Human spleen dihydroorotate dehydrogenase: A study of inhibition of the enzyme

BIOCHEMICAL

MEDICINE

34, 60-69 (1985)

Human Spleen Dihydroorotate Dehydrogenase: A Study of Inhibition of the Enzyme ANNETTE M. GERO,* WILLIAM J. O’SULLIVAN,* ANDDESMONDJ. BROWN-t *School ofBiochemistv?;, University of New South WaleA. P.0. SOS I. lkrtsingtm, &Sk+ 203.<. Austrulia. and fMedica1 Chemistry Group. John Curtbl School oj’ Medical Rescouc~lr. Australian National University. Canberra, ACT 2600. Austruiic~ Received February 22. 1984

Dihydroorotate dehydrogenase (DHO-DHase; t.-5,6-dihydroorotate oxygen oxidoreductase, EC 1.3.3.1) catalyzes the oxidation of L-dihydroorotic acid to erotic acid, the fourth step in the de no~o biosynthesis of pyrimidines. In the few mammalian tissues examined the enzyme activity is associated with the mitochondria and is linked to the respiratory chain via ubiquinone (l-3). Little attempt has been made to study directly the effects of pyrimidine analogs on mammalian DHO-DHase [4], although extensive inhibitor studies have been carried out on the cytoplasmic degradative enzyme from the anaerobic bacterium. Clostridium (Zymobacterium) orotic‘nm (S-7). It was considered that the characterization of the pattern of inhibition of pyrimidine analogs on human spleen DHO-DHase could be used as a starting point in the comparison of the enzyme in neoplastic cells or parasitic protozoa (4,8-l 1). Such comparison would provide a basis for the design of potential chemotherapeutic agents. Characterization of human spleen DHO-DHase, including confirmation of its mitochondrial location, is described in Ref. (12). The present paper describes the inhibition of this enzyme by a number of pyrimidine analogs. The analogs were tested against the enzyme activity in whole mitochondria, as it was considered that this would best approximate the in V~VOsituation. Those compounds found to be inhibitors of the human DHO-DHase were also tested for their effect on the two sequential enzymes, orotate phosphoribosyltransferase (OPRTase; EC 2.4.2.10) and orotidine-5’-monophosphate decarboxylase (ODCase: EC 4. I. I .23) from human spleen; as the potency of compounds which may simultaneously inhibit more than one enzyme of the pathway raises a greater possibility of usefulness in chemotherapy. MATERIALS AND METHODS

Materials [carboxy-‘4C]Orotic acid (41.25 mCi/mmole), and [carboxy-‘4C]orotidine-5’-monophosphate 0006-2944185 $3 .OO Copyright All tights

0 1985 by Academtc Press. Inc of reproduction in any form rexned.

60

[4-%]orotic acid (9.36 mCi/mmole). ([carboxy-‘4C]OMP. 20-40 mCi,

INHIBITION

OF HUMAN

DHO DHase

61

mmole) were obtained from New England Nuclear Corporation. L-5,6-Dihydro[carboxy-‘4C]orotic acid (L-[carboxy-14ClDHO), and L-5,6-dihydro[4-14C]orotic acid (L-[4-14C]DHO) were synthesized from [carboxy-‘4C]orotic acid and [4-‘4C]orotic acid, respectively, as previously reported (13). N-2-Hydroxyethylpiperazine-N’2-ethanesulfonic acid (Hepes), 5-phosphoribosyl-1-pyrophosphate (P-Rib-PP), LDHO, erotic acid (Na salt), OMP, UMP, and bovine serum albumin were obtained from Sigma Chemical Company. Hydroxide of hyamine (10X) and 3MM chromatography paper were purchased from Packard Instrument Company and Whatman, respectively. All inorganic chemicals were analytical reagent grade and were used without further purification. Distilled and deionized water was used for all preparative and experimental purposes. The sources or preparative methods for other compounds are indicated by references in Table 1.

