Covalent Ternary Complex Research Articles

Caught in Action: X-ray Structure of Thymidylate Synthase with Noncovalent Intermediate Analog.

Methylation of 2-deoxyuridine-5'-monophosphate (dUMP) at the C5 position by the obligate dimeric thymidylate synthase (TSase) in the sole de novo biosynthetic pathway to thymidine 5'-monophosphate (dTMP) proceeds by forming a covalent ternary complex with dUMP and cosubstrate 5,10-methylenetetrahydrofolate. The crystal structure of an analog of this intermediate gives important mechanistic insights but does not explain the half-of-the-sites activity of the enzyme. Recent experiments showed that the C5 proton and the catalytic Cys are eliminated in a concerted manner from the covalent ternary complex to produce a noncovalent bisubstrate intermediate. Here, we report the crystal structure of TSase with a close synthetic analog of this intermediate in which it has partially reacted with the enzyme but in only one protomer, consistent with the half-of-the-sites activity of this enzyme. Quantum mechanics/molecular mechanics simulations confirmed that the analog could undergo catalysis. The crystal structure shows a new water 2.9 Å from the critical C5 of the dUMP moiety, which in conjunction with other residues in the network, may be the elusive general base that abstracts the C5 proton of dUMP during the reaction.

Structures, fabrication mechanisms, and emulsifying properties of self-assembled and spray-dried ternary complexes based on lactoferrin, oat β-glucan and curcumin: A comparison study

Protein–polyphenol–polysaccharide non–covalent ternary complexes possess many unique structural and functional properties. However, rare work is available to fabricate the neutral polysaccharide–based ternary complexes. Herein, the ternary complexes composed of lactoferrin (LF), oat β-glucan (OG), and curcumin (Cur) with three binding sequences were successfully developed through self–assembly technique and spray drying technique, respectively. Spray drying could enhance the extent of the intermolecular associations among LF, OG, and Cur, leading to the formation of ternary complexes with smaller particle sizes and lower turbidities. Cur can be loaded in LF-OG complexes to form an amorphous complex through the intermolecular interactions (mainly hydrophobic interactions and hydrogen bonding). The ternary complexes can be used as potential emulsifiers to stabilize oil-in-water Pickering emulsions. The emulsifying capacity (to enhance physical stability) of the complexes was in the order as follows: the spray-dried ternary complexes > the spray-dried LF-OG complexes > the self–assembled ternary complexes > the self–assembled LF-OG complexes. The structural and functional properties (e.g., emulsifying property) of OG-based ternary complexes can be controlled by adjusting the binding sequences. These results will broaden our current understanding of protein–polyphenol–polysaccharide ternary complexes and provide more applications of OG in food, cosmetics, and pharmaceutical industries.

Novel 4-(4-substituted amidobenzyl)furan-2(5H)-one derivatives as topoisomerase I inhibitors

In this study, two series of novel 4-(4-substituted amidobenzyl)furan-2(5H)-one derivatives containing an α,β-unsaturated lactone fragment were synthesized and screened for Topo I inhibition and antitumor activity. The topoisomerase I inhibitory activities and cytotoxicities against three human cancer cell lines (MCF-7,Hela,A549) were evaluated. The results revealed that series 2, compounds bearing an exocyclic double bond on the furanone ring, generally showed more potent activity than series 1, compounds lacking an exocyclic double bond. Several compounds of series 2 possess significant Topo I inhibitory activity and potent antiproliferative activity against cancer cell lines. Further mechanism studies of the most active compound of series 2 (B-15) indicated that synthetic compounds can not only stabilize the drug-enzyme-DNA covalent ternary complex as well as camptothecin, but also interfere with the binding between Topo I and DNA. The binding patterns of these compounds with Topo I and structure-activity relationships are discussed.

