N-Methylformycins. Reactivity with adenosine deaminase, incorporation into intracellular nucleotides of human erythrocytes and L 1210 cells and cytotoxicity to L 1210 cells.

The N-methyl derivatives of the C-nucleoside, formycin (7-amino-3(β-d-ribofurano-syl)pyrazolo[4, 3-d]pyrirnidine) were compared to formycin and adenosine with regard to their substrate activity with human erythrocytie adenosine deaminase (ADA), their ability to form intracellular nucleotides and their cytotoxicity to L1210 cells. Only 2-methylformycin (K_m = 6.1 mM, relative V_max = 396) and N^? -methylformycin (K_m = 0.1 mM, relative V_max = 3) showed substrate activity with ADA (corresponding kinetic parameters for adenosine were: K_m = 0.025 mM, relative V_max = 100). In contrast to previous hypotheses, these results suggest that the conformation (either syn or anti) of an adenosine analog is not a major factor in determining substrate activity with ADA. Neither 4-methylformycin nor 6-methylformycin formed their corresponding nucleotides when incubated with human erythrocytes, whereas both 1-methylfor-mycin and 2-methylformycin formed large amounts of their corresponding mono-, di- and triphosphate nucleotides. Inhibition of ADA by pretreatment of the erythrocytes with the potent ADA inhibitor, 2'-deoxycoformycin, had no effect on the incorporation of 1-methylformycin into erythrocytic nucleotides but greatly increased the incorporation of 2-methylformycin and N⁷-methylformycin. The conversion of both 1-methylformycin and 2-methylformycin into nucleotides was almost complete after 18 hr of incubation (in the presence of 2'-deoxycoformycin in the case of 2 methylformycin), whereas that of N⁷-methylformycin was only partially complete in the presence of 2'-deoxycoformycin. With both 1-methylformycin and N⁷-methylformycin, transient accumulation of the corresponding nucleoside 5'-monophosphate derivative was observed prior to the accumulation of the triphosphate nucleotide. Results, qualitatively similar to those found with erythrocytes, were obtained when the effects of 2'-deoxycoformycin on the incorporation of 1-methyl- and 2-methylformycins into the nucleotide pools of L 1210 cells in vitro were examined. Compounds capable of forming analog nucleotides in human erythrocytes or L1210 cells if deamination is prevented either by the molecular structure of the analog or by pretreatment of the cells with 2'-deoxycoformycin, also showed marked cytotoxicity to L1210 cells in culture, i.e. 1-methyl-, 2-methyl- and N⁷-methylformycin exhibited id₅₀ values of 0.5 to 2 μM, whereas 4-methyl- and 6-methylformyein were not significantly growth inhibitory. The potential usefulness of the various N-methyl derivatives of formycin (alone or in combination with an ADA inhibitor) as cytotoxic or antiviral agents is discussed.     

Biochemical properties of the nucleoside of 3-amino-1,5-dihydro-5-methyl-1,4,5,6,8-pentaazaacenaphthylene (NSC-154020)

