1 mm concentrations of S-adeno- A9145C, which are very strong inhibitors of viral sylhomocysteine itself (Hibasami & Pegg, 1978b). mRNA (guanine-7) methyltransferase and mRNA The inhibitory potential of the sulphoxide derivative (nucleoside-2') methyltransferase (Pugh et al., 1978; of decarboxylated S-adenosylhomocysteine might Borchardt & Pugh, 1979), but inhibition of poly- have been expected, since the polar sulphoxide could amine synthesis may also occur with compound be thought of as analogous to the sulphonium centre. A9154C and with SIBA, which is closely related to This compound therefore may resemble the normal 5 ' -methylthioadenosine. decarboxylated S-adenosylmethionine substrate. Although it may appear at first sight that those However, the very strong inhibitory action of the compounds requiring 1 mm concentration to achieve sulphone of S-adenosylhomocysteine and its de- significant inhibition are not sufficiently active to carboxylated derivative remains to be explained. produce a substantial decrease in polyamine syn- S-Adenosyl-L-homocysteine sulphone and its decar- thesis in vivo, it should be remembered that boxylated derivative and 5'-methylthiotubercidin concentrations of decarboxylated S-adenosylmeth- may be of value in lowering cellular polyamine con- ionine in vivo are very low [about 1-2nmol/g wet centrations, particularly if given in conjunction with wt. (Pegg & Hibasami, 1979; H. Hibasami, J. L. an ornithine decarboxylase inhibitor. The sulphone Hoffman & A. E. Pegg, unpublished work)]. derivatives are chemically stable but inhibit some Therefore the concentration of decarboxylated S- 1980 INHIBITION OF SPERMIDINE SYNTHASE AND SPERMINE SYNTHASE 427 adenosylmethionine used in the enzyme assays Borchardt. R. T. & Pugh, C. S. G. (1979) in Trans- described in the present paper may be 20-40 times methvlation (Usdin, E., Borchardt, R. T. & Creveling. higher than that present in the cell. The complex C. R., eds.), pp. 197-206. Elsevier/North-Holland, kinetics of the aminopropyltransferase reaction New York renders extrapolation of our results to other sub- Borchardt, R. T. & Wu, Y. S. (1974) J. Med. Chem. 17. 862-868 strate concentrations difficult, but it is probable that Borchardt, R. T. & Wu, Y. S. (1975) J. Med. Chem. 18. inhibition by most of the compounds tested would be 300-304 much greater at physiological substrate concen- Borchardt. R. T. & Wu, Y. S. (1976) J. Med. Chem. 19. trations. Therefore it is possible that both S-adeno- 197-198 sylmethionine and S-adenosylhomocysteine could Borchardt, R. T., Huber, J. A. & Wu, Y. S. (1974) J. influence the synthesis of spermidine and spermine Med. Chem. 17.868-873 under some conditions. Borchardt, R. T., Huber. J. A. & Wu, Y. S. (1976a) J. Finally, the very strong product inhibition by Med. Chem. 19. 1094-1099 5'-methylthioadenosine raises the question as to Borchardt, R. T.. Wu, Y. S.. Huber. J. A. & Wycpalek. whether aminopropyltransferase activity might be A. F. (1976b)J. Med. Chem. 19. 1104-1 1 10 Borchardt, R. T., Wu. Y. S. & Wu. B. S. (1978a)J. Med. influenced by this nucleoside. Although there are Chem. 21, 1307-1310 several metabolic routes to 5'-methylthioadenosine Borchardt, R. T.. Wu. Y. S. & Wu, B. S. (1978b) (Ferro, 1979). it is conceivable that the pathway Biochemistriv 17. 4145-4153 thought of as the polyamine-biosynthetic pathway Bowman, W. H., Tabor. C. W. & Tabor, H. (1973) J. might have as its major function the production of Biol. Chein. 248. 2480-2486 this substance. 5'-Methylthioadenosine (Ferro, Cornforth. J. W.. Reichard. S. A., Talalay, P., Carrell, 1979) or its breakdown products have been sug- H. L. & Glusker. J. P. (1977) J. Am. Chem. Soc. 99. gested as important for cell growth (Toohey, 1977. 7292-7300 1978). There are at present no measurements of Coward, J. K.. Motola. N. C. & Moyer, J. D. (1977) J. Med. Chem. 20. 500-505 cellular 5'-methylthioadenosine content, but it is Ferro. A. J. (1979) in Transmethvlation (Usdin. E.. clearly much lower than that of polyamines (Rhodes Borchardt. R. T. & Creveling, C. R., eds.). pp. & Williams-Ashman, 1964; Pegg & Williams- 117-126. Elsevier/North-Holland. New York Ashman, 1969b). This is undoubtedly due to the Garbers, D. L. (1978) Biochim. Bioph.vs. Acta 523. activity of 5'-methylthioadenosine phosphorylase, a 82-93 widespread enzyme (Pegg & Williams-Ashman, Hannonen. P., Janne. J. & Raina, A. (1972) Biochem. 