Fluorine-19 nuclear magnetic resonance studies of binary and ternary complexes of thymidylate synthase utilizing a fluorine-labeled folate analogue

Traditional fluorine-19 nuclear magnetic resonance (19F NMR) studies of thymidylate synthase (TS) have utilized the fluorine substituent of 5-fluorodeoxyuridine 5'-monophosphate (FdUMP), a mechanism-based inhibitor of the enzyme, in complexes with various folate and folate analogues in order to establish a paradigm for the formation of binary and ternary complexes. In order to extent and confirm this paradigm, complexes of thymidylate synthase (TS) and the N-10-(fluoroethyl)quinazolinylfolate analogue CB3731 with either deoxyuridine 5'-monophosphate (dUMP), deoxythymidine 5'-monophosphate (dTMP), or FdUMP were examined from the perspective of the folate analogue using 19F NMR. The spectrum of the free folate analogue gave rise to a multiplet centered at -57.0 ppm, which was broadened by approximately 50% upon incubation with the enzyme. The use of FdUMP with CB3731 afforded us the opportunity to compare the spectrum obtained for the folate with that of the nucleotide. This comparison led to the assignment of the resonance at -53.5 ppm as representing the noncovalent TS:FdUMP:CB3731 ternary complex, while a new resonance at -52.0 ppm corresponded to the species in which the nucleotide is covalently attached to the enzyme and the folate is noncovalently associated. Ternary complexes consisting of TS, CB3731, and either dUMP or dTMP displayed a resonance at -53.5 ppm which represented the noncovalent TS-nucleotide adduct. None of the TS:nucleotide:CB3731 ternary complexes, however, was stable to native polyacrylamide gel electrophoresis.     

Inactivation of 3-hydroxy-3-methylglutaryl-CoA synthase and other Acyl-CoA-utilizing enzymes by 3-Oxobutylsulfoxyl-CoA

3-Oxobutylsulfoxyl-CoA has been produced by oxidation of S-3-oxobutyl-CoA, the thioether analog of acetoacetyl-CoA. Avian hydroxymethylglutaryl-CoA (HMG-CoA) synthase is inactivated by oxobutylsulfoxyl-CoA in a time-dependent fashion. Protection against inactivation is afforded by the substrate, acetyl-CoA, suggesting that inactivation involves modification of the enzyme's active site. Pretreatment of HMG-CoA synthase with the inactivator blocks the enzyme's ability to form Michaelis and acetyl-S-enzyme intermediates, supporting the hypothesis that modification is active-site directed. Incubation of enzyme with oxobutylsulfoxyl-[32P]CoA followed by precipitation with trichloroacetic acid indicates that inactivation correlates with stoichiometric formation of a covalent adduct between enzyme and a portion of the inactivator that includes the CoA nucleotide. The observation of reagent partitioning suggests that HMG-CoA synthase catalyzes conversion of oxobutylsulfoxyl-CoA into a reactive species that modifies the protein. Treatment of inactivated enzyme with DTT or other mercaptans restores enzyme activity and reverses the covalent modification with release of CoASH. Oxobutylsulfoxyl-CoA inactivates beta-ketothiolase and HMG-CoA lyase in a process that is also reversed by DTT. These three enzymes all contain active site cysteines, suggesting that inactivation results from disulfide formation between a cysteine and the CoA moiety of the inhibitor. The data are consistent with the hypothesis that enzymatic cleavage of oxobutylsulfoxyl-CoA results in the transient formation of a sulfenic acid derivative of CoA which subsequently reacts to form a stable disulfide linkage to protein.     