Novel securinine derivatives as topoisomerase I based antitumor agents

DNA topoisomerase I (Topo I) has been validated as a target for anticancer agents. In this study, a series of novel securinine derivatives bearing β′-hydroxy-α,β-unsaturated ketone moiety were designed and synthesized via a Baylis-Hillman reaction for screening as Topo I inhibitors and antitumor agents. Their topoisomerase I inhibitory activity as well as their cytotoxicity against four human cancer cell lines (A549, HeLa, HepG2, SH-SY5Y) were evaluated, and two pairs of diastereomers 4a-1 and 4a-6 with significant Topo I inhibitory activity and potent anti-proliferative activity against cancer cell lines were identified. The diastereomers were separated, and absolute configurations of five pairs of diastereomers were identified based on X-ray crystallographic analysis and circular dichroism (CD) spectra analysis. Further mechanism studies of the most active compounds 4a-1-R and 4a-1-S indicated that this kind of securinine derivative exhibits a different inhibitory mechanism from that of camptothecin, an established Topo I inhibitor. Unlike camptothecin, compounds 4a-1-R and 4a-1-S specifically inhibits the combination of Topo I and DNA rather than forming the drug-enzyme-DNA covalent ternary complex. In addition, molecular docking and molecular dynamic studies revealed the binding patterns of these compounds with Topo I.

Abietane diterpenes induce cytotoxic effects in human pancreatic cancer cell line MIA PaCa-2 through different modes of action

Abietane diterpenes, especially those containing quinone moieties, are often reported to have cytotoxic effects on cancer cell lines. They deserve greater attention because several cancer chemotherapeutic agents also possess the quinone structural feature. To date, very little is known about their cytotoxic molecular modes of action. In the present study, five diterpenes, 7 alpha-acetoxyroyleanone, horminone, royleanone, 7-ketoroyleanone and sugiol which have been previously isolated from the medicinal plant Peltodon longipes were shown to possess cytotoxic activity against the human pancreatic cancer cell line MIA PaCa-2. 7 alpha-Acetoxyroyleanone, horminone and royleanone were demonstrated to possess alkylating properties using the nucleophile 4-(4-nitrobenzyl)pyridine. However, no clear correlation between the alkylating properties and cytotoxicity of these diterpenes was observed. Furthermore, the relaxation activity of human DNA topoisomerases I and II was found to be influenced by these compounds, with 7-ketoroyleanone and sugiol being the most active. These two diterpenes preferentially inhibited topoisomerase I and exhibited lower IC50 values than the classical topoisomerase I inhibitor camptothecin. Molecular docking studies revealed possible interactions of diterpenes with topoisomerase I, indicating that these compounds do not form the drug–enzyme–DNA covalent ternary complex as observed with camptothecin. A binding pocket located at the surface of the DNA-interaction site was proposed. Moreover, the ability of the five diterpenes to generate DNA-strand breaks in single cells was confirmed using the alkaline comet assay. As expected, these diterpenes also influenced cell cycle progression and arrested cells in different phases of the cell cycle, primarily the G1/G0 and S-phases. Interestingly, the diterpenes only exhibited a slight ability to induce apoptotic cell death and failed to generate intracellular reactive oxygen species. These results provide additional understanding of the cytotoxic effects of abietane diterpenes. Depending on their functional groups, we propose that abietane diterpenes utilise different mechanisms to induce cell death.

A novel DNA topoisomerase I inhibitor with different mechanism from camptothecin induces G2/M phase cell cycle arrest to K562 cells.

DNA topoisomerase I (Top1) is an essential nuclear enzyme and a validated target for anticancer agent screening. In a previous study, we found that indolizinoquinoline-5,12-dione derivatives show significant biological activity against several human cancer cell lines. To understand their mechanism of inhibition of cancer cell growth, one indolizinoquinoline-5,12-dione derivative, CY13II, was further studied as lead. Our present results indicate that CY13II shows more potent antiproliferative activity against K562 cells than camptothecin. Additionally, K562 cells were arrested in G2/M, and their growth rate decreased after treatment with CY13II at micromolar concentration. Biochemical Top1 assays indicate that CY13II exhibits a different inhibitory mechanism from camptothecin. Unlike camptothecin, CY13II specifically inhibits the catalytic cleavage activity of Top1 instead of forming the drug-enzyme-DNA covalent ternary complex.

