Inhibition of cellular transformation by berry extracts.

Recent studies have examined and demonstrated the potential cancer chemopreventive activity of freeze-dried berries including strawberries and black raspberries. Although ellagic acid, an abundant component in these berries, has been shown to inhibit carcinogenesis both in vivo and in vitro, several studies have reported that other compounds in the berries may also contribute to the observed inhibitory effect. In the present study, freeze-dried strawberries (Fragara ananassa, FA) or black raspberries (Rubus ursinus, RU) were extracted, partitioned and chromatographed into several fractions (FA-F001, FA-F003, FA-F004, FA-F005, FA-DM, FA-ME from strawberries and RU-F001, RU-F003, RU-F004, RU-F005, RU-DM, RU-ME from black raspberries). These extracts, along with ellagic acid, were analyzed for anti-transformation activity in the Syrian hamster embryo (SHE) cell transformation model. None of the extracts nor ellagic acid by themselves produced an increase in morphological transformation. For assessment of chemopreventive activity, SHE cells were treated with each agent and benzo[a]pyrene (B[a]P) for 7 days. Ellagic acid, FA-ME and RU-ME fractions produced a dose-dependent decrease in transformation compared with B[a]P treatment only, while other fractions failed to induce a significant decrease. Ellagic acid, FA-ME and RU-ME were further examined using a 24 h co-treatment with B[a]P or a 6 day treatment following 24 h with B[a]P. Ellagic acid showed inhibitory ability in both protocols. FA-ME and RU-ME significantly reduced B[a]P-induced transformation only when co-treated with B[a]P for 24 h. These results suggest that a methanol extract from strawberries and black raspberries may display chemopreventive activity. The possible mechanism by which these methanol fractions (FA-ME, RU-ME) inhibited cell transformation appear to involve interference of uptake, activation, detoxification of B[a]P and/or intervention of DNA binding and DNA repair.

New cytotoxic 1-azaanthraquinones and 3-aminonaphthoquinone from the stem bark of Goniothalamus marcanii.

Guided by brine shrimp toxicity and human tumor cell toxicity, fractionation of the alcoholic extract from the stem bark of Goniothalamus marcanii led to the isolation of four new 1-azaanthraquinones: marcanines B (3), C (4), D (5), and E (6), along with two known derivatives: marcanine A and dielsiquinone. A new 5-hydroxy-3-amino-2-aceto-1,4-naphthoquinone (7), a possible 1-azaanthraquinone biosynthetic precursor, was also isolated. The structures of the compounds were elucidated by spectroscopic analyses, mainly 1D and 2D NMR techniques ((1)H, (13)C, NOEDS, COSY, HMQC, and HMBC), as well as comparison with literature data. All the compounds except 6 were evaluated for cytotoxic activity. They exhibited significant cytotoxicity against several human tumor cell lines, A-549, HT-29, MCF7, RPMI, and U251 with the ED(50) in the range of 0.04-3.03 microM.

Topoisomerase II inhibition by aporphine alkaloids.

The aporphine alkaloids (+)-dicentrine and (+)-bulbocapnine are non-planar molecules lacking features normally associated with DNA binding by intercalation or minor groove binding. Surprisingly, dicentrine showed significant activity as a topoisomerase II (EC 5.99.1.3) inhibitor and also was active in a DNA unwinding assay. The DNA unwinding suggests DNA intercalation, which could explain the inhibition of topoisomerase II. Bulbocapnine, which differs from dicentrine only by the presence of a hydroxyl group at position 11 and the absence of a methoxyl group at position 9, was inactive in all assays. Molecular modeling showed that dicentrine can attain a relatively planar conformation, whereas bulbocapnine cannot, due to steric interaction between the 11-hydroxyl group and an oxygen of the methylenedioxy ring. These observations suggest that dicentrine is an "adaptive" DNA intercalator, which can bind DNA only by adopting a somewhat strained planar conformation. The requirement of a suboptimal conformation to achieve DNA binding appears to make dicentrine a weaker topoisomerase II inhibitor than the very planar oxoaporphine alkaloid liriodenine. These results suggest that it may be possible to modulate DNA binding and biologic activity of drugs by modifications affecting their ability to adopt planar conformations.

DNA polymerase and topoisomerase II inhibitors from Psoralea corylifolia.