1969b; Garbers, 1977; Toohey, 1978). Until the Biophvs. Res. Commun. 46. 341-348 extent to which this activity lowers 5'-methyl- Hibasami. H. & Pegg. A. E. (1978a) Biochem. J. 169. thioadenosine concentrations within the cell is 709-712 known. the possible inhibition of polyamine syn- Hibasami. H. & Pegg. A. E. (1978b) Biochem. Bioph's. thesis cannot be evaluated. Also, it must be noted Res. Cominuni. 81. 1398-1405 was a potent Janne. J.. Poso. H. & Raina. A. (1978) Biochiin. Biophis. that, although 5'-methylthioadenosine Acta 473. 241-293 inhibitor of rat prostate spermine synthase in our Leboy. P. S.. Glick. J. M.. Steiner. F. G.. Haney. S. & experiments, it was still less active than reported by Borchardt. R. T. (1978) Biochiin. Biophvs. Acta 520. Pajula & Raina (1979) for highly purified bovine 153-163 brain spermine synthase. This could be a species Mamont. P. S.. Duchesne. M.-C.. Joder-Ohlenbusch. difference, and it appears that another difference was A.-M. & Grove. J. (1978) in Enzi.me-Activated that the rat prostate enzyme was much more Irretversible Inhibitors (Seiler. N.. Jung. M. J. & strongly inhibited by S-adenosylmethionine. How- Koch-Weser. J.. eds.). pp. 43-54. Elsevier/North- ever, it is also possible that the greater degree of Holland, New York purification achieved by Pajula & Raina (1979) Montgomery. J. A.. Shortnacy. A. T. & Thomas. H. J. renders the enzyme more sensitive to 5'-methyl- (1974)J. Med. Cheln. 17. 1197-1207 thioadenosine. Pajula. R.-L. & Raina. A. (1979) FEBS Lett. 99. 343-345 This research was supported in part by grants Pajula. R.-L.. Raina. A. & Kekoni. J. (1978) FEBS Lett. GM26290. CA18138, CA18450, NS10198, MH18038 90. 153-156 and CA 10748 from the National Institutes of Health. Pegg. A. E. (1978)J. Biol. Chem. 253. 539-542 U.S. Department of Health, Education and Welfare. Pegg. A. E. (1979) J. Biol. Chem. 254. 3249-3253 and by Established Investigatorships to R. T. B. and Pegg. A. E. & Hibasami. H. (1979) in Transmethivlation A. E. P. from the American Heart Association. (Usdin. E.. Borchardt. R. T. & Creveling. C. R.. eds.). References pp. 105-116. Elsevier/North-Holland. New York Borchardt. R. T. (1977) in The Biochemistry of S- Pegg. A. E. & Williams-Ashman. H. G. (1969a) J. Biol. Adenosulmnethionine (Salvatore. F., Borek, E., Zappia. Chein. 244. 682-693 V.. Williams-Ashman, H. G. & Schlenk, F.), pp. Pegg. A. E. & Williams-Ashman. H. G. (1969b) Biochein. 151-17 1. Columbia University Press, New York J.115.241-247 Vol. 187 428 H. HIBASAMI AND OTHERS

Pegg, A. E. & Williams-Ashman, H. G. (1970) Arch. Bio- Tabor, C. W. & Tabor, H. (1976) Annu. Rev. Biochem. chem. Biophvs. 137, 156-165 45, 285-306 Poso, H., Hannonen, P. & Janne, J. (1976) Acta Chem. Toohey, J. I. (1977) Biochem. Biophys. Res. Commun. Scand. Ser. B 30, 807-811 78. 1273-1280 Pugh, C. S. G., Borchardt, R. T. & Stone, H. 0. (1978)J. Toohey, J. I. (1978) Biochem. Biophys. Res. Commun. Biol. Chem. 253, 4075-4077 83, 27-35 Robert-Gero, M., Blanchard, P., Lawrence, F., Pierre, A., Williams-Ashman, H. G. & Canellakis, Z. N. (1979) Vedel, M., Vuilhorgne, M. & Lederer, E. (1979) in Perspect. Biol. Med. 22,421-453 Transmethylation (Usdin, E., Borchardt, R. T. & A. (1972) Bio- Creveling, C. R., eds.), pp. 207-214, Elsevier/North- Williams-Ashman, H. G. & Schenone, Holland, New York chem. Biophys. Res. Commun. 46, 288-295 Rhodes, J. B. & Williams-Ashman, H. G. (1964) Med. Williams-Ashman, H. G., Janne, J., Coppoc, G. L., Exp. 10, 281-285 Geroch, M. E. & Schenone, A. (1972) Adv. Enzyme Russell, D. H. & Durie, B. G. M. (1978) Prog. Cancer Regul. 10, 225-245 Res. Ther. 8, 1-178 Williams-Ashman, H. G., Corti, A. & Tadolini, B. (1976) Seiler, N., Jung, M. J. & Koch-Weser, J. (1978) in Ital. J. Biochem. 25, 5-32 Enzyme-Activated Irreversible Inhibitors (Seiler, N., Zappia, V., Oliva, A., Cacciapuoti, G., Galletti, P., Jung, M. J. & Koch-Weser, J., eds.), pp. 55-71. Mignucci, G. & Carteni-Farina, M. (1978) Biochem. J. Elsevier/North-Holland, New York 175, 1043-1050

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