Inhibition of the de novo pyrimidine biosynthesis pathway limits ribosomal RNA transcription causing nucleolar stress in glioblastoma cells.

Glioblastoma is the most common and aggressive type of cancer in the brain; its poor prognosis is often marked by reoccurrence due to resistance to the chemotherapeutic agent temozolomide, which is triggered by an increase in the expression of DNA repair enzymes such as MGMT. The poor prognosis and limited therapeutic options led to studies targeted at understanding specific vulnerabilities of glioblastoma cells. Metabolic adaptations leading to increased synthesis of nucleotides by de novo biosynthesis pathways are emerging as key alterations driving glioblastoma growth. In this study, we show that enzymes necessary for the de novo biosynthesis of pyrimidines, DHODH and UMPS, are elevated in high grade gliomas and in glioblastoma cell lines. We demonstrate that DHODH's activity is necessary to maintain ribosomal DNA transcription (rDNA). Pharmacological inhibition of DHODH with the specific inhibitors brequinar or ML390 effectively depleted the pool of pyrimidines in glioblastoma cells grown in vitro and in vivo and impaired rDNA transcription, leading to nucleolar stress. Nucleolar stress was visualized by the aberrant redistribution of the transcription factor UBF and the nucleolar organizer nucleophosmin 1 (NPM1), as well as the stabilization of the transcription factor p53. Moreover, DHODH inhibition decreased the proliferation of glioblastoma cells, including temozolomide-resistant cells. Importantly, the addition of exogenous uridine, which reconstitutes the cellular pool of pyrimidine by the salvage pathway, to the culture media recovered the impaired rDNA transcription, nucleolar morphology, p53 levels, and proliferation of glioblastoma cells caused by the DHODH inhibitors. Our in vivo data indicate that while inhibition of DHODH caused a dramatic reduction in pyrimidines in tumor cells, it did not affect the overall pyrimidine levels in normal brain and liver tissues, suggesting that pyrimidine production by the salvage pathway may play an important role in maintaining these nucleotides in normal cells. Our study demonstrates that glioblastoma cells heavily rely on the de novo pyrimidine biosynthesis pathway to generate ribosomal RNA (rRNA) and thus, we identified an approach to inhibit ribosome production and consequently the proliferation of glioblastoma cells through the specific inhibition of the de novo pyrimidine biosynthesis pathway.

The inhibition of thymidine phosphorylase can reverse acquired 5FU-resistance in gastric cancer cells.

BACKGROUND: 5FU can be converted to its active metabolite fluoro-deoxyuridine monophosphate (FdUMP) through two pathways: the orotate phosphoribosyl transferase-ribonucleotide reductase (OPRT-RR) pathway and the thymidine phosphorylase-thymidine kinase (TP-TK) pathway. We investigated the mechanism underlying 5FU-resistance, focusing on the changes in the 5FU metabolisms. METHODS: MKN45 and 5FU-resistant MKN45/F2R cells were treated with 5FU or fluoro-deoxyuridine (FdU) in combination with hydroxyurea (HU) or tipiracil (TPI). The amount of FdUMP was determined by the density of the upper band of thymidylate synthase on Western blotting. RESULTS: The MKN45/F2R cells exhibited 5FU resistance (37.1-fold) and showed decreased OPRT and increased TP levels. In both cells, the FdUMP after treatment with 5FU was decreased when RR was inhibited by HU but not when TP was inhibited by TPI. A metabolome analysis revealed the loss of intracellular deoxyribose 1-phosphate (dR1P) in both cells, indicating that FdUMP was synthesized from 5FU only through the OPRT-RR pathway because of the loss of dR1P. After the knockdown of TK, the FdUMP after treatment with FdU was decreased in MKN45 cells. However, it was not changed in MKN45/F2R cells. Furthermore, TP inhibition caused an increase in FdUMP after treatment with 5FU or FdU and reversed the 5FU resistance in MKN45/F2R cells, indicating that FdUMP was reduced through the TP-TK pathway in MKN45/F2R cells. CONCLUSIONS: In MKN45/F2R cells, the reduction of FdUMP through the TP-TK pathway caused 5FU resistance, and the inhibition of TP reversed the resistance to 5FU, suggesting that the combination of 5FU and TPI is a promising cancer therapy.