Variation in human thymidylate synthase is associated with resistance to 5-fluoro-2'-deoxyuridine

Two human colorectal tumor cell lines are differentially sensitive to growth inhibition by 5-fluorodeoxyuridine (FdUrd); cell line RCA is less sensitive to FdUrd than is cell line C. Thymidylate synthase (TS), a target of FdUrd, has been purified to homogeneity from both cell lines. Because of differences in the avidity for a folate ligand affinity matrix, TS forms from the cells were purified by two different procedures. Relative to the enzyme from C cells, the enzyme from RCA cells demonstrated higher Km values for the substrates deoxyuridylate and 5,10-methylene-tetrahydrofolate, a lower rate of association of the inhibitor 5-fluorodeoxyuridylate (FdUMP), a similar rate of FdUMP dissociation, and lower enhancement of covalent FdUMP binding by folate derivatives. The activities of the enzymes in situ and the catalytic efficiencies of the purified enzymes were similar. Thus, a cell line that is naturally resistant to FdUrd has been identified that expresses a TS with reduced affinity for FdUMP and 5,10-methylenetetrahydrofolate, relative to the enzyme expressed in a FdUrd-sensitive cell line.     

Isolation and characterization of thymitaq (AG337) and 5-fluoro-2-deoxyuridylate-resistant mutants of human thymidylate synthase from ethyl methanesulfonate-exposed human sarcoma HT1080 cells

Thymidylate synthase plays an essential role in the synthesis of DNA. Recently, several new and specific thymidylate synthase inhibitors that occupy the folate binding site, including Tomudex(R), BW1843U89, and Thymitaq, have demonstrated therapeutic activity in patients with advanced cancer. In order to find drug-resistant forms of human thymidylate synthase for gene therapy applications, human sarcoma HT1080 cells were exposed to ethyl methanesulfonate and Thymitaq selection. Thymitaq-resistant clonal derived sublines were established, and analysis indicated that both gene amplification and point mutations contributed to drug resistance. Eight mutant cDNAs that were identified from Thymitaq-resistant sublines were generated by site-directed mutagenesis and transfected into thymidylate synthase-negative cells. Only K47E, D49G, or G52S mutants retain enzyme activity. Moreover, cytotoxicity studies demonstrated that D49G and G52S transfected cells, besides displaying resistance to Thymitaq with IC50 values 40- and 12-fold greater than wild-type enzyme transfected cells, respectively, also lead to fluorodeoxyuridine resistance (26- and 97-fold in IC50 values, respectively) but not to Tomudex or BW1843U89. Characterization of the purified altered enzymes obtained from expression in Escherichia coli is consistent with the cell growth inhibition results. We postulate that the D49G or G52S mutation leads to the structural perturbation of the highly conserved Arg50 loop, decreasing the binding of thymidylate synthase to the inhibitors, Thymitaq and fluorodeoxyuridylate.     

Thymidylate synthase is localized to the nuclear periphery in the yeast Saccharomyces cerevisiae

To date, the organization of DNA precursor synthesis within eukaryotic cells remains unresolved. Previous studies have suggested the existence of a multienzyme complex that is responsible for DNA precursor synthesis and is associated with sites of replication within the nucleus. Contrasting this, other studies have proposed that DNA precursor synthesis occurs outside the nucleus. To further these studies, we have addressed the location where thymidylate synthase resides in yeast. Subcellular fractionation experiments indicate thymidylate synthase is associated with purified nuclei. Consistent with this, immunofluorescence analysis suggests that thymidylate synthase is situated at the nuclear periphery.     

Mode of action of site-directed irreversible folate analogue inhibitors of thymidylate synthase