No. 1 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

TABLE 1 Inhibition of Human Spleen Dihydroorotate Dehydrogenase by Pyrimidine Compounds ~~..~. --~. Anionic % Inhibition Compound at 1 mM PK -- .___ .~ Orotic acid [I]*“,’ 2.1 100 5Methylorotic acid [l] 2.5 100 5Bromoorotic acid [I]* 2.3 100 5-Nitrobarbituric acid [l] 100 (4) 6-Thiobarbituric acid [2]* (ca. 3) 100 5-Azaorotic acid (2-carboxy-4,6-dihydroxy-1,3,5triazine [3]* 100 (<2) Dihydro-S-azaorotic acid (2-carboxy-4,6dihydroxydihydro-I ,3,5triazine) [3] 100 (
62

No.

GERO, O’SULLIVAN.

AND BROWN

Compound .~~ ~~-

33 45Diaminopyrimidine [lo] 34 I .3-Dimethylbarbituric acid [ 181 35 S-Methyluraci! (thymine) 1171 36 2.4-Diam~no-&-chforo-I-methylpyr~mid~n~um chloride 1191 37 1,3-Dimethyfuracil i 171 38 4,SDicarboxypyrimidine [ZOJ” 39 Amytal (5-Ethyl-S-isoamylbarbituric acid) [ 11 40 4,5-Diamino-2-hydroxypyr;midine [2i] 41 4-Amino-~-bromo-6-hydroxypyrjmidine 122] 42 4-Amino-2,fSdimethylpyrimidine 1231 43 6-Diethoxymethyluracil [24] 44 2-Thiobarbituric acid [ I I* 45 2-Benzy~-4,6-dihydroxy-S-nitropyrimidine [25j” 46 2-Aminopyrimidine [17] 47 5,6-Dihydroxy-1 ,Z&triazine 1261 4X 4-Aminopyrimidine [IO] 49 5-Bromo-1.3-dimethyluracil 1271 50 Xarboxyuracil (isoorotic acid) [ 171 5 1 h-Methyluracil [ I71 52 SMethyluracil 1281 53 2-Hydroxypyrimidine [29] 54 ~-Bromo-~-methyl-~,6-dihydrouracil [30]* 55 4-Hydroxypyrimidine [ 171 56 2-Mercaptopyrimidine [ 17]* 57 SFluorouracil [I]* 58 4-Amino-S-bromo-2-hydroxypyrimidine [2?] 59 ~-Methyluracil 1301 60 2-Amino-4.6-dimethylpyrimidine [3 11 6 1 2,4-Diamino-S-bromo- I-methylpyrimidinium chloride [ 191 62 4-Amino-2-carboxy-6-hydroxypyrimidine [XI] 63 2-Amino-4.6-bisdimethylaminopyrimidine [33] 64 2-D~methylamino-4.6-dihydro~ypyr~midine /34] 65 SBromouracil [ 171 66 IS-Dimethylpyrimidin-2-one 1321 67 4-Methoxy-I-methylpy~m~d~ne-2-one [36J 68 2-Amino-4-carboxy-5-chloropyrimidine [17] 69 5-Amino-4-chloro-6-hydroxy-2-methylpyrimidine [37] 70 6-Aminouracil 1171 71 Control ” Assayed by the high-voltage electrophoresis technique indicating that the compound was &own to inhibit the yeast OPRTase-ODCase complex. ” pk values in parentheses are estimated by the method of Barlin and Perrm. Q, Nrb,. (‘ht,m. SW. 20, 75 (1966). ’ References indicate source or prepardt~on for each compound. ‘Compounds found to inhibit the yeast OPRTase-ODCase coupling enzymes. [I] Sigma Chemical Co. [2] H. C. Koppel, R. H. Springer, R. K. Robins, and C. C. Cheng, J. Org. Chmf. 26, 792 (1961). [3] Gift from Dr. G. B. Elion. Wellcome Research Laboratories, Research Triangle Park. N .f. 1 ‘ontinued