Nonconserved Residues Ala287 and Ser290 of the Cryptosporidium hominis Thymidylate Synthase Domain Facilitate Its Rapid Rate of Catalysis,

Cryptosporidium hominis TS-DHFR exhibits an unusually high rate of catalysis at the TS domain, at least 10-fold greater than those of other TS enzymes. Using site-directed mutagenesis, we have mutated residues Ala287 and Ser290 in the folate-binding helix to phenylalanine and glycine, respectively, the corresponding residues in human and most other TS enzymes. Our results show that the mutant A287F, the mutant S290G, and the double mutant all have reduced affinities for methylene tetrahydrofolate and reduced rates of reaction at the TS domain. Interestingly, the S290G mutant enzyme had the lowest TS activity, with a catalytic efficiency approximately 200-fold lower than that of the wild type (WT). The rate of conformational change of the S290G mutant is approximately 80 times slower than that of WT, resulting in a change in the rate-limiting step from hydride transfer to covalent ternary complex formation. We have determined the crystal structure of ligand-bound S290G mutant enzyme, which shows that the primary effect of the mutation is an increase in the distance between the TS ligands. The kinetic and crystal structure data presented here provide the first evidence explaining the unusually fast TS rate in C. hominis.

Catalytic Mechanism and Structure of Viral Flavin-dependent Thymidylate Synthase ThyX

By using biochemical and structural analyses, we have investigated the catalytic mechanism of the recently discovered flavin-dependent thymidylate synthase ThyX from Paramecium bursaria chlorella virus-1 (PBCV-1). Site-directed mutagenesis experiments have identified several residues implicated in either NADPH oxidation or deprotonation activity of PBCV-1 ThyX. Chemical modification by diethyl pyrocarbonate and mass spectroscopic analyses identified a histidine residue (His53) crucial for NADPH oxidation and located in the vicinity of the redox active N-5 atom of the FAD ring system. Moreover, we observed that the conformation of active site key residues of PBCV-1 ThyX differs from earlier reported ThyX structures, suggesting structural changes during catalysis. Steady-state kinetic analyses support a reaction mechanism where ThyX catalysis proceeds via formation of distinct ternary complexes without formation of a methyl enzyme intermediate.

Chemical mechanism and substrate binding sites of NADP-dependent aldehyde dehydrogenase from Streptococcus mutans

Non-phosphorylating glyceraldehyde 3-phosphate dehydrogenase from Streptococcus mutans (GAPN) belongs to the aldehyde dehydrogenase (ALDH) family, which catalyzes the irreversible oxidation of a wide variety of aldehydes into acidic compounds via a two-step mechanism: first, the acylation step involves the formation of a covalent ternary complex ALDH-cofactor-substrate, followed by the oxidoreduction process which yields a thioacyl intermediate and reduced cofactor and second, the rate-limiting deacylation step. Structural and molecular factors involved in the chemical mechanism of GAPN have recently been examined. Specifically, evidence was put forward for the chemical activation of catalytic Cys-302 upon cofactor binding to the enzyme, through a local conformational rearrangement involving the cofactor and Glu-268. In addition, the invariant residue Glu-268 was shown to play an essential role in the activation of the water molecule in the deacylation step. For E268A/Q mutant GAPNs, nucleophilic compounds like hydrazine and hydroxylamine were shown to bind and act as substrates in this step. Further studies were focused at understanding the factors responsible for the stabilization and chemical activation of the covalent intermediates, using X-ray crystallography, site-directed mutagenesis, kinetic and physico-chemical approaches. The results support the involvement of an oxyanion site including the side-chain of Asn-169. Finally, given the strict substrate-specificity of GAPN compared to other ALDHs with wide substrate specificity, one has also initiated the characterization of the G3P binding properties of GAPN. These results will be presented and discussed from the point of view of the evolution of the catalytic mechanisms of ALDH.

In Vivo Clearance of Ternary Complexes of Vitronectin-Thrombin-Antithrombin Is Mediated by Hepatic Heparan Sulfate Proteoglycans