A study of 3-amino-1, 5-dihydro-5-methyl-l-β-d-ribofuranosyl-1, 4, 5,6,8- pentaazaacenaphthylene (NSC-154020), a “tricyclic” nucleoside with activity against certain experimental tumors, was undertaken to determine if it differed in biochemical properties from structurally related7- deazapurine nucleosides with established biological activity, such as sangivamycin. In cultured L1210 cells, [¹⁴C-methyl]-NSC-154020 was converted to a single metabolite with the properties of a 5'-monophosphate as shown by (a) similarity to AMP in migration on paper chromatograms and in retention time when subjected to high pressure liquid chromatography (h.p.l.c.) on an ion exchange column and (b) conversion to a compound with the properties of NSC-154020 upon treatment with alkaline phosphatase or 5'-nucleotidase. In cultured H.Ep.-2 cells, the principal metabolite of NSC-154020 was also the monophosphate. H.Ep.-2 cells contained in addition a variable amount of a second metabolite which also had the retention time (on h.p.l.c. analysis) of a monophosphate and which was converted by the action of alkaline phosphatase or 5'-nucleotidase to a compound that migrated like NSC-154020 upon chromatography in three solvent systems. This second metabolite is as yet unidentified. In crude extracts of L1210 cells, addition of adenosine or 6-(methylthio)purine ribonucleoside decreased the phosphorylation of NSC-154020. NSC-154020 was a substrate for adenosine kinase 110-fold purified from H.Ep.-2 cells; the K_m was 215 μM and the V_max was 1.8 times greater than that of adenosine. No ¹⁴C from labeled NSC-154020 was found in the polynucleotides of either H.Ep.-2 cells or L1210 cells grown for 24 hr in the presence of the labeled nucleoside. Several different studies failed to reveal any selective sites of action for NSC-154020. In cultured L1210 cells it inhibited synthesis of DNA, RNA and protein and reduced ribonucleotide pools, but with little selectivity. The incorporation of [¹⁴C]formate into polynucleotides was inhibited more severely than that of hypoxanthine; this is evidence for a blockade of purine synthesis de novo, an effect also produced by many other analogs of purines and nucleosides. Sangivamycin produced generally similar inhibitions of incorporation of formate and hypoxanthine. However, the cytotoxicity of NSC-154020 and sangivamycin to L1210 cells was not prevented or reversed by 5-amino-4-imidazolecarboxamide (AIC), adenine, guanine, hypoxanthine, uridine, or AIC + uridine; therefore, inhibition of de novo synthesis of purine and pyrimidine nucleotides is not a primary site of action of these compounds. Although the loci of action of NSC-154020 are not yet defined, the fact that it is not metabolized to polyphosphates indicates that its mechanism of action probably differs significantly from those of the related compounds, tubercidin and sangivamycin, which are converted to polyphosphates and incorporated into RNA and DNA.     

Inosine analogs as substrates for adenosine kinase--influence of ionization of the N-1 proton on the rate of phosphorylation.

This study was undertaken to attempt to rationalize previously obtained and apparently conflicting findings that although adenosine kinase (EC 2.7.1.20) from H.Ep. # 2 cells did not accept inosine as a substrate, these cells became resistant to an inosine analog, 8-azainosine, only when activities of both hypoxanthine (guanine) phosphoribosyltransferase [H(G)PRTase] and adenosine kinase were lost. No evidence could be found for the presence of inosine kinase in H.Ep. # 2 cells: crude supernatants from these cells converted ring-labeled inosine to IMP, but the conversion was prevented by the addition of hypoxanthine and therefore apparently was achieved by the alternative pathway involving the action of H(G)PRTase on hypoxanthine. An investigation of inosine and inosine analogs as substrates for adenosine kinase revealed that certain inosine analogs were substrates and that the substrate activity could be correlated with the degree of ionization of the N-1 proton. Of four 6-oxo and 6-thio nucleosides studied. 8-aza-6-thioinosine had the lowest pK_a (6.75) and was the only one that was a good substrate at pH 7.0; the K_m was 210μM and the V_max was about 1.5 times that of adenosine. The rate of phosphorylation of 8-aza-6-thioinosine increased markedly as the pH of the reaction mixture was increased in the pH range 6.0 to 7.0. Phosphorylation of 8-azainosine (pK_a 7.45) and 6-thioinosine (pK_a 7.60) was much poorer and could be demonstrated at pH 7.0 only after overnight incubation with the kinase. The fact that 8-azainosine and 8-aza-6-thioinosine are substrates whereas ionsine is not, can be rationalized by the facts that (a) the substitution of an N-atom for the C-8 atom of the nucleoside lowers the pK_a so that the N-1 proton is more strongly ionized at physiological pH; and (b) the ionized form of a 6-oxo or 6-thio nucleoside resembles adenosine with respect to the bond structure at the 1- and 6-positions of the purine ring whereas the unionized form does not.     