INHIBITION

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TABLE

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63

l-Continued

141 P. Bitterli and H. Erlenmeyer. He/v. C&n. ACM 34, 835 (1951). [Sl A. Albert, D. J. Brown, and H. C. S. Wood, J. Chem. Sot. 3832 (1954). [6] D. J. Brown, J. Chern. Sot. 2312 (1956). [7] J. M. Sprague. L. W. Kissinger, and R. M. Lincoln, J. Amer. Chem. Sot. 63, 3028 (1941). [8] R. B. Barlow and A. D. Welch, J. Amer. Chem. Sot. 78, 1258 (1956). [9] J. Evans and T. B. Johnson, J. Amer. Chem. Sot. 52, 4993 (1930). [lo] D. J. Brown, J. Sot. Chem. Med. (London) 69, 353 (1950). [l l] F. F. Blicke and H. C. Godt, J. Amer. Chem. Sot. 76, 2798 (1954). [I21 M. Ishidate and H. Yuki, Chem. Pharmaco(. Bzcli. (Tokyo] 8, 137 (1960). 1131 M. Ishidate and H. Yuki, Pharmacol. Bull. (Toliyo) 5, 241 (1957). [14] W. ‘I’. Caldwell and H. B. Kime, J. Amer. Chem. Sot. 62, 236.5 (1940). [IS] D. J. Brown, J. Appl. C’hem. (London) 5, 1.58 (1955). [lh] D. Davidson and 0. Baudisch. Ber. Drsch. Chem. Gex. 58, 1685 (1925). 1171 A. G. Fluka. iIS] P. A. Leermakers and W. A. Hoffman, J. Amer. Chem. Sot. 80, 5663 (1958). 1191 D. J. Brown and T. Teitei, J. Chem. Sot. 755 (1965). [203 S. Gabriel and J. Colman, Ber. Dtsch. Chem. Ges. 37, 3643 (1904). [?I] D. J. Brown, J. Appl. Chem. (London) 7, IO9 (1957). [22] D. J. Brown and J. S. Harper, J. Chem. Sot. 1298 (1961). [23] EGA-Chemie. 1241 T. B. Johnson and E. F. Schroeder, J. Amer. Chem. Sot. 53, 1989 (1931). I251 M. E. C. Biffin, D. J. Brown, and T.-C. Lee, J. fhem. Sot. C 573 (1967). [26] D. J. Brown and R. L. Jones, Ausf. J. Chem. 25. 772 1 (1972). [27] G. E. Hilbert. J. Amer. Chem. Sot. 56, 190 (1934). [28] C. W. Whitehead, J. Amer. Chem. Sot. 74, 4261 (1952). [29] D. J. Brown, Nurure Ilondon) 165, 1010 (1950). [30] D. J. Brown. E. Hoerger, and S. F. Muson, J. Chem. SM. 211 (1955). 13l] Koch-Light Laboratories Ltd. 1321 D. J. Brown, B. T. England, and J. M. LyalI, J. Chem. Sot. C 226 (1966). [33] D. J. Brown and J. S. Harper, J. Chem. SW. 1276 (1963). [34] W. R. Boon. J. Chem. Sot. 1532 (1952). 1351 D. J. Brown and T.-C. Lee. Ausf. J. Chem. 21, 243 (1968). [36] G. E. Hilbert and T. B. Johnson, J. Amer. Chem. Sac. 52, 2001 (1930). [37] F. L. Rose and D. J. Brown, J. Chem. Sot. 19.53(1956).