Thrombin is inhibited by its cognate plasma inhibitor antithrombin, through the formation of covalent thrombin-antithrombin (TAT) complexes that are found as ternary complexes with vitronectin (VN-TAT). To determine whether the metabolism of VN-TAT ternary complexes is different from that previously reported for binary TAT complexes, plasma clearance studies were done in rabbits using human VN-TAT. 125I-VN-TAT was shown to be cleared rapidly from the circulation (t1/2alpha = 3.8 min) in a biphasic manner mainly by the liver. 125I-TAT had a similar initial clearance (t1/2alpha = 5.3 min) but had a significantly faster beta-phase clearance (t1/2beta = 42.8 min versus 85.4 min for VN-TAT; p = 0.005). Protamine sulfate and heparin abolished the rapid initial alpha-phase of 125I-VN-TAT clearance and reduced its liver-specific association and in vivo degradation. Heparin also reduced the alpha-phase clearance of 125I-TAT and was associated with the appearance of high molecular weight complexes, suggesting enhanced complex formation between VN and TAT. 125I-VN-TAT binding to HepG2 cells was reduced by competition with VN-TAT or heparin but to a much lesser extent in the presence of TAT. The binding of VN-TAT to HepG2 cells was not inhibited by competition with the low density lipoprotein receptor-related protein ligand, methylamine-alpha2-macroglobulin. 125I-VN-TAT binding was also inhibited by treating HepG2 cells with heparinase or by growing the cells in the presence of beta-D-xyloside. Finally, both heparin and chloroquine, but not methylamine-alpha2-macroglobulin, reduced the internalization and degradation of VN-TAT by HepG2 cells. Taken together, these data indicate the importance of VN in TAT metabolism and demonstrate that VN-TAT binds to liver-associated heparan sulfate proteoglycans, which mediate its internalization and subsequent intracellular degradation.

D221 in thymidylate synthase controls conformation change, and thereby opening of the imidazolidine.

In thymidylate synthase (TS), the invariant residue Asp-221 provides the only side chain that hydrogen bonds to the pterin ring of the cofactor, 5,10-methylene-5,6,7,8-tetrahydrofolate. All mutants of D221 except cysteine abolish activity. We have determined the crystal structures of two ternary complexes of the Escherichia coli mutant D221N. In a complex with dUMP and the antifolate 10-propargyl-5,8-dideazafolate (CB3717), dUMP is covalently bound to the active site cysteine, as usual. CB3717, which has no imidazolidine ring, is also bound in the usual productive orientation, but is less ordered than in wild-type complexes. The side chain of Asn-221 still hydrogen bonds to N3 of the quinazoline ring of CB3717, which must be in the enol form. In contrast, the structure of D221N with 5-fluoro-dUMP and 5,10-methylene-5,6,7, 8-tetrahydrofolate shows the cofactor bound in two partially occupied, nonproductive binding sites. In both binding modes, the cofactor has a closed imidazolidine ring and adopts the solution conformation of the unbound cofactor. In one of the binding sites, the pterin ring is turned around such that Asn-221 hydrogen bonds to the unprotonated N1 instead of the protonated N3 of the cofactor. This orientation blocks the conformational change required for forming covalent ternary complexes. Taken together, the two crystal structures suggest that the hydrogen bond between the side chain of Asp-221 and N3 of the cofactor is most critical during the early steps of cofactor binding, where it enforces the correct orientation of the pterin ring. Proper orientation of the cofactor appears to be a prerequisite for opening the imidazolidine ring prior to formation of the covalent steady-state intermediate in catalysis.

Aspartate 221 of thymidylate synthase is involved in folate cofactor binding and in catalysis.

Structural studies indicate that Asp 221 of Lactobacilluscasei thymidylate synthase forms a hydrogen bond network with the 2-amino and 3-imino groups of the folate [Matthews, D. A. (1990) J. Mol. Biol. 214, 937-948; Finer-Moore, J. S. (1990)Biochemistry 29, 6977-6986] that has been proposed to participate in catalysis. We prepared a complete replacement set of 19 mutants at position 221 of L. casei thymidylate synthase. Of these, the only one with sufficient activity to complement growth of a thymidylate synthase-deficient host was the Cys mutant. To further elucidate the function of the Asp 221 side chain, seven thymidylate synthase 221 mutants were studied in detail with regard to catalysis of dTMP formation and of thymidylate synthase partial reactions. Most of the mutants bound the nucleotide substrate dUMP with only moderate loss of binding affinity, indicating that the Asp side chain does not contribute to dUMP binding. Most of the mutants catalyzed the cofactor-independent dehalogenation of 5-bromodUMP; hence, the Asp side chain of TS is not essential for addition of the catalytic Cys residue to the nucleotide substrate. Mutants showed decreased affinity for the folate cofactor, but those with side chains capable of hydrogen bond formation were less severely affected. Some of the mutants were capable of forming covalent thymidylate synthase-5-fluorodUMP-methylenetetrahydrofolate complex; hence, the Asp side chain is not essential for steps leading to the covalent complex. We conclude that the hydrogen bond network between Asp 221 and the folate cofactor contributes to cofactor binding and a catalytic step after formation of the covalent ternary complex intermediate.