Studies with a 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine (DDMP)-resistant L1210 leukemia cell line without cross-resistance to methotrexate

An L1210 leukemia cell line resistant to 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine (DDMP) (L1210/DDMP) was developed in vivo by treatment of tumor-bearing mice. Resistance to DDMP was confirmed by subsequent in vivo survival experiments and by in vitro dose-response curves. The L1210/DDMP line demonstrated little cross-resistance to another folate analog, methotrexate (MTX). This was confirmed both in vivo, with survival experiments, and in vitro, using dose-response curves. A statistical analysis of the in vivo data confirmed DDMP resistance with lack of MTX cross-resistance. Dihydrofolate reductase (DHFR) activity in the L1210/DDMP/R₅ line was no greater than in the parent cell line (L1210/S). and the K_m of DHFR for dihydrofolate was the same in the L1210/DDMP/R₅ and L1210/S lines. The K_i for DHFR of the L1210/DDMP/R₅ cell line versus the L1210/S cell line was increased 3.0-fold for MTX and 3.5-fold for DDMP. Total accumulation of [¹⁴C]DDMP was identical in the two cell lines. The explanation for the lack of MTX cross-resistance in the L1210/DDMP/R₅ line is unknown.     

C(2′)-substituted purine nucleoside analogs: Interactions with adenosine deaminase and purine nucleoside phosphorylase and formation of analog nucleotides

Four C(2′)-substituted 2′-deoxyadenosines were examined as substrates for human erythrocytic adenosine deaminase and for formation of intracellular nucleotide analogs in human erythrocytes, lymphocytes and murine Sarcoma 180 cells: 9-(2′-deoxy-2′-fluoro-β-D-ribofuranosyl)adenine, 9-(2′-deoxy-2′-fluoro-β-D-arabinofuranosyl)adenine, 9-(2′-azido-2′-deoxy-β-D-ribofuranosyl)adenine (2′-N₃-riboA) and 9-(2′-azido-2′-deoxy-β-D-arabinofuranosyl)adenine. All four adenosine analogs were substrates of human erythrocytic adenosine deaminase, but the corresponding inosine analogs (synthesized by the adenosine deaminase reaction) were highly resistant to cleavage by human erythrocytic purine nucleoside phosphorylase. Only 9-(2′-deoxy-2′-fluoro-β-D-ribofuranosyl)hypoxanthine underwent very slow phosphorolysis, and no inhibition of inosine phosphorolysis was detected when a 30 μM concentration of any studied inosine analog was added to a reaction mixture containing 30 μM inosine (the K_m concentration). Kinetic parameters were determined for the deamination of the adenosine analogs. The greatest affinity for adenosine deaminase was found with 2′-N₃-riboA (K_i=2μM), but the reaction velocity was highest with the F-substituted analogs. All four adenosine analogs formed triphosphate nucleotides after incubation with human erythrocytes, murine Sarcoma 180 cells, or human lymphocytes (tested only with the F analogs) in the presence of deoxycoformycin.     

Enzyme kinetic modelling as a tool to analyse the behaviour of cytochrome P450 catalysed reactions: application to amitriptyline N-demethylation

1To determine kinetic parameters (V_max, K_m) for cytochrome P450 (CYP) mediated metabolic pathways, nonlinear least squares regression is commonly used to fit a model equation (e.g., Michaelis Menten [MM]) to sets of data points (reaction velocity vs substrate concentration). This method can also be utilized to determine the parameters for more complex mechanisms involving allosteric or multi-enzyme systems. Akaike''s Information Criterion (AIC), or an estimation of improvement of fit as successive parameters are introduced in the model ( F-test), can be used to determine whether application of more complex models is helpful. To evaluate these approaches, we have examined the complex enzyme kinetics of amitriptyline (AMI) N-demethylation in vitro by human liver microsomes. 2For a 15-point nortriptyline (NT) formation rate vs substrate (AMI) concentration curve, a two enzyme model, consisting of one enzyme with MM kinetics (V_max=1.2 nmol min⁻¹ mg⁻¹, K_m=24 μm) together with a sigmoidal component (described by an equation equivalent to the Hill equation for cooperative substrate binding; V_max=2.1 nmol min⁻¹ mg⁻¹, K′=70 μm; Hill exponent n=2.34), was favoured according to AIC and the F-test. 3Data generated by incubating AMI under the same conditions but in the presence of 10 μm ketoconazole (KET), a CYP3A3/4 inhibitor, were consistent with a single enzyme model with substrate inhibition (V_max=0.74 nmol min⁻¹ mg⁻¹, K_m=186 μm, K₁=0.0028 μm⁻¹). 4Sulphaphenazole (SPA), a CYP2C9 inhibitor, decreased the rate of NT formation in a concentration dependent manner, whereas a polyclonal rat liver CYP2C11 antibody, inhibitory for S-mephenytoin 4′-hydroxylation in humans, had no important effect on this reaction. 5Incubation of AMI with 50 μm SPA resulted in a curve consistent with a two enzyme model, one with MM kinetics (V_max=0.72 nmol min⁻¹ mg⁻¹, K_m=54 μm) the other with ‘Hill-kinetics’ (V_max=2.1 nmol min⁻¹ mg⁻¹, K′=195 μm; n=2.38). 6A fourth data-set was generated by incubating AMI with 10 μm KET and 50 μm SPA. The proposed model of best fit describes two activities, one obeying MM-kinetics (V_max=0.048 nmol min⁻¹ mg⁻¹, K_m=7 μm) and the other obeying MM kinetics but with substrate inhibition (V_max=0.8 nmol min⁻¹ mg⁻¹, K_m=443 μm, K₁=0.0041 μm⁻¹). 7The combination of kinetic modelling tools and biological data has permitted the discrimination of at least three CYP enzymes involved in AMI N-demethylation. Two are identified as CYP3A3/4 and CYP2C9, although further work in several more livers is required to confirm the participation of the latter.     