An ethanol extract of Psoralea corylifolia caused strong DNA polymerase inhibition in a whole cell bioassay specific for inhibitors of DNA replication enzymes. Bioassay-directed purification of the active compounds led to the isolation of the new compound corylifolin (1) and the known compound bakuchiol (2) as DNA polymerase inhibitors. On the basis of the structures of 1 and 2, resveratrol (3) was tested and found to be active as a DNA polymerase inhibitor in this bioassay. Neobavaisoflavone (4) was isolated as a DNA polymerase inhibitor, daidzein (5) as a DNA polymerase and topoisomerase II inhibitor, and bakuchicin (6) as a topoisomerase II inhibitor.

Inhibition of topoisomerase II by liriodenine.

The cytotoxic oxoaporphine alkaloid liriodenine, isolated from Cananga odorata, was found to be a potent inhibitor of topoisomerase II (EC 5.99.1.3) both in vivo and in vitro. Liriodenine treatment of SV40 (simian virus 40)-infected CV-1 cells caused highly catenated SV40 daughter chromosomes, a signature of topoisomerase II inhibition. Strong catalytic inhibition of topoisomerase II by liriodenine was confirmed by in vitro assays with purified human topoisomerase II and kinetoplast DNA. Liriodenine also caused low-level protein-DNA cross-links to pulse-labeled SV40 chromosomes in vivo, suggesting that it may be a weak topoisomerase II poison. This was supported by the finding that liriodenine caused topoisomerase II-DNA cross-links in an in vitro assay for topoisomerase II poisons. Verapamil did not increase either liriodenine-induced protein-DNA cross-links or catalytic inhibition of topoisomerase II in SV40-infected cells. This indicates that liriodenine is not a substrate for the verapamil-sensitive drug efflux pump in CV-1 cells.

Posttranslational acylation of the transferrin receptor in LSTRA cells with myristate, palmitate and stearate: evidence for distinct acyltransferases.

When incubated with [3H]myristate or [3H]palmitate, LSTRA cells, a murine T cell line, incorporated radiolabel into a protein of 95 kDa as analyzed by SDS-polyacrylamide gel electrophoresis. This dually acylated protein was identified as the transferrin receptor by immunoprecipitation with a monoclonal anti-transferrin receptor antibody. Acylation of the transferrin receptor was posttranslational and occurred via ester or thioester linkages. Analysis of radiolabeled transferrin receptor protein from [3H]myristate-labeled cells by acid hydrolysis followed by thin layer chromatography revealed the exclusive presence of [3H]myristate. Labeled transferrin receptor protein from [3H]palmitate-labeled cells contained predominantly [3H]stearate and smaller amounts of [3H]palmitate. This is in contrast to the protein-tyrosine kinase p56lck, which in [3H]palmitate-treated LSTRA cells, incorporated primarily [3H]palmitate. An analog of myristic acid, 5-nonanyloxyfuran-2-carboxylic acid, inhibited the incorporation of [3H]myristate, but not [3H]palmitate or [3H]stearate into transferrin receptor protein, suggesting that these acylation events are distinct. These studies indicate that the murine transferrin receptor is acylated posttranslationally with myristate, palmitate and stearate and suggest that more than one acyltransferase activity is responsible for its acylation.

Mechanism of action of the antileukemic xanthone psorospermin: DNA strand breaks, abasic sites, and protein-DNA cross-links.

Psorospermin, a cytotoxic dihydrofuranoxanthone isolated from Psorospermum febrifugum, produced aberrant simian virus 40 DNA replication intermediates when added to lytically infected CV-1 monkey kidney cells. The aberrant viral intermediates showed dose-dependent DNA strand breaks and protein-DNA cross-links, as well as decreased electrophoretic mobility. Simian virus 40 DNA from psorospermin-treated cells was shown to contain numerous abasic (apyrimidinic/apurinic) sites. The density of abasic sites was a function of the psorospermin dose. We conclude that psorospermin causes extensive loss of DNA bases in vivo. Primary amine groups of cellular proteins are known to react with abasic sites to form covalent protein-DNA cross-links and DNA strand breaks. Cytochrome c cross-linked spontaneously to viral DNA prepared from psorospermin-treated cells but not to DNA from untreated cells. This suggests that the protein-DNA cross-links and many of the DNA strand breaks observed in vivo result from reactions between abasic sites and chromosomal proteins. It is likely that the protein-DNA cross-links and DNA strand breaks contribute to the cytotoxicity and antineoplastic activity of psorospermin.

Synthesis of myristoyl CoA analogues and myristoyl peptides as inhibitors of myristoyl CoA:protein N-myristoyltransferase.