Cloning, Expression, Purification, and Characterization of 2′-Deoxyuridylate Hydroxymethylase from Phage SPO1

2′-Deoxyuridylate hydroxymethylase (dUMP-hmase) from phage SPO1 has been cloned and expressed in Escherichia coli. In crude extracts, the enzyme represents about 25% of the soluble protein and has a higher specific activity than the most purified preparation yet reported, The enzyme was purified to homogeneity by ion-exchange and hydrophobic chromatography. The subunits of dUMP-hmase are 45 kDa by SDS-PAGE and form dimers with a molecular mass of 89.2 kDa by analytical centrifugation. In addition to the normal reaction, dUMP-hmase catalyzes the 5,10-methylene-5,6,7,8-tetrahydrofolate (CH 2H 4folate)-independent tritium exchange of [5- 3H]dUMP for protons of water and dehalogenation of 5-bromo-2′-deoxyuridine-5′-monophosphate; the enzyme also forms a covalent binary adduct with pyridoxal 5′-monophosphate and a covalent ternary complex with 5-fluoro-2′-deoxyuridine-5′-monophosphate and CH 2H 4folate. Folic acid inhibits the tritium release catalyzed by dUMP-hmase in the presence of cofactor but has no effect an the catalysis of cofactor-independent tritium exchange.

Fluorine-19 nuclear magnetic resonance and biochemical characterization of fluorotyrosine-labeled-thymidylate-synthetase

Fluorotyrosine has been incorporated into thymidylate synthetase from Lactobacillus casei by growth of the bacterium in media containing 3-fluorotyrosine. The enzyme exhibited a specific activity 70% of that of the normal enzyme and formed a covalent binary complex with pyrimidine nucleotides, as well as a covalent ternary complex with 5-fluorodeoxyuridylate and 5,10-methylenetetrahydrofolate. 19F nuclear magnetic resonance spectroscopy has been used to follow the formation of these complexes. 5-Fluorodeoxyuridylate, dUMP, dTMP and dCMP produced identical conformational changes in the enzyme as monitored by the fluorotyrosyl resonances. Ternary complex formation of the fluorotyrosine-containing enzyme with 5-fluorodeoxyuridylate and 5,10-methylenetetrahydrofolate resulted in further spectral changes.

Purification and characterization of recombinant mouse thymidylate synthase

Recombinant mouse thymidylate synthase (TS) expressed at high levels in Escherichia coli was purified to homogeneity in greater than 70% yield by a rapid three-step procedure. Both 0.1% Triton X-100 and 10% glycerol were required to stabilize the enzyme whose activity remained unchanged after 1 month when stored at −20°C. Thermal inactivation of the enzyme was a first-order process at 37°C, with t 1 2 values of 6.9, 15.6 and 3.0 min at pH 5.5, 7.0 and 8.5, respectively. The presence of saturating levels of dUMP at pH 8.5 increased the t 1 2 of inactivation to 38 min. The pH profile for enzyme activity showed a narrow optimum region centered at pH 7.0, which was mirrored by the shape of the K m, dUMP/ V max plot. The pH dependence of K d for the covalent inhibitory ternary complex of enzyme, 5-fluoro-2′-deoxyuridylate and 5,10-methylenetetrahydrofolate exhibited a broad minimum between pH 5.5 and 8.5, and ranged between 3.1, 0.8 and 1.1 nM at pH 5.5, 7.0 and 8.5, respectively. The UV/VIS spectrum of the native enzyme exhibited a maximum at 280 nm ( ϵ = 98 200 M −1 cm −1), while that of the inhibitory ternary complex showed an additional maximum at 320 nm. The 19F-NMR spectrum of the mouse enzyme: FdUMP binary complex revealed two new resonances at −2.8 and −34.8 ppm. The most deshielded resonance represented the noncovalent binary complex while the other resonance was assigned to the nucleotide covalently bound to the enzyme. The alteration of nucleotide binding equilibria produced by addition of H 4 folate was exemplified by both an increase in intensity and a 5 ppm deshielding of the resonance attributed to the covalent FdUMP-enzyme complex. Addition of formaldehyde to the latter mixture produced the covalent ternary complex which resulted in the collapse of the resonances at −2.8 and −39.5 ppm and the appearance of a new resonance at −12.4 ppm.