Antipyrimidine effects of five different pyrimidine de novo synthesis inhibitors in three head and neck cancer cell lines.

The pyrimidine de novo nucleotide synthesis consists of 6 sequential steps. Various inhibitors against these enzymes have been developed and evaluated in the clinic for their potential anticancer activity: acivicin inhibits carbamoyl-phosphate-synthase-II, N-(phosphonacetyl)-L- aspartate (PALA) inhibits aspartate-transcarbamylase, Brequinar sodium and dichloroallyl-lawsone (DCL) inhibit dihydroorotate-dehydrogenase, and pyrazofurin (PF) inhibits orotate-phosphoribosyltransferase. We compared their growth inhibition against 3 cell lines from head-and-neck-cancer (HEP-2, UMSCC-14B and UMSCC-14C) and related the sensitivity to their effects on nucleotide pools. In all cell lines Brequinar and PF were the most active compounds with IC50 (50% growth inhibition) values between 0.06-0.37 µM, Acivicin was as potent (IC50s 0.26-1 µM), but DCL was 20-31-fold less active. PALA was most inactive (24-128 µM). At equitoxic concentrations, all pure antipyrimidine de novo inhibitors depleted UTP and CTP after 24 hr exposure, which was most pronounced for Brequinar (between 6-10% of UTP left, and 12-36% CTP), followed by DCL and PF, which were almost similar (6-16% UTP and 12-27% CTP), while PALA was the least active compound (10-70% UTP and 13-68% CTP). Acivicin is a multi-target inhibitor of more glutamine requiring enzymes (including GMP synthetase) and no decrease of UTP was found, but a pronounced decrease in GTP (31-72% left). In conclusion, these 5 inhibitors of the pyrimidine de novo nucleotide synthesis varied considerably in their efficacy and effect on pyrimidine nucleotide pools. Inhibitors of DHO-DH were most effective suggesting a primary role of this enzyme in controlling pyrimidine nucleotide pools.

Synthesis of bis-Phosphate Iminoaltritol Enantiomers and Structural Characterization with Adenine Phosphoribosyltransferase.

Phosphoribosyl transferases (PRTs) are essential in nucleotide synthesis and salvage, amino acid, and vitamin synthesis. Transition state analysis of several PRTs has demonstrated ribocation-like transition states with a partial positive charge residing on the pentose ring. Core chemistry for synthesis of transition state analogues related to the 5-phospho-α-d-ribosyl 1-pyrophosphate (PRPP) reactant of these enzymes could be developed by stereospecific placement of bis-phosphate groups on an iminoaltritol ring. Cationic character is provided by the imino group and the bis-phosphates anchor both the 1- and 5-phosphate binding sites. We provide a facile synthetic path to these molecules. Cyclic-nitrone redox methodology was applied to the stereocontrolled synthesis of three stereoisomers of a selectively monoprotected diol relevant to the synthesis of transition-state analogue inhibitors. These polyhydroxylated pyrrolidine natural product analogues were bis-phosphorylated to generate analogues of the ribocationic form of 5-phosphoribosyl 1-phosphate. A safe, high yielding synthesis of the key intermediate represents a new route to these transition state mimics. An enantiomeric pair of iminoaltritol bis-phosphates (L-DIAB and D-DIAB) was prepared and shown to display inhibition of Plasmodium falciparum orotate phosphoribosyltransferase and Saccharomyces cerevisiae adenine phosphoribosyltransferase (ScAPRT). Crystallographic inhibitor binding analysis of L- and D-DIAB bound to the catalytic sites of ScAPRT demonstrates accommodation of both enantiomers by altered ring geometry and bis-phosphate catalytic site contacts.

Development of new promising antimetabolite, DFP-11207 with self-controlled toxicity in rodents.