Analog specific aberrancies in antifolate inhibition of L1210 cell dihydrofolate reductase

During studies with L1210 cells and a variety of folate analogs, large discrepancies were revealed between data on membrane transport, on inhibition of dihydrofolate reductase in cell-free extracts, and on inhibition of growth in culture for 10-oxa-, 10-benzyl- and 10-phenethyl-aminopterin, and for 3-deaza, 10-methyl-aminopterin. While aminopterin, 10-methyl (methotrexate)-, 10-ethyl- and 10-propyl-aminopterin were tight binding inhibitors (K_i: 2–3 × 10^?12M) of dihydrofolate reductase in cell-free extracts from L1210 cells, the other four analogs were only weak competitive inhibitors (K_i = 3–300 × 10^?8M). Similar differences among analogs were observed for inhibition of dihydrofolate reductase in cell-free extracts from Sarcoma 180 and Ehrlich cells, but not for this enzyme in microbial cell-free extracts. There were only small differences in the transport of all of the analogs by L1210 cells. Inhibition of L1210 cell growth in culture by 10-oxa-, 10-benzyl- and 10-phenethyl-aminopterin and by 3-deaza, 10-methyl-aminopterin, in contrast to the other analogs, was several orders of magnitude greater than that predicted from the data on dihydrofolate reductase inhibition. The extent of binding of 10-oxa-, 10-benzyl- and 10-phenethyl-aminopterin, and of 3-deaza and 10-methyl-aminopterin to dihydrofolate reductase in intact L1210 cells, in contradistinction to that seen for the cell-free enzyme preparations, approached that observed for methotrexate; these estimates of drug-enzyme interaction in situ were more predictive of the extent of inhibition by these analogs of L1210 cell growth in culture.     

Failure of 6-thioGMP to inhibit guanylate kinase in intact cells.