5,8-Dideazafolate analogues are tight binding but not irreversible inhibitors of thymidylate synthase (TS). However, when a chloroacetyl (ClAc) group is substituted at the N10-position of 2-desamino-2-methyl-5,8-dideazafolate (DMDDF), the resulting compound, ClAc-DMDDF, although still a reversible inhibitor (KI = 3.4 x 10(-3) M), gradually inactivates thyA-TS irreversibly at a rate of 0.37 min-1. The corresponding iodoacetyl derivative alkylated the enzyme somewhat slower (k3 = 0.15 min-1 ) than ClAc-DMDDF but was bound more tightly (KI = 1.4 x 10(-5) M), resulting in a second-order rate constant (k3/KI) of inactivation that was 100-fold greater than that of ClAc-DMDDF. A tryptic digest of the ClAc-DMDDF-inactivated enzyme yielded a peptide on HPLC, which revealed that cysteine-146, the residue at the active site that is intimately involved in the catalytic process, had reacted with ClAc-DMDDF to form a covalent bond. This derivative was confirmed indirectly by Edman analysis and more directly by mass spectrometry. Deoxyuridine 5'-monophosphate, a substrate in the catalytic reaction, protected against inactivation. Similar to previously described Lactobacillus casei TS inhibition studies with sulfhydryl reagents [Galivan, J., Noonan, J., and Maley, F. (1977) Arch. Biochem. Biophys. 184, 336-345], the kinetics of inhibition suggested that complete inhibition occurs on reaction of only one of the two active site cysteines, although sequence and amino acid analysis revealed that iodoacetate and ClAc-DMDDF had reacted with both active site cysteines. These studies demonstrate that a sulfhydryl reactive compound that is directed to the folate binding site of TS may diffuse to the active site cysteine, and form a covalent bond with this residue. How this inhibition comes about is suggested in a stereoscopic view of the ligand when modeled to the known crystal structure of Escherichia coli TS.     

Inactivation of pyridoxal phosphate dependent enzymes by mono- and polyhaloalanines

beta,beta-Dichloro- and beta,beta,beta-trifluoroalanine irreversibly inactivate a number of pyridoxal phosphate dependent enzymes which catalyze beta- or gamma-elimination reactions. The inactivation is time dependent and the rate of inactivation is first order in enzyme concentration. This suggests that inactivation is due to covalent modification of the enzyme by a species generated at the active site from the polyhaloalanine (i.e., suicide inactivation). Monohaloalanines are substrates and do not inactivate. For gamma-cystathionase, covalent and stoichiometric attachment of [1-14C]beta,beta,beta-trifluoroalanine was shown. It is proposed that the mechanism of inactivation involves Schiff base formation between inactivator and enzyme-bound pyridoxal and subsequent elimination of HC1 from dichloroalanine or HF from trifluoroalanine. This results in the formation of a beta-halo-alpha,beta unsaturated imine, an activated Michael acceptor. Michael addition of a nucleophile at the active site leads to covalent labeling of the enzyme and inactivation. Alanine racemase is also inactivated by the two polyhaloalanines. Glutamate-pyruvate and gultamate-oxaloacetate transaminase are inactivated by monohaloalanines but not by polyhaloalanines.     

Transcription and activity of 5-fluorouracil converting enzymes in fluoropyrimidine resistance in colon cancer in vitro

Cellular resistance to 5-fluorouracil (5-FU) is not completely understood. Since 5-FU shares the pyrimidine pathway with the physiological pyrimidines, we investigated the relationship between fluoropyrimidine metabolism, nucleic acid uptake and cytotoxicity of 5-FU in eight colon tumour cell lines including 5-FU-resistant subclones. The cytotoxicity of 5-FU was increased up to 423-fold when the anabolites 5-fluorouridine (FUrd), 5-fluorodeoxyuridine (FdUrd), and 5-fluorodeoxyuridine monophosphate (FdUMP) were compared with the parent drug in vitro. The enzymes uridine phosphorylase and thymidine phosphorylase were predictive for the cytotoxicity of 5-FU in 5/7 cell lines. Inhibition of uridine phosphorylase and thymidine phosphorylase by antisense strategies effectively antagonised 5-FU, abolishing 84% and 79% of its toxicity. The importance of thymidine phosphorylase was supported by a highly restricted enzyme activity in 5-FU-resistant cells. In 5-FU naive cells, a stimulating effect of 5-FU on thymidylate synthase mRNA and ribonucleotide reductase mRNA expression was observed. In these cells, antisense oligonucleotides to ribonucleotide reductase significantly reduced cell growth. Downregulation of ribonucleotide reductase mRNA in 5-FU-resistant subclones suggests different mechanisms in primary and secondary resistance to 5-FU. Most of the intracellular 5-FU was selectively incorporated into RNA (range: 45-91%) and generally spared DNA (range: 0.2-11%). In synthesising our data, we conclude that drug resistance could be overwhelmed through bypassing limiting steps in the activation of 5-FU. In the majority of colonic tumours, the activity of uridine phosphorylase and thymidine phosphorylase may have prognostic relevance for the cytotoxicity of 5-FU in vitro.     