To reduce 5-fluorouracil (5-FU)-induced serious toxicities without loss of antitumor activity, we have developed DFP-11207, a novel fluoropyrimidine, which consists of 1-ethoxymethyl-5-fluorouracil (EM-FU; a precursor form of 5-FU), 5-chloro-2,4-dihydroxypyridine (CDHP; an inhibitor of 5-FU degradation), and citrazinic acid (CTA; an inhibitor of 5-FU phosphorylation). In vitro studies of DFP-11207 indicated that it strongly inhibited the degradation of 5-FU by dihydropyrimidine dehydrogenase (DPD) in homogenates of the rat liver, and also inhibited the phosphorylation of 5-FU by orotate phosphoribosyltransferase (OPRT) in tumor tissues in a similar magnitude of potency by CDHP and CTA, respectively. Especially, DFP-11207 inhibited the intracellular phosphorylation of 5-FU in tumor cells in a dose-dependent manner whereas CTA alone did not protect intracellular 5-FU phosphorylation. These results postulate that DFP-11207 rapidly entered into the cell and the free CTA produced from DFP-11207 inhibited the phosphorylation of 5-FU in the cell. Furthermore, following oral administration of DFP-11207, CTA was found to be highly retained in the gastrointestinal (GI) tract compared to other tissues in rats. Interestingly, EM-FU, the prodrug of 5-FU was found to specifically produce 5-FU by various species of liver microsomes. When DFP-11207 was administered to rats, the plasma level of 5-FU was persisted for a long-time with lower Cmax and longer half-life than that from other 5-FU prodrugs. The antitumor activity of DFP-11207 was evaluated in human tumor xenografts in nude rats and found that DFP-11207 showed an antitumor activity in a dose-dependent fashion and its efficacy is equivalent to reference 5-FU drugs. In striking contrast, DFP-11207 manifested no or less 5-FU-related toxicities, such as a decrease in body weights, GI injury, and myelosuppression, especially thrombocytopenia. Taken together, the preclinical evaluation of DFP-11207 strongly indicates that DFP-11207 be a potential new version of the oral fluoropyrimidine prodrug for further clinical development.

Physiologic Medium Rewires Cellular Metabolism and Reveals Uric Acid as an Endogenous Inhibitor of UMP Synthase.

A complex interplay of environmental factors impacts the metabolism of human cells, but neither traditional culture media nor mouse plasma mimic the metabolite composition of human plasma. Here, we developed a culture medium with polar metabolite concentrations comparable to those of human plasma (human plasma-like medium [HPLM]). Culture in HPLM, relative to that in traditional media, had widespread effects on cellular metabolism, including on the metabolome, redox state, and glucose utilization. Among the most prominent was an inhibition of de novo pyrimidine synthesis-an effect traced to uric acid, which is 10-fold higher in the blood of humans than of mice and other non-primates. We find that uric acid directly inhibits uridine monophosphate synthase (UMPS) and consequently reduces the sensitivity of cancer cells to the chemotherapeutic agent 5-fluorouracil. Thus, media that better recapitulates the composition of human plasma reveals unforeseen metabolic wiring and regulation, suggesting that HPLM should be of broad utility.

Investigation of vital pathogenic target orotate phosphoribosyltransferases (OPRTase) from Thermus thermophilus HB8: Phylogenetic and molecular modeling approach.