In vitro and in vivo inhibition of thymidylate synthase of human colon cancer by 5-fluorouracil.

Thymidylate synthase (TS) is a key enzyme in de novo synthesis of TMP (Fig. 1). TS catalyzes the conversion of dUMP to TMP for which 5, 10-methylenetetrahydrofolate (CH2THF) acts as the methyl-donor; the Km for dUMP is about 1–5 μM (1–5). Inhibition of TS by FdUMP is one of the main mechanisms for the action of 5-fluorouracil (5FU). Without preincubation FdUMP acts as a potent competitive inhibitor of TS with a Ki of about 1 nM. The Km/Ki ratio is about 1000. The inhibition of TS by FdUMP is mediated by the formation of a tight-binding covalent ternary complex of FdUMP with TS and CH THF. Retention of the inhibition of TS is mainly related to the FdUMP/dUMP ratio (6), the FdUMP binding to TS and the stabilization of the ternary complex by CH2 THF or one of its polyglutama-tes (6, 7, 9). In vitro, resistance against 5FU or its analog 5-fluoro-2′-deoxyuridine (FUdR) has been related to altered kinetics of TS with respect to Km values for dUMP and FdUMP binding (2, 5, 8), disturbed folate pools (9), and the level of enzyme before treatment (6, 8). Gene amplification of TS has been demonstrated for FUdR-resistant sub-cell lines (10, 11). Recently evidence for gene amplication has also been obtained in a patient with colon cancer who developed resistance against 5FU (12), while in breast cancer patients binding of FdUMP and the effect of CH2THF decreased during development of resistance (13).KeywordsPyrimidine NucleosidePrimary Colorectal TumorPotent Competitive InhibitorTritium Release AssayFdUMP BindingThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Effect of polyglutamylation of 5,10-methylenetetrahydrofolate on the binding of 5-fluoro-2'-deoxyuridylate to thymidylate synthase purified from a human colon adenocarcinoma xenografT

CH 2-H 4PteGlu and H 4PteGlu exist in human colon adenocarcinoma xenografts predominantly in the form of polyglutamate species at concentrations of < 3 μM. The interaction of polyglutamates of [6 R]CH 2,-H 4PteGlu in the formation and stability of [6- 3H]FdUMP-thymidylate synthase-CH 2-H 4PteGlu n ternary complexes has therefore been examined using enzyme purified from a human colon adenocarcinoma xenograft. Dissociation of these complexes was first-order and was dependent upon the concentration of folate. [6 R]CH 2-H 4PteGlu 3–6 (0.9 to 1.6 μM) were >200-fold and [6 R]CH 2-H 4PteGlu 2 (18.2 μm) was 18-fold more effective than [6 R]CH 2-H 4PteGlu 1 (335 μM) at stabilizing ternary complexes for a T 1 2 for dissociation of 100 min. Polyglutamylation of CH 2-H 4PteGlu also increased the affinity of binding of [6- 3H]FdUMP to thymidylate synthase as determined by Scatchard analysis at folate concentrations of 10 μM, where the K d in the presence of [6 R]CH 2-H 4PteGlu 1 was in the order of 4.0 × 10 −8 M, and for [6 R]CH 2-H 4PteGlu 3–5 was between 3.7 and 5.5 × 10 −9 M. To examine whether this effect was due to differences in the rates at which [6- 3H]FdUMP was bound ( k on) or dissociated ( k off) from the enzyme, the apparent rate of [6- 3H]FdUMP binding was determined in the presence of [6 R]CH 2H 4PteGlu 1, [6 R]CH 2-H 4PteGlu 3 and [6 R]CH 2-H 4PteGlu 4. The k on values were similar and were in the range of 1.7 to 2.3 × 10 6M −1 min −1 for 10 or 20 μM folate concentrations. Differences in binding affinity determined for [6 R]CH 2-H 4PteGlu 1 and longer polyglutamate forms of [6 R]CH 2-H 4PteGlu were thus due to differences in k off. The V max for the initial velocity of [6- 3H]FdUMP binding was achieved at 10 μM folate. Consequently, at concentrations of CH 2-H 4PteGlu polyglutamates present in tumors, inhibition of thymidylate synthase by FdUMP in vivo would be expected to be transient, based upon the concentration of [6 R]CH 2-H 4PteGlu n required for maximal formation and stability of the covalent ternary complex. It would be advantageous for modulation of CH 2-H 4PteGlu n pools to increase the concentrations of the longer polyglutamate species (n ≥ 3) to maximize the interaction between FdUMP, thymidylate synthase and CH2-H 4PteGlu.