To develop inhibitors of myristoyl CoA:protein N-myristoyltransferase (NMT), a series of myristoyl coenzyme A analogues and myristoyl peptides were synthesized, including S-(2-oxopentadecyl)-CoA (1), S-(2-hydroxypentadecyl)-CoA (2), S-(2-oxopentadecyl)-pantetheine (3), Myr-N-Gly-(L)-Phe (4), Myr-N-Gly-(L)-Tyr (5), and Myr-N-Gly-(L)-Asn-Ala- Ala-Ser-Ala-Arg-(NH2) (6). Biological evaluation of these compounds in an in vitro NMT enzyme assay revealed that the nonhydrolyzable acyl CoA analogue 1 was the most potent inhibitor [inhibitor dissociation constant (Ki) = 24 nM]. A preliminary structure-activity relationship study showed that the adenosine moiety and the 2-keto group in this nonhydrolyzable analogue were necessary for inhibitory activity. A possible mechanism for the inhibition of NMT by 1 was proposed, in which 1 might block the reaction at the stage of an acyl-CoA-NMT-peptide complex. Product analogues such as the myristoylated peptides 4-6 were poor inhibitors of NMT.

Ohioensins and pallidisetins: novel cytotoxic agents from the moss Polytrichum pallidisetum.

Bioassay-directed fractionation of an EtOH extract of the moss Polytrichum pallidisetum (Polytrichaceae) led to the isolation of three novel benzonaphthoxanthenones, 1-O-methylohioensin B [6], 1-O-methyldihydroohioensin B [7] and 1,14-di-O-methyldihydroohioensin B [8], and two novel cinnamoyl bibenzyls, pallidisetin A [9] and pallidisetin B [10]. Their structures and relative stereochemistry were established by spectral analyses and chemical correlation. Compounds 6-10 exhibited cytotoxic activity against the human tumor cell lines RPMI-7951 melanoma and U-251 glioblastoma multiforme. These two types of compounds could hypothetically be derived from cinnamic acid and bibenzyls through different biogenetic pathways.

Treatment of T cells with 2-hydroxymyristic acid inhibits the myristoylation and alters the stability of p56lck.

N-Myristoylation of p56lck, a member of the Src family of protein-tyrosine kinases, is essential for its proper targeting to the plasma membrane. 2-Hydroxymyristic acid (HMA) is an analog of myristic acid that becomes metabolically activated in cells to form 2-hydroxymyristoyl-CoA, a potent inhibitor of myristoyl-CoA:protein N-myristoyltransferase (NMT), the enzyme that catalyzes protein N-myristoylation [Paige, L. A., Zheng, G.-q., DeFrees, S. A., Cassady, J. M., & Geahlen, R. L. (1990) Biochemistry 29, 10566]. In the presence of HMA, LSTRA cells, which overexpress p56lck, synthesized nonmyristoylated p56lck, which displayed a reduced electrophoretic mobility on SDS-polyacrylamide gels identical to that of a nonmyristoylated Gly2-->Ala2 mutant of p56lck. Treatment with myristic acid, 2-hydroxypalmitic acid, or 2-fluoromyristic acid did not result in the synthesis of nonmyristoylated p56lck. In contrast to the membrane-associated, myristoylated p56lck, nonmyristoylated p56lck was cytosolic. Although nonmyristoylated p56lck retained tyrosine kinase activity, it was not labeled in vivo with [32P]orthophosphate, indicating that a change in subcellular location altered its state of phosphorylation. A pulse-chase analysis revealed that cytosolic, nonmyristoylated p56lck was less stable than the myristoylated enzyme. In cell lines that do not overexpress p56lck, HMA treatment resulted in a reduction in the levels of both newly synthesized and total p56lck. Treatment of CD4+ cells with HMA caused a corresponding decrease in the amount of CD4-associated p56lck. Thus, chemical inhibition of protein N-myristoylation with HMA is an effective method for reducing the amount of p56lck available at the plasma membrane for signal transduction.

Reversible palmitoylation of the protein-tyrosine kinase p56lck.