On the mechanism of 5-enolpyruvylshikimate-3-phosphate synthase

5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the condensation of shikimate 3-phosphate (S3P) and phosphoenolpyruvate (PEP) to form EPSP, a precursor for the aromatic amino acids. This paper examines a recent claim [Studelska, D. R., McDowell, L. M., Espe, M. P., Klug, C. A., and Schaefer, J. (1997) Biochemistry 36, 15555-15560] that the mechanism of EPSP synthase involves two covalent enzyme-intermediates, in complete contrast to a large body of literature that has already proven the involvement of a single noncovalent intermediate. The evidence in the paper of Studelska et al. is examined closely, and unequivocal proof is provided that those authors' NMR assignments to covalent structures are in error, and that in fact the species they observed were simply the product EPSP and a side-product EPSP ketal. Since those authors used rotational-echo double-resonance (REDOR) solid-state NMR to measure intermolecular and intramolecular distances in the proposed covalent intermediates, we have used REDOR to measure the same distances in enzyme-free and enzyme-bound preparations of purified EPSP, and enzyme-free preparations of purified EPSP ketal. The distance between the shikimate ring phosphorus atom and C8 in enzyme-free EPSP is 6.6 +/- 0.1 A, which lengthens to 7.4 +/- 0.1 A in the presence of the enzyme, and in enzyme-free EPSP ketal is 5.6 +/- 0.1 A. These are entirely consistent with those measured by Studelska et al., which were 7.5 +/- 0.5 A for a putative enzyme-enolpyruvyl species and 6.1 +/- 0.3 A for a putative enzyme-ketal species.     

Random sequence mutagenesis and resistance to 5-fluorouridine in human thymidylate synthases

Thymidylate synthase (TS) catalyzes the methylation of dUMP to dTMP and is the target for the widely used chemotherapeutic agent 5-fluorouracil. We used random sequence mutagenesis to replace 13 codons within the active site of TS and obtain variants that are resistant to 5-fluorodeoxyuridine (5-FdUR). The resulting random library was selected for its ability to complement a TS-deficient Escherichia coli strain, and sequence analysis of survivors found multiple substitutions to be tolerable within the targeted region. An independent selection of the library was carried out in the presence of 5-FdUR, resulting in a more limited spectrum of mutations. One specific mutation, C199L, was observed in more than 46% of 5-FdUR-resistant clones. A 5-FdUR-resistant triple mutant, A197V/L198I/C199F, was purified to apparent homogeneity. Kinetic studies with the substrate dUMP indicate that this mutant is similar to the wild type in regards to kcat and Km values for dUMP and the cosubstrate CH2H4-folate. In contrast, equilibrium binding studies with the inhibitor, FdUMP, demonstrate that the dissociation constant (Kd) for FdUMP binding into the ternary complex was 20-fold higher than values obtained for the wild-type enzyme. This 5-FdUMP-resistant mutant, or others similarly selected, is a candidate for use in gene therapy to render susceptible normal cells resistant to the toxic effects of systemic 5-fluorouracil.     

Serum interleukin 4 and interleukin 10 levels in patients with chronic hepatitis C virus infection

