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The in Vitro Inhibition of Xanthine Dehydrogenase by 8@@Azaguanine*

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The in Vitro Inhibition of Xanthine Dehydrogenase by 8@@Azaguanine* The in Vitro Inhibition of Xanthine Dehydrogenase by 8@@Azaguanine* PHILIP FEIGELSON AND JACK DAVIDSON (Departments of BiOChSSZÃœLrJJandMedieine, Collegeof Physicians and Surgeons, Columbia University, and Francis Delafield Hospital, New York 85, N.Y.) The biochemical basis underlying the cytotoxic linear initial slopes were taken as a measure of en and carcinostatic effects of 8-azaguanine is still un zyme activity. The assay conditions were arranged certain. The initial proposal that this compound so that the reaction rates were linear functions of acts as a guanine antagonist now seems less likely the enzyme concentration. Control flasks in which in view of recent findings that 8-azaguanine does the enzyme was omitted were run, and in no case @ not diminish the rate of incorporation of guanine did significant changes in E500 occur. A Beckman C'4 into tissue nucleic acids (3). Observations that DU Spectrophotometer and a Cary Model 11 Re small amounts of isotopically labeled 8-azaguanine cording Spectrophotometer were employed in these are incorporated into tissue nucleic acids have led kinetic studies. All analyses were conducted at some investigators to the belief that these fraudu room temperature. The experimental points pre lent nucleic acids containing 8-azaguanine may be sented are the means of duplicate determinations. responsible for the growth-inhibitory effects The straight lines through these points were de (1@, 18). rived by the method of least squares (@0). Recent studies in our laboratory concerning the Stock 0.03 M solutions of 8-azaguanine (Lederle) mode of action of 8-azaguanine have explored the were prepared by the addition of NaOH until a alternative hypothesis that 8-azaguanine inhibits pH of 9.5 was attained, and final 0.006 M 8-aza one or more of the enzymes concerned with purmne guanine solutions were prepared by dilution with interconversions. The present report concerns our water. findings that even subtherapeutic concentrations of 8-azaguanine significantly inhibit xanthine de RESULTS hydrogenase activity in vitro. A hypothesis is pre Chart 1 depicts the in ritro effects of 8-azagua sented integrating these findings with other pre nine on xanthine dehydrogenase activity. It is ap viously established phenomena induced by 8-aza parent that a 50 per cent enzyme inhibition oc guanine. curred at a concentration of approximately 5 X MATERIALS AND METHODS 5M 8-azaguanine, and that the extent of inhibi tion was proportional to the drug concentration. The xanthine dehydrogenase assays employed The enzyme activity was never observed to de were as previously described (6), wherein the en crease to below @25per cent of the initial activity, zyme catalyzes the transfer of hydrogen atoms even in the presence of drug concentrations several from xanthine to @,6-dichlorophenol indophenol. times higher than the highest concentration de Xanthine dehydrogenase inhibition measurements picted in this figure. These findings suggest that a were routinely carried out by the addition of the part of the catalytic activity, possibly one of the indicated amounts of 8-azaguanine to spectropho active centers of the enzyme, is less sensitive to tometer cuvettes containing the appropriate reac this inhibitor. tion mixtures. The enzyme, cream xanthine oxi To elucidate the nature of the 8-azaguanine dase, was added last, and the rate of dye reduction xanthine dehydrogenase interaction, a series of was followed by noting the decrease in optical kinetic experiments were undertaken. Enzyme ac density at 600 m@s (E@oo @,,)at 1-minute intervals. tivities were determined over a wide range of sub.. The extinctions were plotted against time, and the strate concentrations in the absence and presence @ S This investigation was supported in part by research of 5.6 X —M8-azaguanine. In Chart the data grants C 2046 and C 2332, from the National Cancer Institute obtained are plotted as the reciprocal of the en of the National Institutes of Health, Public Health Service. zyme activity (@) versus the reciprocal of the Received for publication December 4, 1955. substrate concentration (k). The upper line oh 35@ Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1956 American Association for Cancer Research. FEIGELSON AND DAviDsoN—Xanthine Dehydrogenase Inhibition by 8-Azaguanine 858 tamed in the presence of 8-azaguanine clearly effects of 8-azaguanine upon the former reaction shows both an elevated ordinate intercept and an were studied directly. With hypoxanthine as a increased slope when compared with the lower line substrate the disappearance of optical density at obtained in the absence of the inhibitor. This in @55m@ upon incubation with cream xanthine dicates that high concentrations of substrate do dehydrogenase was measured in the absence and not reverse the 8-azaguanine-induced inhibition presence of 8-azaguanine. The drug was found to and that the inhibition is noncompetitive in na inhibit this activity of the enzyme to a