Biosynthesis pathways of pyrimidine and purine are shown to play an important role in regular cellular activities. The biosynthesis can occur either through de novo or salvage pathways based on the requirement of the cell. The pyrimidine biosynthesis pathway has been linked to several disorders and various autoimmune diseases. Orotate phosphoribosyl transferase (OPRTase) is an important enzyme which catalyzes the conversion of orotate to orotate monophosphate in the fifth step of pyrimidine biosynthesis. Phylogenetic analysis of 228 OPRTase sequences shows the distribution of proteins across different living forms of life. High structural similarities between Thermusthermophilus and other organisms kindled us to concentrate on OPRTase as an anti-pathogenic target. In this study, a homology model of OPRTase was constructed using 2P1Z as a template. About 100 ns molecular dynamics simulation was performed to investigate the conformational stability and dynamic patterns of the protein. The amino acid residues (Met1, Asp2, Glu43, Ala44, Glu47, Lys51, Ala157 and Leu158) lining in the binding site were predicted using SiteMap. Further, structure based virtual screening was performed on the predicted binding site using ChemBridge, Asinex, Binding, NCI, TosLab and Zinc databases. Compounds retrieved from the screening collections were manually clustered. The resultant protein-ligand complexes were subjected to molecular dynamics simulations, which further validates the binding modes of the hits. The study may provide better insight for designing potent anti-pathogenic agent.

Pyrimidin-2(1H)-ones based inhibitors of Mycobacterium tuberculosis orotate phosphoribosyltransferase.

Tuberculosis (TB) is an ancient human chronic infectious disease caused mainly by Mycobacterium tuberculosis. The emergence of strains resistant to first and second line anti-TB drugs, associated with the increasing number of TB cases among HIV positive subjects, and the large number of individuals infected with latent bacilli have urged the development of new strategies to treat TB. Enzymes of nucleotide metabolism pathways provide promising molecular targets for the development of drugs, aiming at both active and latent TB. The orotate phosphoribosyltransferase (OPRT) enzyme catalyzes the synthesis of orotidine 5'-monophosphate from 5'-phospho-α-d-ribose 1'-diphosphate and orotic acid, in the de novo pyrimidine synthesis pathway. Based on the kinetic mechanism and molecular properties, here we describe the design, selection and synthesis of substrate analogs with inhibitory activity of M. tuberculosis OPRT (MtOPRT) enzyme. Steady-state kinetic measurements were employed to determine the mode of inhibition of commercially available and chemically derived compounds. The 6-Hydroxy-2-oxo-1,2-dihydropyridine-4-carboxylic acid (6) chemical compound and its derivative, 3-Benzylidene-2,6-dioxo-1,2,3,6-tetrahydropyridine-4-carboxylic acid (13), showed enzyme inhibition constants in the submicromolar range. Isothermal titration calorimetry data indicated that binding of both compounds to MtOPRT have negative enthalpy and favorable Gibbs free energy probably due to their high complementarity to the enzyme's binding pocket. Improvement of compound 13 hydrophobic character by addition of an aromatic ring substituent resulted in entropic optimization, reflected on a thermodynamic discrimination profile characteristic of high affinity ligands. These inhibitors represent lead compounds for further development of MtOPRT inhibitors with increased potency, which may be tested as anti-TB agents.

Electrophilic aromatic selenylation: new OPRT inhibitors.

2-Ethoxyethaneseleninic acid reacts with electron-rich aromatic substrates to deliver, by way of the selenoxides, the (2-ethoxyethyl)seleno ethers, which can in turn be transformed into a diverse set of aryl-selenylated products. Among these, a family of 5-uridinyl derivatives shows submicromolar inhibition of human and malarial orotate phosphoribosyltransferase.

Pyrophosphate interactions at the transition states of Plasmodium falciparum and human orotate phosphoribosyltransferases.

Orotate phosphoribosyltransferases from Plasmodium falciparum and human sources (PfOPRT and HsOPRT) use orotidine as a slow substrate in the pyrophosphorolysis reaction. With orotidine, intrinsic kinetic isotope effects (KIEs) can be measured for pyrophosphorolysis, providing the first use of pyrophosphate (PPi) in solving an enzymatic transition state. Transition-state structures of PfOPRT and HsOPRT were solved through quantum chemical matching of computed and experimental intrinsic KIEs and can be compared to transition states solved with pyrophosphate analogues as slow substrates. PfOPRT and HsOPRT are characterized by late transition states with fully dissociated orotate, well-developed ribocations, and weakly bonded PPi nucleophiles. The leaving orotates are 2.8 A distant from the anomeric carbons at the transition states. Weak participation of the PPi nucleophiles gives C1'-O(PPi) bond distances of approximately 2.3 A. These transition states are characterized by C2'-endo ribosyl pucker, based on the beta-secondary [2'-(3)H] KIEs. The geometry at the 5'-region is similar for both enzymes, with C3'-C4'-C5'-O5' dihedral angles near -170 degrees . These novel phosphoribosyltransferase transition states are similar to but occur earlier in the reaction coordinate than those previously determined with orotidine 5'-monophosphate and phosphonoacetic acid as substrates. The similarity between the transition states with different substrate analogues supports similar transition state structures imposed by PfOPRT and HsOPRT even with distinct reactants. We propose that the transition state similarity with different nucleophiles is determined, in part, by the geometric constraints imposed by the catalytic sites.