Enzyme Soarces

Fresh human following routine in Ref. ( 12). The was used as the ODCase enzyme Company.

spleens were obtained from Prince of Wales Hospital, Sydney, splenectomy. Mitochondria were isolated at 4°C as described 100,OOOg supernatant following the isolation of the mitochondria source of human OPRTase-ODCase activity. Yeast OPRTasemixture and yeast ODCase were obtained from Sigma Chemical

Enzyme Assays DHO-L?Hase (coupled enzyme assay). DHO-DHase activity was routinely assessed by the conversion of L-~ca~~o~y-i4C]DH0 into UMP and “C0, using a coupied enzyme assay (13). The assay mixture was modified for the inhibition studies to contain: Hepes-KOH, pH 8.0 (10 mM); P-Rib-PP (0.5 mM); MgC& (5 mM); 2 munit OPRTase-ODCase enzyme mixture; the pyrimidine inhibitor to be tested (1 mM); I.-[carboxy-14C]DH0 (approximately 600 dpmkrmole) (8 FM;

64

GERO, O'SULLIVAN,i\ND

BROWN

protein (0.4 mgf in a total volume of f ,O -f&7,, L-DHO) (13) and mitochondrial ml. The reactants were incubated at 30°C for IO min. The activity of the enzyme was expressed as nanomoles 14C02 formed per milligram protein per hour. Dab-DBASE amigo-voltage ~~~~trup~~~es~~s~. As the above DHO-DHase coupted enzyme assay is dependent on the addition of excess yeast OPRTase and ODCase, any compound screened as a potential inhibitor of the DHO-DHase by this assay was reassayed with the omission of the coupling enzymes. DHO-DHase activity was determined by the conversion of I.-L~-‘~C]DHO to /4-‘3C]orotate and the dihydroorotate and orotate separated by high-voltage electrophoresis ( 14). The assay conditions were as follows: Hepes-KOH, pH 8.0 (10 mM); L-[4-?I’]DHO protein (0.1 mg) in a (8 PM; -&,, L-D&IO); inhibitor (I mM) and mitochondrial final volume of 200 ~1. The reaction was carried out at 30°C for 10 min as described previously (14). OPRTuse-ODCase. Compounds which were inhibitors of the coupled enzyme system were assayed for their ability to inhibit yeast and human spleen OPRTasc and ODCase enzymes according to the procedure of Fox et ul. (15). The reaction mixture contained Hepes-KOH, pH 8 (10 mM); P-Rib-PP (0.5 mM): MgCI, (S mM); 2 munit OPRTase-ODCase yeast enzyme mixture (or 4 mg protein, human spleen I~,~Og supernatant); the compound to be tested (1 rnM) and [cu&o,~~“C]orotic acid (2600 dpmlnmole; 16 PM - K,,, erotic acid) in a total volume of 1.0 ml. The reaction was run for 10 min at 30°C. High-voltage electrophoresis was also used in some cases to separate erotic acid. OMP, and UMP (141. ODCase. ODCase activity in yeast and in the human spIeen I~,~~ supernatant was also assessed by the conversion of [c.urhox?f-‘4C]OMP (12 PM: e-K,,, OMP) to UMP and 14C0, (15).

Kj values were determined for those compounds established as effective inhibitors of DHO-DHase. The substrate concentration ranged from 5 to 50 ,LLMI,-DHO and two concentrations of the inhibitor were used, usualty 10 and 20 phj. The kinetic data were analyzed by computer analysis using a linear least-squares regression procedure (16). Protein. Protein concentrations were determined by the method of Lowry t~f al. (171, using bovine serum albumin as the standard. RESULTS The inhibition of human spleen DHO-DHase produced by each pyrimidinc analog at 1 mM concentration is summarized in Table I, together with the anionic pK, value for each compound. Of the 71 analogs examined, 9 exhibited an inhibition of 70% or greater at 1 mM and were subsequently selected for the determination of inhibition constants (Table 2). Other compounds which were found to inhibit human spleen DHO-DHase less than 70% at 1 mM were not investigated further unless they had been shown to be inhibitors of the yeast coupling enzymes. These compounds are discussed below.