The antiproliferative effect of 5-fluorouracil (5-FU) in colon cancer can be enhanced by interferons (IFN-alpha and IFN-gamma). The mechanisms by which IFNs modulate 5-FU activity are not completely elucidated. IFN-alpha may elevate the levels of the active 5-FU metabolite 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) in the cell, possibly leading to increased inhibition of the target enzyme thymidylate synthase (TS), which might enhance DNA damage. It has been shown that IFN-gamma can prevent 5-FU induced overexpression of TS. We studied IFN modulation in three colon cancer cell lines (SW948, WiDr, human; C26-10, murine) and the sublines WiDr/F and C26-10/F, which were adapted to low folate levels. A 1.5-fold increase in 5-FU sensitivity was observed in C26-10 and C26-10/F (by murine IFN-alpha, beta); in SW948, WiDr and WiDr/F (by human IFN-gamma) and in SW948 and WiDr/ F (by human IFN-alpha). In none of the cell lines did human IFN-alpha, IFN-gamma or murine IFN-alpha, beta increase FdUMP levels after exposure to 5-FU. TS activity, indirectly measured by incorporation of [6-3H]-deoxyuridine into DNA, was inhibited by 5-FU, but the IFNs did not enhance inhibition. DNA damage was measured as a drug-induced decrease of double-stranded (dss) DNA compared to control cells. After 5-FU exposure, dss DNA decreased to 60-75% in WiDr, WiDr/F and SW948 cells. Human IFN-alpha alone caused minimal DNA damage (95% dss DNA), but increased 5-FU-induced effects to 35-50% dss DNA. IFN-gamma did not cause DNA damage and did not enhance 5-FU-mediated DNA damage. Expression of TS protein, analysed by ELISA, was increased after 5-FU exposure of SW948 cells, but this increase was not affected by addition of either IFN-alpha or IFN-gamma. It is concluded that one of the mechanisms involved in modulation of 5-FU activity is the effect of IFN-alpha on 5-FU-mediated DNA damage, but for IFN-gamma no mechanism of action was found.     

Turnover number of Escherichia coli F0F1 ATP synthase for ATP synthesis in membrane vesicles

The rate of ATP synthesized by the ATP synthase (F0F1-ATPase) is limited by the rate of energy production via the respiratory chain, when measured in everted membrane vesicles of an Escherichia coli atp wild-type strain. After energization of the membranes with NADH, fractional inactivation of F0F1 by the covalent inhibitor N,N'-dicyclohexylcarbodiimide allowed the rate of ATP synthesis/mol remaining active ATP synthase complexes to increase; the active ATP synthase complexes were calculated using ATP hydrolysis rates as the defining parameter. In addition, variation of the assay temperature revealed an increase of the ATP synthesis rate up to a temperature of 37 degrees C, the optimal growth temperature of E. coli. In parallel, the amount of F0F1 complexes present in membrane vesicles was determined by immunoquantitation to be 3.3 +/- 0.3% of the membrane protein for cells grown in rich medium and 6.6 +/- 0.3% for cells grown in minimal medium with glycerol as sole carbon and energy source. Based on these data, a turnover number for ATP synthesis of 270 +/- 40 s(-1) could be determined in the presence of 5% active F0F1 complexes. Therefore, these studies demonstrate that the ATP synthase complex of E. coli has, with respect to maximum rates, the same capacity as the corresponding enzymes of eukaryotic organells.     

Combined therapeutic efficacy of the thymidylate synthase inhibitor ZD1694 (Tomudex) and the immunotoxin B43(anti-CD19)-PAP in a SCID mouse model of human B-lineage acute lymphoblastic leukemia

The quinazoline antifolate N-(5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N- methylamino]-2-thenoyl)-L-glutamic acid (ZD1694; Tomudex) is a potent inhibitor of thymidylate synthase and causes cell death through disruption of DNA synthesis and repair by blocking the obligatory thymidine nucleotide synthesis. B43(anti-CD19)-PAP immunotoxin is a potent inhibitor of protein synthesis in CD19+ B-lineage acute lymphoblastic leukemia (ALL) cells and causes apoptosis. In this model, 100% of SCID mice challenged with 1 x 10(6) human NALM-6 B-lineage ALL cells develop overt and invariably fatal leukemia. All of the 22 control SCID mice treated with phosphate-buffered saline died of disseminated human leukemia between 31 and 61 days with a median survival of 41.2 days. Treatment with ZD 1694 resulted in improved leukemia-free survival with a median survival of 69.2 days (P < 0.001, log-rank test). B43-PAP treatment was more effective than ZD1694 (P=0.026) and resulted in 51.0% long-term leukemia-free survival with a median survival of 187.5 days (P < 0.0001. log-rank test). The combination of ZD1694 and B43-PAP was more effective than either agent alone and resulted in 100% long-term leukemia-free survival. To our knowledge, this preclinical study is the first to demonstrate the feasibility and therapeutic advantage of combining an anti-leukemia immunotoxin with a thymidylate synthase inhibitor.     