Kinetic benefits and thermal stability of orotate phosphoribosyltransferase and orotidine 5'-monophosphate decarboxylase enzyme complex in human malaria parasite Plasmodium falciparum.

We have previously shown that orotate phosphoribosyltransferase (OPRT) and orotidine 5'-monophosphate decarboxylase (OMPDC) in human malaria parasite Plasmodium falciparum form an enzyme complex, containing two subunits each of OPRT and OMPDC. To enable further characterization, we expressed and purified P. falciparum OPRT-OMPDC enzyme complex in Escherichia coli. The OPRT and OMPDC activities of the enzyme complex co-eluted in the chromatographic columns used during purification. Kinetic parameters (K(m), k(cat) and k(cat)/K(m)) of the enzyme complex were 5- to 125-folds higher compared to the monofunctional enzyme. Interestingly, pyrophosphate was a potent inhibitor to the enzyme complex, but had a slightly inhibitory effect for the monofunctional enzyme. The enzyme complex resisted thermal inactivation at higher temperature than the monofunctional OPRT and OMPDC. The result suggests that the OPRT-OMPDC enzyme complex might have kinetic benefits and thermal stability significantly different from the monofunctional enzyme.

'Irreversible' slow-onset inhibition of orotate phosphoribosyltransferase by an amidrazone phosphate transition-state mimic.

A mimic of the putative transition-state intermediate has been synthesized and found to be a very slow-onset inhibitor of yeast orotate phosphoribosyltransferase. The mechanism of inhibition may involve a rate-determining isomerization of the enzyme to a form receptive to the inhibitor, which then remains tightly bound.

Diamine oxidase, a plasma biomarker in rats to GI tract toxicity of oral fluorouracil anti-cancer drugs.

Diamine oxidase (DAO; EC 1.4.3.6), which catabolizes a variety of substrates including histamine and diamines, is the degradative enzyme of the catabolic pathway of polyamines found in high activity in the mature upper villus cells of the rat intestinal mucosa [Luk, G.D., Bayless, T.M., Baylin, S.B., 1983. Plasma post-heparin diamine oxidase. Sensitive provocative test for quantitating length of acute intestinal mucosal injury in the rat. J. Clin. Invest. 71, 1308-1315; Wolvekamp, M.C.J., de Bruin, R.W.F., 1994. Diamine oxidase: an overview of historical, biochemical and functional aspects. Dig. Dis. 12, 2-14]. Rats were given 1-week repeated oral administration of anti-cancer drugs S-1, containing FT+CDHP+Oxo, and FCD, containing FT+CDHP, and the ameliorating effect of Oxo on the rat gastrointestinal (GI) tract toxicity from 5-FU was evaluated by measuring plasma DAO activity which is related to the enzyme located in the rat intestinal mucosa. Plasma DAO activity in the FCD-treated group was significantly less than that in the S-1-treated group while the jejunal mucosal area in the FCD group was significantly smaller than that in the S-1 group. In addition the histopathological findings in the FCD group showed villus atrophy in the jejunal mucosa which was not observed in the S-1 group. The degree of these findings correlated with the plasma DAO levels. Therefore, the protective effect of Oxo on 5-FU-induced GI tract toxicity was clarified by measuring plasma DAO activity in rats. In summary, DAO is a very sensitive plasma biomarker and will be useful for the quantitative evaluation of the small intestinal mucosal lesions induced by the anti-cancer drug, 5-FU, in rats.