Fluorouracil: biochemistry and pharmacology.

Fluorouracil (5FU) is still considered the most active antineoplastic agent in the treatment of advanced colorectal cancer. The drug needs to be converted to the nucleotide level in order to exert its effect. It can be incorporated into RNA leading to interference with the maturation of nuclear RNA. However, its conversion to 5-fluoro-2'deoxy-5' monophosphate (FdUMP) leading to inhibition of thymidylate synthase (TS) and subsequently of DNA synthesis, is considered to be its main mechanism of action. In the presence of a folate cofactor a covalent ternary complex is formed, the stability of which is the main determinant of the action of 5FU. Resistance against 5FU can be mainly attributed to aberrations in its metabolism or to alterations of TS, eg, gene amplification, altered kinetics in respect to nucleotides or folates. Biochemical modulation of 5FU metabolism can be applied to overcome resistance against 5FU. A variety of normal purines, pyrimidines, and other antimetabolites have been studied in this respect, but only some of them have been clinically successful. Delayed administration of uridine has recently been shown to "rescue" mice and patients from toxicity, while pretreatment with leucovorin is the most promising combination to enhance the therapeutic efficacy. 5FU is frequently administered in an intravenous (IV) injection, and shows a rapid distribution and a triphasic elimination. The nonlinearity of 5FU pharmacokinetics is related to saturation of its degradation. Continuous infusion of 5FU led to different kinetics. Regional administration, such as hepatic artery infusion, offers a way to achieve higher drug concentrations in liver metastases and is accompanied by lower systemic concentration. The current status of the biochemical and pharmacokinetic data is reviewed.

Linear free energy relationship studies of enzyme active site binding: thymidylate synthase.

The requirements for active-site binding of thymidylate synthase from three sources, Lactobacillus casei, murine leukemia L1210, and human lymphoblast (Molt/4F), were investigated by analyzing the binding of a series of 5-(p-substituted phenyl)-2'-deoxyuridylates (N1-substituted 5-aryl-2, 4-dioxopyrimidines) to the enzyme. Multiple regression analysis revealed that an increase in electron density of the heterocyclic ring and hydrophobic substituents enhance affinity. Correlations of biological results with spectral data indicated that higher electron densities at the oxygen atoms are responsible for increase in binding. These results support the presence of both a cationic binding site and a hydrophobic region. In addition, the results revealed an unusual reversal of electronic requirements for binding and catalysis. The formation of the binary complex is enhanced by electron-donating substituents, while the initial catalytic reaction, formation of the covalent ternary complex, is promoted and stabilized by electron-withdrawing substituents.

Chemical modelling of the thymidylate synthase reaction: evidence for the formation of an exocyclic methylene intermediate from analogues of the covalent ternary complex formed by intramolecular thiol addition to C(6) of 5-aminomethyluracil derivatives.

The intramolecular thiol addition to C(6) in 1-(3-mercaptopropyl)-5-aminomethyluracil derivatives in protic media leads to the formation of bicyclic; 5,6-dihydrouracil derivatives, which maybe regarded as models of the covalent ternary complex in the thymidylate synthase reaction. Evidence is presented for the formation of an exocyclic methylene intermediate from these model compounds.