The myristoylated protein-tyrosine kinase, p56lck, is expressed predominantly in T cells where it is believed to play a role in T cell activation. We observed a 56-kDa protein that became metabolically labeled in intact T lymphoid cells that were incubated with either [3H]myristate or [3H]palmitate. This protein was identified as p56lck based on its specific immunoprecipitation with polyclonal antisera to p56lck, by induction of a shift in its electrophoretic mobility following treatment of cells with 12-O-tetradecanoylphorbol-13-acetate and by co-chromatography with p56lck on protamine-agarose. Characterization of the two acylation events revealed that, in contrast to the p56lck-associated radioactivity from [3H]myristate-labeled cells, the p56lck-associated radioactivity from [3H]palmitate-labeled cells was susceptible to cleavage by neutral hydroxylamine and was not blocked by inhibitors of protein synthesis. Pulse-chase analyses revealed that the labeling of p56lck with [3H]palmitate, but not [3H]myristate, was reversible. The presence of covalently attached palmitate on p56lck from [3H]palmitate-labeled cells was verified by thin-layer chromatography following acid hydrolysis of the acylated protein. 2-Hydroxymyristate, which is metabolically activated to form a potent inhibitor of protein myristoylation, specifically inhibited the acylation of p56lck with [3H]myristate without affecting its labeling with [3H]palmitate. These studies indicate that p56lck is both a cotranslationally myristoylated and post-translationally palmitoylated protein.

Effects of synthetic and naturally occurring flavonoids on metabolic activation of benzo[a]pyrene in hamster embryo cell cultures.

Biochanin A, an isoflavone, has previously been shown to inhibit the metabolic activation of the carcinogen benzo[a]pyrene (B[a]P) to metabolites that bind to DNA in hamster embryo cells and are mutagenic in Chinese hamster V79 cells. To determine the structural features required for this activity and to attempt to find more effective inhibitors, a series of synthetic and naturally occurring flavonids were tested for their ability to modulate B[a]P metabolism in hamster embryo cell cultures. The observed structure-activity relationships indicate that the structural features of flavonoids important for effective inhibition of B[a]P metabolism in hamster embryo cells are the presence of two hydroxyl, two methoxyl, or methyl and hydroxyl substituents at the 5- and 7-positions and a 2,3-double bond. Flavones are slightly better inhibitors of B[a]P metabolism than the corresponding isoflavones. A substituent at the 4'-position is not essential for inhibition of B bdP metabolism. The presence of a hydroxyl group at position 3 slightly enhances activity. Apigenin, acacetin and kaempferide are effective inhibitors of B[a]P-induced mutagenesis in a hamster embryo cell-mediated V79 cell mutation assay. However, apigenin is cytotoxic at the inhibitory dose, whereas acacetin and kaempferide are not. These results suggest that acacetin and kaempferide are promising candidates for in vivo testing as potential chemopreventive agents.

Isolation of potential cancer chemopreventive agents from Eriodictyon californicum.

Activity-based fractionation of Eriodictyon californicum resulted in the isolation of 12 flavonoids that inhibit the metabolism of the carcinogen benzo[a]pyrene by hamster embryo cells in tissue culture. One was identified as a new flavanone, 3'-methyl-4'-isobutyryleriodictoyol [1], on the basis of spectroscopic analysis and alkaline hydrolysis. The seven other active flavanones were identified as eriodictyol [2], homoeriodictyol [3], 5,4'-dihydroxy-6,7-dimethoxyflavanone [4], pinocembrin [5], sakuranetin [6], 5,7,4'-trihydroxy-6,3'-dimethoxyflavanone [7], and naringenin 4'-methyl ether [8]. Four active flavones were also isolated: cirsimaritin [9], chrysoeriol [10], hispidulin [11], and chrysin [12]. The high inhibition of benzo[a]pyrene metabolism and the activation of benzo[a]pyrene to ultimate carcinogenic DNA-binding metabolites by cirsimaritin and chrysoeriol at a concentration of only 10 micrograms/ml indicates that these flavones warrant further investigation in vivo as potential chemopreventive agents.

Effects of biochanin A on metabolism, DNA binding and mutagenicity of benzo[a]pyrene in mammalian cell cultures.

The search for potential chemopreventive agents from higher plants based upon alteration of benzo[a]pyrene (B[a]P) metabolism in cell cultures resulted in isolation of the isoflavone biochanin A. The mechanisms by which biochanin A inhibits the metabolic activation of B[a]P were investigated in hamster embryo cell cultures. Biochanin A treatment inhibited the metabolism of B[a]P to water-soluble metabolites. B[a]P-9,10-diol and B[a]P-7,8-diol by 44, 60 and 52% respectively. Biochanin A inhibited the formation of glucuronide conjugates from 3-OH-B[a]P and 9-OH-B[a]P. Biochanin A also inhibited, in a dose-dependent manner, oxidation of B[a]P by homogenate (S-9) of Aroclor 1254-induced rat liver. Exposure of hamster embryo cells to biochanin A and [3H]B[a]P resulted in a decrease in the total level of [3H]B[a]P bound to DNA compared with the control groups at all time points studied between 24 and 120 h. This decrease was due to reduction in the formation of DNA adducts from both (+)-anti-B[a]P-diolepoxide and (+)-syn-B[a]P-diolepoxide. In a hamster embryo cell-mediated V79 cell mutation assay, biochanin A treatment resulted in a dose-dependent reduction in the number of B[a]P-induced mutants. These results indicate that biochanin A inhibits metabolic activation of B[a]P to mutagenic intermediates and warrants further investigation as a potential chemopreventive agent.