Changes in global stability and local structure of cytochrome c upon substituting phenylalanine-82 with tyrosine

We have examined the F82Y;C102T variant of Saccharomyces cerevisiae iso-1-cytochrome c using high-resolution proton nuclear magnetic resonance spectroscopy, chemical denaturation, and differential scanning calorimetry. Comparison of proton chemical shifts, paramagnetic shifts, and nuclear Overhauser effects indicates structural changes are localized to the vicinity of position 82. One alteration involves the rearrangement of the side chain of leucine-85. Using many more proton assignments than were available in the initial report [G. J. Pielak, R. A. Atkinson, J. Boyd, and R. J. P. Williams, Eur. J. Biochem. 177, 179-185 (1988)], a second alteration involving an interaction between arginine-13 and tyrosine-82 is observed. The interaction appears to involve a hydrogen bond with the eta-protons of arginine's guanido group acting as donor and tyrosine's phenolic eta-oxygen as acceptor. In spite of this potentially-stabilizing interaction, the free energy of denaturation decreases by approximately 2.4 kcal mol-1. Results are discussed with respect to alterations in the native and denatured states.     

Synergistic effect of 5-fluorodeoxyuridine and quinazoline antifolates on murine leukemia self-cultured in vitro

The effect of thymidylate synthase inhibitors, fluorodeoxyuridine (FdUrd) and its two sulphonamide derivatives was examined in the culture of murine leukemia cells -- 5178Y (parental subline) and its fluorodeoxyuridine resistant subline 5178Y/F. A synergistic effect of the antimetabolites on cell survival was observed on exposure of the culture of either line to a slightly inhibitory concentration of FdUrd (1 nM) in combination with 2-desamino-2-methyl-10-propargyl-5,8-dideaza-pteroylsulphogluta mate or 2-desamino-2-methyl-10-propargyl-5,8-dideaza-pteroylsulphoglyci ne. This effect was accompanied by a marked reduction, in both cell lines of intracellular concentration of 5,10-methylenetetrahydro-pteroyl-polyglutamate, although its concentration in the resistant subline was 3 times as high as in the parental line. The inhibitory effect of combined drugs on the cellular pool of folates in 5178Y line depended also on the sequence of drug addition, whereas in the FdUrd resistant line this sequence was without any effect. The results obtained strongly suggest that under certain conditions inhibition of thymidylate synthesis by antifolates is intensified by a prior use of FdUrd.     

A role of the amino acid residue located on the fifth position before the first aspartate-rich motif of farnesyl diphosphate synthase on determination of the final product

Farnesyl diphosphate (FPP) synthase catalyzes consecutive condensations of isopentenyl diphosphate with allylic substrates to give FPP, C-15 compound, as a final product and does not catalyze a condensation beyond FPP. Recently, it was observed that, in Bacillus stearothermophilus FPP synthase, a replacement of tyrosine with histidine at position 81, which is located on the fifth amino acid before the first aspartate-rich motif, caused the mutated FPP synthase to catalyze geranylgeranyl diphosphate (C-20) synthesis (Ohnuma, S.-i., Nakazawa, T., Hemmi, H., Hallberg, A.-M., Koyama, T., Ogura, K., and Nishino, T. (1996) J. Biol. Chem. 271, 10087-10095). Thus, we constructed 20 FPP synthases, each of which has a different amino acid at position 81, and analyzed them. All enzymes except for Y81P can catalyze the condensations of isopentenyl diphosphate. The final products and the product distributions are different from each other. Y81A, Y81G, and Y81S can produce hexaprenyl diphosphate (C-30) as their final product. The final product of Y81C, Y81H, Y81I, Y81L, Y81N, Y81T, and Y81V are geranylfarnesyl diphosphate (C-25), and Y81D, Y81E, Y81F, Y81K, Y81M, Y81Q, and Y81R cannot produce polyprenyl diphosphates more than geranylgeranyl diphosphate. Substitution of tryptophan does not affect the product specificity of FPP synthase. The average chain length of products is inversely proportional to the accessible surface area of substituted amino acid. However, no significant relation between the final chain length and the kinetic constants Km and Vmax are observed. These observations strongly indicate that the amino acid does not come into contact with the substrates but directly contacts the omega-terminal of an elongating allylic product. This interaction must prevent further condensation of isopentenyl diphosphate.     