INHIBITION

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DHO DHase

65

Inhibition constants for the active pyrimidine inhibitors of human spleen DHODHase are collected in Table 2. All inhibitors were competitive with respect to L-DHO. The most effective were dihydro-Sazaorotate and 6-thiobarbiturate, whose Ki values, 3 and 4.7 FM, respectively, were lower than the K,,,, 5.3 PM, for L-DHO (13). Orotate and carbamyl-ix-aspartate, the substrate of the previous enzyme, dihydroorotase (DHOase, EC 3.5.2.3), were also competitive inhibitors with Kj values of 6.5 and 13.0 PM, respectively (12). Inhibition of OPRTase and ODCase Yeast enzymes. The DHO-DHase coupled enzyme assay is advantageous for its rapidity for testing large numbers of potential inhibitors. However, due to the inherent use of the yeast OPRTase-ODCase enzymes in the assay, any compound found to be an inhibitor of the DHO-DHase assay had also to be assessed for its possible inhibition of the yeast coupling enzymes. Those compounds which were found to inhibit the yeast OPRTase-ODCase coupling enzymes during the assays performed in Table 1 (indicated by an asterisk) are shown in Table 3. They were reassayed to determine whether the inhibition was of the OPRTase-ODCase system or of the ODCase alone. These compounds were also tested as inhibitors of human spleen OPRTase and ODCase (Table 3). Of the effective inhibitors of the DHO-DHase (>70%) (Table I), 6-thiobarbiturate, 5-azaorotate, 5-bromoorotate, and barbiturate were also found to inhibit the yeast OPRTase-ODCase coupling enzymes (Table 3). Whereas supplementary assays with the yeast ODCase demonstrated that 6-thiobarbiturate effectively inhibited the ODCase activity, the other three compounds appear primarily to act at the level of the yeast OPRTase. The data do not allow a definitive conclusion to be drawn on the degree of inhibition of the OPRTase by 6-thiobarbiturate; if the TABLE 2 of Human Spleen DHO-DHase: Comparison of K, Values for Some Pyrimidine Analogs that Act as Effective Inhibitors of the Enzyme ___.__ Compound K, (PM) ~~~ .~~..~ 3 Dihydro-5azaorotate 6-Thiobarbiturate*” 4.7 Orotate* 6.5 (8.4)h 5Methylorotate IO 5Bromoorotate* I? 5-Nitrobarbiturate 18 Barbiturate* 48 t56jb 5-Azaorotate* 50 5-Aminoorotate 75 130 N-Carbamyl-or-aspartate L-DHO’ 5.3

Inhibition

-~-

” The Michaelis kinetics of those compounds marked with an asterisk were determined by the high-voltage electrophoresis technique. A Values in parenthesis represent comparative inhibition constants for rat liver mitochondrial DHODHase (2). ’ K,

value

(13).

66

GERO, O’SULLIVAN.

AND BROWN

decarboxylase is fully inhibited. the transferase may appear to be inhibited due to accumulation of OMP (18). Such comments also apply to the results obtained with 2-mercaptopyrimidine, 4-amino-6-hydroxy-2-mercaptopyrimidine. and 2thiobarbiturate, which were inhibitors of yeast ODCase. These compounds were ineffective as inhibitors of human DHO-DHase. Humun spleen enzymes. Of the compounds which inhibited human DHODHase. Zkazaorotate, $bromoorotate, and barbiturate were good inhibitors of human OPRTase. 6-Thiobarbiturate had no effect. None of these pyrimidinc analogs were efficient inhibitors of human ODCase (Table 3). Of the compounds tested that had no effect on DHO-DHase. only Lmercaptopyrimidine was shown to be an effective inhibitor of the human OPRTase. These results are in accord with the significant differences between the yeast and human OPRTase and ODCase enzymes. DISCUSSION Nine pyrimidine analogs, dihydro-5-azaorotate, 6-thiobarbiturate. orotate, 5methylorotate, 5bromoorotate, 5-nitrobarbiturate, barbiturate, 5-azaorotate, and 5-aminoorotate have been shown to be good inhibitors of human spleen mitochondrial DHO-DHase with apparent Ki values ranging from 3 to 75 ~.RI (Table 2). Values for the inhibition constants for barbiturate and orotate are in good agreement with those reported for rat liver mitochondrial DHO-DHase (app K,, 56 and 8.4 PM. respectively (2)). However. the value reported for barbiturate for crude brain homogenate is much higher with an apparent K, of 41.2 rnM ( 19). The analog of the substrate L-DHO, dihydro-5-azaorotate, and 6-thiobarbiturate appear to be specific inhibitors of the human DHO-DHase as neither of these Inhibitors of