Effects of synthetic and naturally occurring flavonoids on benzo[a]pyrene metabolism by hepatic microsomes prepared from rats treated with cytochrome P-450 inducers.

Activity-directed fractionation of Trifolium pratense resulted in isolation of the isoflavone biochanin A, a potent inhibitor of metabolic activation of the carcinogen benzo[a]pyrene (B[a]P) in cells in culture. To determine the structural features required for maximal inhibition of cytochrome P-450 mediated metabolism of B[a]P, the inhibitory potencies of 23 flavonoids on metabolism of B[a]P to water-soluble derivatives were examined in liver S-9 homogenate from rats induced with Aroclor 1254. Flavones were much more efficient inhibitors than their corresponding isoflavone or flavanone analogs. Most flavonols were as effective inhibitors as their flavone analogs with the exception of kaempferide. Flavones with two hydroxyl or two methoxyl groups at positions 5 and 7 were the most active. Although all eight flavonoids tested effectively inhibited B[a]P metabolism by beta-naphthoflavone-induced microsomes, none were very effective inhibitors of B[a]P metabolism by phenobarbitol-induced microsomes, and only three were effective inhibitors of B[a]P metabolism by microsomes from non-induced rats. These results indicate that flavones or flavonols that contain free 5- and 7-hydroxyls are potent inhibitors of P-450 induced by beta-naphthoflavone (P-450IA1 and/or P-450IA2) and may potentially be useful as chemopreventive agents against hydrocarbon-induced carcinogenesis.

Metabolic activation of 2-substituted derivatives of myristic acid to form potent inhibitors of myristoyl CoA:protein N-myristoyltransferase.

The importance of myristoylation for the proper biological functioning of many acylated proteins has generated interest in the enzymes of the myristoylation pathway and their interactions with substrates and inhibitors. Previous observations that S-(2-oxopentadecyl)-CoA, a nonhydrolyzable methylene-bridged analogue of myristoyl-CoA, was a potent inhibitor of myristoyl-CoA:protein N-myristoyltransferase (NMT) [Paige, L. A., Zheng, G.-q., DeFrees, S. A., Cassady, J. M., & Geahlen, R. L. (1989) J. Med. Chem. 32, 1665] prompted a closer examination of the effect of substituents at the 2-position on the interactions of myristic acid and myristoyl-CoA analogues with NMT. As an initial approach, three myristic acid derivatives bearing different substituents at the 2-position, 2-fluoromyristic acid, 2-bromomyristic acid, and 2-hydroxymyristic acid, were selected for study. Both 2-bromomyristic acid and 2-hydroxymyristic acid were available commercially; 2-fluoromyristic acid was prepared synthetically. All three compounds were found to be only weak inhibitors of NMT in vitro. Of the three, 2-bromomyristic acid was the most potent (Ki = 100 microM). In cultured cells, however, 2-hydroxymyristic acid was by far the more effective inhibitor of protein myristoylation. Neither 2-hydroxymyristic acid nor 2-bromomyristic acid significantly inhibited protein palmitoylation in cultured cells, indicating that inhibition was not occurring at the level of acyl-CoA synthetase. Activation of the 2-substituted myristic acid derivatives to their corresponding acyl-CoA thioesters by acyl-CoA synthetase resulted in inhibitors of greatly increased potency. The 2-substituted acyl-CoA analogues, 2-hydroxymyristoyl-CoA, 2-bromomyristoyl-CoA, and 2-fluoromyristoyl-CoA, were synthesized and shown to be competitive inhibitors of NMT in vitro (Ki's = 45, 450, and 200 nM, respectively). These data suggested that the enhanced inhibitory potency of 2-hydroxymyristic acid seen in cells was most probably a result of its metabolic activation to the CoA thioester. The presence of substituents at the 2-position also affected the ability of the acyl group to be transferred by NMT to a peptide substrate. Of the three acyl-CoA analogues, only 2-fluoromyristoyl-CoA served as a substrate for NMT.