Metabolic effects of novel N-1-sulfonylpyrimidine derivatives on human colon carcinoma cells.

Novel N-1-sulfonylpyrimidine derivatives have a strong antiproliferative activity and an ability to induce apoptosis in treated tumor cells. The purpose of this study was to elucidate the effects of two N-1-sulfonylpyrimidine nucleobases on catalytic activity of tumor cells' enzymes involved in DNA and RNA synthesis, and in de novo and salvage pyrimidine and purine syntheses. Investigations were performed in vitro on colon carcinoma cells (Caco2). The biosynthetic activity of the tumor cells' enzymes was determined using sensitive radio-assays. Enzyme activity in treated cells was calculated relative to untreated control cells. Both of the investigated compounds, 1-(p-toluenesulfonyl) cytosine (TsC) and 5-bromo-1-(methanesulfonyl) uracil (BMsU) inhibited activities of specific enzymes involved in nucleic acid synthesis. BMsU strongly inhibited activities of DNA polymerase alpha (53%), thymidine kinase (68%), thymidilate synthase (43%), and ribonucleotide reductase (46%). De novo biosynthesis of pyrimidine and purine was reduced by 20%. TsC was able to inhibit RNA polymerase (37%), orotate phosphoribosyltransferase (39%), uridine kinase (44%), ribonucleotid reductase (47%), and de novo purine synthesis (61%). Antitumor activity of 1-(p-toluenesulfonyl) cytosine (TsC) and 5-bromo-1-(methanesulfonyl) uracil (BMsU) is closely associated with their inhibitory activity on enzymes that play an important role in the metabolism of tumor cells.

A novel enzyme complex of orotate phosphoribosyltransferase and orotidine 5'-monophosphate decarboxylase in human malaria parasite Plasmodium falciparum: physical association, kinetics, and inhibition characterization.

Human malaria parasite, Plasmodium falciparum, can only synthesize pyrimidine nucleotides using the de novo pathway, whereas mammalian cells obtain pyrimidine nucleotides from both the de novo and salvage pathways. The parasite's orotate phosphoribosyltransferase (PfOPRT) and orotidine 5'-monophosphate decarboxylase (PfOMPDC) of the de novo pyrimidine pathway are attractive targets for antimalarial drug development. Previously, we have reported that the two enzymes in P. falciparum exist as a multienzyme complex containing two subunits each of 33-kDa PfOPRT and 38-kDa PfOMPDC. In this report, the gene encoding PfOPRT has been cloned and expressed in Escherichia coli. An open reading frame of PfOMPDC gene was identified in the malaria genome database, and PfOMPDC was cloned from P. falciparum cDNA, functionally expressed in E. coli, purified, and characterized. The protein sequence has <20% identity with human OMPDC and four microbial OMPDC for which crystal structures are known. Recombinant PfOMPDC was catalytically active in a dimeric form. Both recombinant PfOPRT and PfOMPDC monofunctional enzymes were kinetically different from the native multienzyme complex purified from P. falciparum. Oligomerization of PfOPRT and PfOMPDC cross-linked by dimethyl suberimidate indicated that they were tightly associated as the heterotetrameric 140-kDa complex, (PfOPRT)2(PfOMPDC)2. Kinetic analysis of the PfOPRT-PfOMPDC associated complex was similar to that of the native P. falciparum enzymes and was different from that of the bifunctional human enzymes. Interestingly, a nanomolar inhibitor of the yeast OMPDC, 6-thiocarboxamido-uridine 5'-monophosphate, was about 5 orders of magnitude less effective on the PfOMPDC than on the yeast enzyme. Our results support that the malaria parasite has unique structural and functional properties, sharing characteristics of the monofunctional pyrimidine-metabolizing enzymes in prokaryotes and bifunctional complexes in eukaryotes.

