Showdomycin, a New Nucleoside Antibiotic'

[CANCER RESEARCH 28, 1106-1610,Aug@at1968] Showdomycin, A New Nucleoside Antibiotic' S. Roy-Burman,P.Roy-Burman,and D. W. Visser Department of Biochemistry, University of Southern California, School of Medii*ie, Los Angeles, California 9008$ SUMMARY Streptococcus pyogenes (14). Showdomycin has antitumor activity against Ehrlich ascites cells both in vitro and in vivo The metabolism and inhibitory effects of the carbon-linked and is active against cultured HeLa cells ( 11, 14, 15) . Its nucleoside antibiotic showdomycin were investigated in cell structure has been established as 3-$-o-ribofuranosylmaleimide free preparatiozis of Ehrlich ascites cells. The antibiotic is not (5), a carbon-linked nucleoside antibiotic. a substrate for nucleoside kinase or nucleoside phosphorylase. The structural similarity of showdomycin, uridine, and Showdomycin shows inhibitory effects on certain enzymes in an pseudouridine are apparent from the formulae (Chart 1). Ehrlich ascites cell preparation, which are involved in uridine Darnall et at. (5) have pointed out that showdomycin may be and orotic acid metabolism. It inhibits Ã¼ridine-5'-monophos viewed as pseudouridine which has lost an -NH group in the phokinase, uridine phosphorylase, and possibly orotidylic acid contraction to a five-membered ring. pyrophosphorylase reactions, but has no effect on the activity The unique structure of showdomycin, its structural simi of uridine kinase or adenosine phosphorylase. Showdomycin larity to uridine, and its biologic properties prompted this in strongly inhibits bovine liver uridine-5'[email protected] vestigation of its metabolism and its effect on various enzymes, dehydrogenase but has no effect on rabbit muscle lactic acid particularly those involved in uridine metabolism. dehydrogenase. The selective inhibition of certain enzymes by showdomycin may be related to the alkylating property of its maleimide structure, which is known to specifically react with MATERIAL AND METHODS sulfhydryl groups. Showdomycin was obtained as a gift from Dr. Ronald K. Robins of the University of Utah, Salt Lake City, Utah, and INTRODUCflON Dr. Haruo Nishimura of Shionogi Research Laboratory, The antibiotic, showdomycin, first isolated by Nishimura Fukushima-Ku, Osaka, Japan. The purity of the compound et at. (15) from Streptomyces showdoensis, inhibits growth of was determined in two descending paper chromatographic several Gram-positive and Gram-negative bacteria and is par systems. The compound migrated as a single ultraviolet ab ticularly active against Streptococcus haemotyticus (15) and sorbing area in ethanol 0.5 M ammonium acetate, pH 6.5 (5:2 v/v) and isopropanol: concentrated HC1: 1120 (65:16.7:18.3 v/v/v) solvent systems, the respective RF values being 0.64 1 This investigation was supported by research grants from and 0.68. Uridine-2-14C and UMP-2-'4C2 were purchased from USPHS (CA 02373-12), the American Cancer Society (E-412) Schwarz BioResearch, Inc. Orotic acid-6-14C was obtained and (in part) by a Dernham Senior Fellowship of the American Cancer Society, California Division (No. D120). from New England Nuclear Corporation. Nucleoside, nucleo Received January 26, 1968; accepted April 21, 1968. tides, ribose-5'-phosphate, and 3-phospho-D-glyceric acid (sodium salt) were obtained from California Corporation for Biochemical Research. A supernatant fraction (100,000 x g) from a hyperdiploid HN strain of Ehrlich ascites tumor cells was prepared as described previously (18, 21). A crude preparation of uridine phosphory 0 _.-@ lase was obtained from Ehrlich ascites cells in the following manner. Ascites cells were washed as described previously (21) and disrupted using a French press at 1800 lb/sq inch at HOC@ HOCH2 0 0-5Â°C in 2 volumes of buffer containing 0.05 M potassium phosphate, pH 7.4, and 0.13 M potassium chloride. An addi tional amount of buffer was added to the disrupted cells to make OH OH OH OH 2 Abbreviations used are : UMP, uridine-5'-monophosphate; UDP, uridine-5'-diphosphate ; UDP-glucose, uridlne-5'-diphoe SHOWDOMYCIN URIDINE PSEUDOURIDINE phate-a-n-glucose ; UTP, uridine-S'-triphosphate ; Tris, tria(hy droxymethyl)aminomethane ; NAD+, nicotinamide adenine di Chart 1. Graphic formulas. nucleotide. AUGUST 1968 1605 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1968 American Association for Cancer Research. S. Roy-Burman, P. Roy-Burman, and D. W. Visser a final concentration of 3 ml of buffer per gm of cells. The omitted from the incubation mixture. The detection limit of suspension was centrifuged for 60 min at 85,000 x g. The the photometric assay procedure is about 10 optical density supernatant enzyme preparation contained 12-14 mg protein/ units which represents about 1% of the added showdomycin. ml and had a