Ansamitocin P-3, a maytansinoid, from Claopodium crispifolium and Anomodon attenuatus or associated actinomycetes.

Guided by cytotoxicity, ansamitocin P-3, a maytansinoid, was isolated in very low yield from two members of the moss family Thuidiaceae, Claopodium crispifolium (Hook.) Ren. & Card. and Anomodon attenuatus (Hedw.) Hueb. Ansamitocin P-3 showed potent cytotoxicity against the human solid tumor cell lines A-549, HT-29. A possible basis for the occurrence of this compound in mosses is discussed.

Natural products as a source of potential cancer chemotherapeutic and chemopreventive agents.

Recent advances in the chemistry of novel bioactive natural products are reported. This research is directed to the exploration of plants with confirmed activity in bioassays designed to detect potential cancer chemotherapeutic and chemopreventive agents. Structural work and chemical studies are reported for several cytotoxic agents from the plants Annona densicoma, Annona reticulata, Claopodium crispifolium, Polytrichum obioense, and Psorospermum febrifugum. Studies are also reported based on development of a mammalian cell culture benzo[a]pyrene metabolism assay for the detection of potential anticarcinogenic agents from natural products. In this study a number of isoflavonoids and flavonoids with antimutagenic activity have been discovered.

Use of a mammalian cell culture benzo(a)pyrene metabolism assay for the detection of potential anticarcinogens from natural products: inhibition of metabolism by biochanin A, an isoflavone from Trifolium pratense L.

Based on the epidemiological evidence for a relationship between consumption of certain foods and decreased cancer incidence in humans, an assay was developed to screen and fractionate plant extracts for chemopreventive potential. This assay measures effects on the metabolism of [3H]benzo(a)pyrene [B(a)P] in hamster embryo cell cultures. Screening of several plant extracts has generated a number of activity leads. The 95% ethyl alcohol extract of one of these actives, Trifolium pratense L. Leguminosae, red clover, significantly inhibited the metabolism of B(a)P and decreased the level of binding of B(a)P to DNA by 30 to 40%. Using activity-directed fractionation by solvent partitioning and then silica gel chromatography, a major active compound was isolated and identified as the isoflavone, biochanin A. The pure compound decreased the metabolism of B(a)P by 54% in comparison to control cultures and decreased B(a)P-DNA binding by 37 to 50% at a dose of 25 micrograms/ml. These studies demonstrate that the hydrocarbon metabolism assay can detect and guide the fractionation of potential anticarcinogens from plants. The ability of the isoflavone biochanin A to inhibit carcinogen activation in cells in culture suggests that in vivo studies of this compound as a potential chemopreventive agent are warranted.

Structure-activity relationships of pyrimidines as dihydroorotate dehydrogenase inhibitors.

The activity of dihydroorotate dehydrogenase (DHO-dehase) has been reported to decrease both in vitro and in vivo in hepatocellular carcinomas. DHO-dehase, the fourth enzyme of the de novo pyrimidine biosynthetic pathway, is a mitochondrial enzyme which is both a potential rate-limiting reaction in the de novo pyrimidine biosynthetic pathway and a potential therapeutic target for tumor inhibitors. This paper reports results on a series of pyrimidine analogs of dihydroorotate (DHO) and orotic acid (OA) as inhibitors of DHO-dehase. The enzyme test results established that the intact amide and imide groups of the pyrimidine ring and the 6-carboxylic acid are required for significant enzyme inhibition. The testing of several functional groups similar in characteristics to that of the carboxylic acid, such as sulfonamide, tetrazole and phosphate, indicated that the carboxylic acid group is preferred by the enzyme. Using various 5-substituted OA and DHO derivatives, it was shown that there is a steric limitation of a methyl group at this position. The compound D,L-5-trans-methyl DHO (7) (Ki of 45 microM) was both an inhibitor and a weak substrate for the enzyme, demonstrating that mechanism-based enzyme inhibitors should be effective. The testing results further suggest that a negatively charged enzyme substituent may be present near the 5-position of the pyrimidine ring and that there may be an enzyme-substrate metal coordination site near the N-1 and carboxylic acid positions of the pyrimidine ring. The combined testing results were then used to define both conformational and steric substrate enzyme binding requirements from which a model was proposed for the binding of DHO and OA to the DHO-dehase active site.