The cyanobacterial toxin cylindrospermopsin inhibits pyrimidine nucleotide synthesis and alters cholesterol distribution in mice.

The hepatotoxin Cylindrospermopsin, a sulfated-guanidinium alkaloid with substituted dioxypyrimidine (uracil) moiety, was isolated from several cyanobacteria species. Our previous studies on the toxicity of cylindrospermopsin and its derivatives suggested that the uracil moiety is crucial for the toxicity and that such toxicity could partly stem from competitive binding of the toxin to a catalytic site(s) involved in the synthesis of pyrimidine nucleotides (i.e., uridine). In the present study we demonstrated that cylindrospermopsin inhibited in a noncompetitive manner the in vitro activity of uridine monophosphate (UMP) synthase complex (responsible for the conversion of orotic acid to UMP) in a cell free liver extract from mice, with an inhibition constant, KI, of 10 microM. Exposure of mice to cylindrospermopsin at subacute concentrations, via drinking water, only slightly affected the in vitro activity of UMP synthase. The typical metabolic disorder associated with the inhibition of UMP synthase activity, known as "orotic aciduria," was not observed under these conditions, but other anomalous metabolic responses related to cholesterol metabolism were developed.

Phase I and pharmacokinetic study of the oral fluoropyrimidine S-1 on a once-daily-for-28-day schedule in patients with advanced malignancies.

PURPOSE: The oral fluoropyrimidine S-1, which consists of a mixture of a 5-fluorouracil (5-FU) prodrug (tegafur), a dihydropyrimidine dehydrogenase inhibitor [5-chloro-2,4-dihydroxypyrimidine (CDHP)], and an inhibitor of orotate phosphoribosyltransferase [potassium oxonate (oxonic acid)], was developed to increase the feasibility and therapeutic index of 5-FU administered orally. The principal objective of this study was to assess the feasibility of administering S-1 on a once-daily-for-28-day schedule every 5 weeks, determine the maximum tolerated dose, characterize the pharmacokinetics of S-1, and seek evidence of anticancer activity. EXPERIMENTAL DESIGN: Patients with advanced solid malignancies were treated with escalating doses of S-1 on a once-daily oral schedule for 28 days every 5 weeks. The maximum tolerated dose was defined as the highest dose in which fewer than two of the first six new patients experienced dose-limiting toxicity. The pharmacokinetic profiles of the tegafur, CDHP, and oxonic acid constituents were characterized. RESULTS: Twenty patients were treated with 72 courses of S-1 at three dose levels ranging from 50 to 70 mg/m(2)/day. Diarrhea, which was often associated with abdominal discomfort and cramping, was the principal dose-limiting toxicity of S-1 on this protracted schedule. Nausea, vomiting, mucositis, fatigue, and cutaneous effects were also observed but were rarely severe. Myelosuppression was modest and uncommon. A partial response and a 49% reduction in tumor size were observed in patients with fluoropyrimidine- and irinotecan-resistant colorectal carcinoma. The pharmacokinetic data suggested potent inhibition of 5-FU clearance by CHDP, with resultant 5-FU exposure at least 10-fold higher than that reported from equitoxic doses of tegafur modulated by uracil in the oral fluoropyrimidine UFT. CONCLUSIONS: The recommended dose for Phase II studies of S-1 administered once daily for 28 consecutive days every 5 weeks is 50 mg/m(2)/day. The pharmacokinetic data indicate substantial modulation of 5-FU clearance by CDHP. Based on these pharmacokinetic data, the predictable toxicity profile of S-1, and the low incidence of severe adverse effects at the recommended Phase II dose, evaluations of S-1 on this schedule are warranted in malignancies that are sensitive to the fluoropyrimidines.

Human malaria parasite orotate phosphoribosyltransferase: functional expression, characterization of kinetic reaction mechanism and inhibition profile.