OPRTdse

TABLE : and ODCase Activrty from Yeast ;mJ Human Spleen ‘i Inhibition of enzyme BCI~\XIL Yca5t

f-iumari \plcr12

Inhibitor at I rnM Compounds which inhibit DHO-DHase (from Table I) h-Thiobarbiturate 5-Azaorotate 5-Bromoorotate Barbiturate Noninhibitors of DHO-DHase 2-Mercaptopyrimidine 4-Amino-6-hydroxy-2-mercaptopyrimidine 2-Thiobarbiturate 4.5Dicarboxypyrimidine S-Bromo-I-methyl-5,6-dihydrouracil 2-Benzyl-4,6-dihydroxy-5-nitropyrimidine 3,.(-Dihydroxy-I .2.4-triazine

U! IY

9s YJX i?

0 0 ICI

INHIBITION

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HUMAN

DHO

DHase

67

compounds inhibited the human spleen OPRTase-ODCase system. Both compounds bound more strongly to the enzyme than did its natural substrate. Further, neither compound had any effect on DHOase, the preceding enzyme of the pathway, although this enzyme would be expected to have a similar binding site (A.M. Gero, unpublished data). However, S-methylorotate, which also does not inhibit the human OPRTase-ODCase complex, has been reported to inhibit DHOase (14). Dihydro-5azaorotate has been previously reported as an inhibitor of DHODHase in Escherichia coli (20), mouse liver (21), and Ehrlich ascites tumor cells (22). The compound 6-thiobarbiturate has not previously been reported as an inhibitor of DHO-DHase. It may also be worthy of consideration as a chemotherapeutic agent since derivatives of barbiturate which are unsubstituted in the 5 position do not have hypnotic or convulsant properties (19). From the results reported in Table 1 there appear to be three criteria for a compound to have reasonable inhibitory activity against DHO-DHase (defined as better than 25% of the best observed): (i) it must be both 4 or 6 substituted: (ii) it must ionize to form an anion from the 4 or 6 position (rather than from the 2 or 5 position); (iii) it must be appreciably (>lO%) ionized at the pH of the assay (7). Thus, compound Nos. I-12 (with the exception of carbamyl aspartate. which is not a pyrimidine compound) are all ionized appreciably at pH 7, are 4, 6 substituted, ionized to form an anion from the 4 or 6 position, and in addition, are 2-OH substituted. The necessity of a negatively charged group on the 6 position of the pyrimidine suggests an attachment of the substrate to a basic center on the dehydrogenase enzyme, as the substitution of the carboxy group by a methyl group, e.g., 6methyluracil (No. 51, Table f), amino group, e.g., 6-aminouracil (No. 70), esterification, e.g., 6-diethoxymethyl uracil (No. 43), or elimination of the carboxy group, decreases any inhibitory activity. In contrast, isoorotate (No. 50) and isobarbiturate (No. 30), which both have a negatively charged group on the 5 position, have little activity as inhibitors. Similarly, those pyrimidine analogs lacking a 2-OH group, e.g., a-dimethylamino4,6-dihydroxypyrimidine (No. 64) and 2-thiobarbiturate (No. 44) have no inhibitory activity. A secondary attachment site appears to be essential for a compound to be able to bind to the enzyme. These observations are substantially similar to those made by Aleman and Handler (7) and Friedmann and Vennesland (5) for the pattern of inhibition of pyrimidine analogs for the degradative cytoplasmic DHO-DHase enzyme of C. oroticum. The results from Table 1 also indicate that large substituents on the 5 position of barbiturate lessen its ability to act as an inhibitor of DHO-DHase, due to probable steric hindrance (i.e., barbiturate = 5-nitro-barbiturate > 5,5-diethylbarbiturate > amytal). The lack of inhibition by diethylbarbiturate and amytal has been noted by others (2). A minimum size may also be necessary for inhibition to occur. This may be the reason for the lesser activity of 5-fluoroorotate than the 5-bromo compound. 5-Fluoroorotate has been shown to act as a substrate for the C. oroticum enzyme (5).