degree ture (@1). The calculated dissociation constant of similar to its inhibition of xanthine oxidation. the enzyme-substrate complex (K,) is @.6X To ascertain the specificity and biological sig 10—iM,and the dissociation constant of the en nificance of the observed inhibition of xanthine zyme-8-azaguanine complex (K1) is 5.6 X 10@ M. dehydrogenase by 8-azaguanine, two approaches This indicates that 8-azaguanine has about one were utilized. It was first determined whether simi half the affinity of xanthine for the enzyme. lar compounds would inhibit this enzyme. Thus the inhibitory influences of guanine and 8-azaxan $00 thine were studied comparatively with isomolar w levels of 8-azaguanine. The results of two such ex @ e0@ periments shown in Table 1 indicate that both 8-azaxanthine and guanine exert but a fraction of @60 the inhibitory activity on xanthine dehydrogenase shown by 8-azaguanine. 0 w 40 .05 w@ H I- 0 H — 20 .04 4 * x @@ 0 12 $6 20 .03 aIO@@N8AZAGUAN$NE -@ .02 CHART 1.—The effect of 8-azaguanine on xanthine dehy drogenase activity. Each experimentalcuvette contained 2.5 ml. 0.1 ai glycyl .0 I glycine (pH 8.0); 0.2 ml. 20 mg. per cent 2,6-dichiorophenol indophenol; 0.3 ml. S X 10@ Mxanthine; 0.1 ml. xanthine oxi dase; the indicated concentrations of 8-azaguanine and water to make the total volume 3.2 ml. *10 CHART 2.—Lineweaver-Burk analysis of the inhibition of To gain further insight into this enzyme-in xanthine dehydrogenase by 8-azaguanine. hibitor relationship the number of moles of 8-aza Each experimental cuvette contained 2.6 ml. 0.1 M glycyl guanine which combine per mole of xanthine de glycine (pH 8.0), 0.2 ml. 20 mg. per cent 2,6-dichiorophenol hydrogenase was estimated from a plot of log indophenol, 0.1 ml. xanthine oxidase, varying concentrations (J; —1) = R log (I) + log @-,where v and v of the substrate xanthine, and water to make the total volume 3.2 ml. A duplicate series of cuvettes containing 0.08 ml. 0.006 are the enzyme activities observed in the absence M 8-azaguanine were run. and presence, respectively, of the inhibitor, 8-aza guanine (I) ; “R―isthe number of moles of in The second test of the specificity of 8-azagua hibitor combining per mole enzyme; K1 is the dis nine inhibitions of xanthine dehydrogenase in sociation constant for the inhibitor-enzyme com volved studies of the effect of 8-azaguanine on a plex (@1). variety of other enzymes. It was found that the The data are depicted in Chart 3. Since the slope highest drug concentration tested (8 X 10@ M) of the line “R―is0.99, one may conclude that 1 exerted no significant inhibition on the following mole of 8@azaguanine combines with 1 mole of en enzyme systems : yeast acid phosphatase, erythro zyme over the entire inhibitor range tested. The cyte phosphohexose isomerase, erythrocyte nucleo calculated K is 4.5 X 10@ M, which is in fair side phosphorylase, and liver succinic oxidase. agreement with K1 of 5.6 X 10@ M derived from Thus, at a drug concentration 100 times that the Lineweaver-Burk analysis. which significantly inhibits xanthine dehydroge Although the reactive center of xanthine de nase, these other enzymes were uninfluenced. It hydrogenase concerned with the oxidation of hy was found, however, that one other enzyme, ade poxanthine has always been considered to be iden nosine deaminase, was inhibited by 8-azaguanine tical to that involved in xanthine oxidation, the in concentrations above 10@ M (7) . These data Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1956 American Association for Cancer Research. 854 Cancer Research clearly indicate that 8-azaguanine shows a high acid biosynthesis in the mammal. This reaction degree of specificity for the enzymes it will inhibit. network is supplied with inosinic acid .arising from the de novo formation of the purine moiety @ DISCUSSION (11), and with preformed purines. Edmonds and To have pharmacological significance, sensitivi LePage have demonstrated that the respective ty of an enzyme to 8-azaguanine must satisfy at free bases, ribosides, and ribotides of purines are least two criteria: inhibition must take place at or in rapid equilibrium (5, 15). It therefore appears below drug concentrations therapeutically achiev probable that the vertical reactions of Chart 4 oc able, and pharmacologically inactive catabolites cur readily. Less is known regarding the horizontal of the drug should be less potent inhibitors of the pathways for interconverting purine moieties. enzyme than the drug itself. An effective carcino.. Abrams and Bentley have recently demonstrated static dose of 8-azaguanine in mice is approximate ly 50 mg/kg body weight/day (18). Assuming an TABLE 1 animal to be approximately 75 per cent water and THE COMPARATiVE INHIBITORY EFFECTS OF 8-AZAGUANINE, 8-AZAXANTHINE, AND .6 GUANINE ON XANTHINE DEHYDROGEN S ASE AcTiviTY .5 X&@imniz DLHTDROO@NA@ACTIVITY 0 (X10' EINMM/5 mm.) TIOR 8-Azsgua- 8-@axan@ -.5 x1o-5 H nine tbine Guanine 0 0 78 73 0 -J -.6 8.2 49 67 5.3 89 65 -.9 10.7 29 55 0 57 57 .12 9.4 28 58 18.8 19 48 s.8 4.2 46 5.0 54 —LOG (I) Caa@ 8.—Thedeterminationof the numberof moles of 8-asaguanine which combine per mole zanthine debydrogenase.
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