Plasmodium falciparum, the causative agent of the most lethal form of human malaria, relies on de novo pyrimidine biosynthesis. A gene encoding orotate phosphoribosyltransferase (OPRT), the fifth enzyme of the de novo pathway catalyzing formation of orotidine 5'-monophosphate (OMP) and pyrophosphate (PP(i)) from 5-phosphoribosyl-1-pyrophosphate (PRPP) and orotate, was identified from P. falciparum (pfOPRT). The deduced amino acid sequence for pfOPRT was compared with OPRTs from other organisms and found to be most similar to that of Escherichia coli. The catalytic residues and consensus sequences for substrate binding in the enzyme were conserved among other organisms. The pfOPRT was exceptional in that it contained a unique insertion of 20 amino acids and an amino-terminal extension of 66 amino acids, making the longest amino acid sequence (281 amino acids with a predicted molecular mass of 33kDa). The cDNA of the pfOPRT gene was cloned, sequenced and functionally expressed in soluble form. The recombinant pfOPRT was purified from the E. coli lysate by two steps, nickel metal-affinity and gel-filtration chromatography. From 1l E. coli culture, 1.2-1.5mg of pure pfOPRT was obtained. SDS-PAGE revealed that the pfOPRT had a molecular mass of 33kDa and analytical gel-filtration chromatography showed that the enzyme activity eluted at approximately 67kDa. Using dimethyl suberimidate to cross-link neighboring subunits of the pfOPRT, it was confirmed that the native enzyme exists in a dimeric form. The steady state kinetics of initial velocity and product inhibition studies indicate that the enzyme pfOPRT follows a random sequential kinetic mechanism. Compounds aimed at the pfOPRT nexus may act against the parasite through at least two mechanisms: by directly inhibiting the enzyme activity, or be processed to an inhibitor of thymidylate synthase. This study provides a working system with which to investigate new antimalarial agents targeted against P. falciparum OPRT.

Synthesis of carbocyclic orotidine analogs as potential orotidine decarboxylase inhibitors.

An asymmetric synthesis of carbocyclic orotidine 15 and its monophosphate 16 were accomplished via the key intermediate cyclopentanone 4, which was prepared from D-gamma-ribonolactone in steps. None of synthesized the compounds inhibited orotidine 5'-monophosphate decarboxylase (EC 4.1.1.23) or orotate phosphoribosyltransferase (EC 2.4.2.10).

Pyrimidine nucleobase ligands of orotate phosphoribosyltransferase from Toxoplasma gondii.

Sixty-seven pyrimidine nucleobase analogues were evaluated as ligands of Toxoplasma gondii orotate phosphoribosyltransferase (OPRTase, EC 2.4.2.10) by measuring their ability to inhibit this enzyme in vitro. Apparent Ki values were determined for compounds that inhibited T. gondii OPRTase by greater than 20% at a concentration of 400 microM. 1-Deazaorotic acid (0.47 microM) and 5-azaorotic acid (2.1 microM) were found to bind better (8.3- and 1.9-fold, respectively) to T. gondii OPRTase than orotic acid, the natural substrate of the enzyme. Based on these results, a structure-activity relationship of ligand binding to OPRTase was formulated using uracil, barbituric acid, and orotic acid as reference compounds. It was concluded that the following structural features of pyrimidine nucleobase analogues were required or strongly preferred for binding: (i) an endocyclic pyridine-type nitrogen or methine at the 1-position; (ii) exocyclic oxo groups at the 2- and 4-positions; (iii) a protonated endocyclic pyridine-type nitrogen at the 3-position; (iv) an endocyclic pyridine-type nitrogen or methine at the 5-position; (v) an exocyclic hydrogen or fluorine at the 5-position; (vi) an endocyclic pyridine-type nitrogen or methine at the 6-position; and (vii) an exocyclic negatively charged or electron-withdrawing group at the 6-position. A comparison of the results from the present study with those from a previous study on mammalian OPRTase [Niedzwicki et al., Biochem Pharmacol 33: 2383-2395, 1984] identified four compounds (6-chlorouracil, 5-azaorotic acid, 1-deazaorotic acid, and 6-iodouracil) that may bind selectively to T. gondii OPRTase.