specific activity of 110-160 (17). This prepara The fractions were also tested with Elson-Morgan's color re tion was used for assay of uridine phosphorylase and adenosine agent, which gives a red color with showdomycin (15). The phosphorylase. results, with and without showdornycin, were identical based Preparation of acid-soluble fractions from the various incu on these assay procedures, showing that showdomycin is not. bation mixtures and separation of phosphorylated derivatives converted to phosphorylated derivatives by enzyme prepara by chromatography on Dowex 1 (formate) were performed tions which converted 50-70% of uridine-'4C to UTP. as described previously (18). Effect of Showdomycin on Uridine Metabolism. The results Uridine diphosphate glucose dehydrogenase was purchased of experiments designed to determine the effect of showdomy from Sigma Chemical Company and was assayed (19) by cin on the conversion of uridine-'4C to phosphorylated deriva following NAD+ reduction. Rabbit muscle lactic dehydrogenase tives are shown in Table 1. The data show that showdomycin was obtained from California Corporation for Biochemical did not inhibit uridine kinase but strongly inhibited the con Research and was assayed by determining the rate of oxidation version of UMP to UDP. The latter effect increased with in of reduced NM) with pyruvate as substrate. creasing concentrations of showdomycin. In the presence of 2.5 @molesshowdomycin per ml reaction mixture, UMP-14C RESULTS represented more than 70% of the radioactive nucleotides, whereas in the absence of showdornycin, about 90% of the Effect of Kinases on Showdomycin. Since inhibitory effects acid-soluble nucleotides was recovered as UTP. Uridine kinase of most nucleoside derivatives are dependent upon a prior activity was not inhibited by showdomycin under the same conversion to phosphorylated derivatives, it was of interest to conditions. determine the susceptibility of showdomycin to kinase activi Since the previous experiments indicated that showdornycin ties. Showdomycin was incubated at pH 7.8, 7.0, and 6.2 with specifically inhibited UMP kinase, the effect of the analog on a supernatant fraction prepared from Ehrlich ascites cells the phosphorylation of UMP-14C was investigated. In the having high levels of uridine, UMP, and UDP kinase activity presence of 1.6 @molesshowdomycin per ml reaction mixture, (18, 21) . Incubation mixtures were treated as described pre UTP formation was inhibited about 50% (Table 2). When viously (18) except that the separation of acid-soluble corn the enzyme mixture was preincubated with the same concen ponents was carried out on a Dowex 1 (chloride) column tration of showdomycin prior to the addition of UMP-14C, using a gradient concentration of hydrochloric acid and sodium UMP kinase activity was completely inhibited, whereas 94% chloride. Initially the mixer contained 1 liter of water and the of UMP was converted to the triphosphate in the absence of reservoir contained 0.01 N hydrochloric acid. After collection showdomycin. Preincubation of showdomycin with the reaction of about 1 liter of eluent, the solution in the mixer was changed mixture prior to the addition of both UMP-'4C and enzyme did to 1 liter of 0.01 N hydrochloric acid and that in the reservoir not increase the inhibitory effect. to 0.2 M NaC1. After collection of about 2 additional liters of Effect of Showdomycin on the Conversion of Orotic Acid eluent, the column was eluted directly with 0.5 M NaCl in 0.01 to Uridine Nucleotides. Showdomycin inhibited the conversion N hydrochloric acid. Since showdomycin in acidic solution has of orotic acid-'4C to uridine nucleotides (Table 3) . At a con @ a characteristically high absorbance at 220 and a very low centration of 0.56 @&moleshowdomycin per ml reaction mixture, absorbance at 260 m@ ( 15) , the absorbance of the effluent was about 50% of the total radioactivity was recovered as erotic measured at 220 m@ and compared with a control which was acid, whereas in the absence of showdomycin, less than 15% handled in an identical manner, except that showdomycin was of the radioactivity remained as orotic acid, the remainder Table 1 %UMPUDP recovered as Showdoinycin phos added phorylated (@unoles)Radioactivityderivatives00.5088.670.180.05.029.305.050.084.310.063.302.720.686.6sugarUDPUTPTotal Effect of showdomycin on uridine-14C phosphorylation. The reaction mixtures (4 ml) con tamed : 20 @molesATP ; 10 @@molesphosphoglycericacid ; 50 @@molesMgCl2; 250 @&moles tris(hydroxymethyl)aminomethane-HC1, pH 7.5; 1 @moleuridine-14C (7.6 x 105 cpm) ; 5 or 10 @imolesshowdomycin ; and 22 ml (29 mg) 100,000 x g supernatant fraction. Incubations were carried out for 10 mm at 37Â°Cand terminated with 1.0 ml 4.0 M perchloric

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