68

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The inhibitors of DHO-DHase, 5-azaorotate, 5-bromoorotate, and barbiturate were also shown to be inhibitors of the human OPRTase (Table 3). 5-Azaorotate has previously been shown to inhibit OPRTase in rat Iiver (23, 24) and rat brain (19), and barbiturate has been reported to inhibit OPRTase in rat brain (19) and Ehrlich ascites cells (18). These pyrimidine compounds had little effect on human spleen ODCase under the conditions of the assay system. However, Potvin et crl. (19) have reported that barbiturate, when preincubated with the reaction mixture in the presence of exogenous P-Rib-PP plus Mg’+. caused a marked inhibition of the ODCase activity. The ribotide of barbiturate has been observed to be a powerful inhibitor of ODCase (19,25), Similarly, 6-aza-UMP and S-bromo-UMP have been reported to be inhibitors of the mammalian ODCase (18, 26. 27). In the i/z viva situation, where endogenous P-Rib-PP is available, the compounds 5-azaorotate, S-bromoorotate. and barbiturate may appear to act as moderate inhibitors and alternate substrates of OPRTase; the ribotides formed in this way being more effective inhibitors of the ODCase (cf. rest&s with allopurinol ( 15)). Thus, these three compounds would be inhibitors of the three subsequent enzymes, DHO-DHase, OPRTase, as we11 as ODCase. Such compounds. which cause inhibition of more than one enzyme of the pathway, would be expected to be more effective as antimetabolites in viw. Eight compounds were effective inhibitors of the yeast OPRTase-ODCase (Table 3). Four of these, 6thiobarbiturate, Ltmercaptopyrimidine, 4-amino-6-hydroxy-2-mercaptopyrimidine, and 2-thiobarbiturate were good inhibitors of the yeast ODCase but were considerably less effective as inhibitors of the human enzyme. The differential effects of the inhibitors on ODCase from the two sources appear to reflect the sensitivity of the yeast enzyme to sulfydryl group inhibitors (28). The inhibition of the yeast OPRTase by these compounds is difficult to assess because the accumulation of OMP leads automatically to inhibition of the OPRTase. It was not pursued in this study (cf. Ref. (18)). SUMMARY Seventy-one pyrimidine analogs have been tested as possible inhibitors of human spleen mitochondrial dihydroorotate dehydrogenase. Of these nine were demonstrated to be effective inhibitors of the enzymic activity. Two compounds, dihydro-5-azaorotate and 6-thiobarbiturate appeared to be specific inhibitors of the DHO-DHase. In addition, three compounds, 5-azaorotate, S-bromoorotate, and barbiturate were also inhibitory against the two subsequent enzymes of the pathway, orotate phosphoribosyltransferase and orotidylate decarboxylase. so that they could act against three enzymes of the mammalian pyrimidine de nmw biosynthetic pathway. ACKNOWLEDGMENTS We thank Dr. G. B. Elion for a gift of dihydro-S-azaorotate and Associate Professor J. Ham and his colleagues at the Prince of Wales Hospital for supplying human spleen material. This work was supported by grants from the Welicome foundation Ltd.. and from Wellcome Australia Limited.

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