Acetylation of amikacin, a new semisynthetic antibiotic, by Pseudomonas aeruginosa carrying an R factor

A clinical isolate Pseudomonas aeruginosa GN315 resistant to amikacin (AK), a new semisynthetic antibiotic, inactivated AK by acetylation. The acetylating enzyme was purified approximately 146-fold from a crude extract of GN315 by affinity chromatography. Fractionated samples obtained by affinity chromatography showed almost the same inactivation curves toward 3',4'-dideoxykanamycin B (DKB) and AK. Partially purified AK-acetylating enzyme inactivated DKB and kanamycin A but could not inactivate gentamicin C(1). The optimal pH for their inactivation was 6.0 to 7.0, and the pH curves for the inactivation of both drugs were almost the same. These facts indicate that AK and DKB are inactivated by the same aminoglycoside-acetylating enzyme. Through elemental analysis, the inactivated AK was found to be a monoacetylated product of AK. A sample of inactivated AK was purified and compared with a synthetic 6'-N-acetyl AK by thin-layer chromatography, and the results indicated that AK was inactivated by acetylation of the 6'-NH(2) group. The ultraviolet, infrared, and nuclear magnetic resonance spectra of the inactivated AK showed that AK was inactivated by the enzyme through acetylation of the amino group of 6'-amino-6'-deoxy-d-glucose moiety of AK. This enzyme, mediated by R factor, is capable of conferring resistance to AK, DKB, kanamycin, gentamicin, and sulfanilamide.     

Antitumor activity of antifolate inhibitors of thymidylate and purine synthesis in human soft tissue sarcoma cell lines with intrinsic resistance to methotrexate

We examined the antitumor effects of two antifolate inhibitors of thymidylate synthesis, N-(5-[N-(3, 4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino ]-2-theno yl-L-glutamic acid (D1694; Tomudex) and 1843U89 as well as a folate-based inhibitor of purine synthesis, 5, 10-dideazatetrahydrofolic acid (DDATHF) on human soft tissue sarcoma cell lines having intrinsic resistance to methotrexate (MTX) due to impaired accumulation of polyglutamates of MTX (HS-16 and HS-42 cells) and to increased levels of dihydrofolate reductase and thymidylate synthase activity (HS-18 cells). Growth inhibition studies showed that ED50 values for D1694 and 1843U89 after a 24-h exposure were 11-19-fold and 22-222-fold lower, respectively, than those for MTX in HT-1080, a MTX-sensitive cell line, and the three MTX-resistant cell lines. In contrast, DDATHF was less cytotoxic than MTX in both the MTX-sensitive and the three resistant sarcoma cell lines. Uptake of D1694, 1843U89, or DDATHF was 2.5-4.5-fold higher than MTX in these sarcoma cell lines. However, D1694 and 1843U89, unlike MTX, accumulate in HS-16 and HS-42 cells as polyglutamate forms, reaching 70% of the total intracellular drug level after 24 h. DDATHF polyglutamates (9.4-24%) were less in the same cell lines. Much lower Km values for D1694 and 1843U89 as compared to MTX for folylpolyglutamate synthase were measured in the sarcoma cell lines, with Vmax values equal to or slightly higher than those obtained with MTX. D1694 and 1843U89 are significantly more cytotoxic than MTX in intrinsically MTX-resistant sarcoma cell lines as a result of extensive formation of polyglutamates. These two thymidylate synthase inhibitors should be evaluated in patients with soft tissue sarcomas.