Experiments have been performed that make necessary a modification of the hypothesis that 6-thioguanine produces cytotoxicity by a sequential blockade of the enzymes, ribosylamine-5-phos-phate: pyrophosphate phosphoribosyltransferase (PRPP-amidotransferase), inosinate dehydrogenase and guanylate kinase. L5178Y murine leukemia cells (in vivo and in vitro) were pretreated with 6-thio-guanine under conditions known to produce significant accumulations of the analog nucleoside 5'-monophosphate, 6-thioGMP, inhibition of purine de novo biosynthesis, and marked cytotoxicity. When these cells were incubated with [8-¹⁴C]guanine, no inhibition in the formation of guanine nucleotides was observed in comparison with control cells not treated with 6-thioguanine. Furthermore, measurement of changes in the intracellular concentrations of GMP, GDP and GTP did not provide evidence for the occurrence of a ‘cross-over’' between GMP and GDP in the presence of 6-thioGMP. Thus, the predicted accumulation of GMP resulting from the postulated blockade of guanylate kinase by 6-thioGMP did not occur. L5178Y cells incubated with guanine for periods of 1 hr or less accumulated concentrations of GDP and GTP that approximated the intracellular levels of ADP and ATP. Time studies were performed with human erythrocytes in which the rate of formation of nucleotides was compared in cells incubated with guanosine or 6-thioguanosine. With guanosine, the rapid formation of guanine nucleotides was observed with the ratio of GTP:GDP:GMP approximating the ratios of the adenine nucleotides of the erythrocytes, i.e. no accumulation of GMP was observed at any time period up to 6 hr. In contrast, with 6-thioguanosine, a rapid initial formation of 6-thioGMP was observed with a gradually accelerating formation of 6-thioGTP. After 2 hr, the concentration of 6-thioGMP decreased whereas the formation of 6-thioGTP achieved a velocity of about 0.007 μmole/ min/ml of erythrocytes. This velocity is about 2 per cent of that expected with saturating levels of GMP, if one assumes the intraerythrocytic activity of guanylate kinase to be 0.3 enzyme unit/ml of cells (enzyme unit: 1 unit catalyzes the conversion of 1 μmole substrate into product/min). These findings are in general agreement with the results of studies with purified guanylate kinase preparations described in the accompanying publication [R.L. Miller, D.L. Adamczyk, T. Spector, K.C. Agarwal, R.P. Miech and R.E. Parks Jr., Biochem. Pharmac. 26, 1573 (1977)], which indicate that the K_m value and V_max value of 6-thioGMP are approximately 2.0 mM and about 3 per cent of the V_max with GMP respectively. Therefore, it may be concluded that administration of 6-thioguanine does not cause significant inhibition of guanylate kinase. However, the poor reactivity of 6-thioGMP with guanylate kinase probably causes the marked intracellular accumulation of this analog nucleotide after administration of 6-thioguanine or its derivatives.     

Inhibition of hypoxanthine-guanine phosphoribosyl transferase.

The affinities of eighteen purines or purine analogs for human erythrocytic hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8; HGPRTase) were compared to assess the feasibility of obtaining active inhibitors of the enzyme. Three compounds appeared to inhibit the utilization of hypoxanthine by L5178Y cells in vitro due to inhibition of the enzyme rather than depletion of the intracellular 5-phosphoribosyl-1-pyrophosphate pool. The three competitive inhibitors and their affinity constants (K_i) using 6-mercaptopurine as substrate were: 6-mercapto-9-(tetrahydro-2-furyl)-purine, 37 μM; 2,6-bis-(hydroxyamino)-9-β-d-ribofuranosyl-purine, 12 μM; and 6-iodo-9-(tetrahydro-2-furyl)-purine, 108 μM. The KInm for 6-mercaptopurine was 9 μM. Thus, the enzyme tolerates bulky substitution at N⁹. 6-Mercapto-9-(tetrahydro-2-furyl)-purine also potentiated the chemotherapeutic effect of azaserine, an inhibitor of de novo purine biosynthesis, in L5178Y ascites tumor-bearing mice. Four 2-substituted, oxazolo-[5, 4-d]-pyrimidine-7-ones and 2-methylthiazolo-[5, 4-d]-pyrimidine-7-one had K_i values in the range of 84–173 μM. Consequently, isosteric substitution at N⁹ may also be a fruitful and logical course to pursue in the design and synthesis of more potent inhibitors of this important enzyme.     

AG2034: a novel inhibitor of glycinamide ribonucleotide formyltransferase

Summary The glycinamide ribonucleotide formyltransferase (GARFT) inhibitor, 4-[2-(2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4-6][1,4]thiazin-6-yl)-(S)-ethyl]-2,5-thienoyl-L-glutamic acid (AG2034), was designed from the X-ray structure of the GARFT domain of the human tri functional enzyme. AG2034 inhibits human GARFT (K_i = 28 nM), has a high affinity for the folate receptor (K _d = 0.0042 nM), and is a substrate for rat liver folylpolyglutamate synthetase (K _m = 6.4 μM, V _max = 0.48 nmole/hr/mg). The IC₅₀ for growth inhibition was 4 nM against L1210 cells and 2.9 nM for CCRF-CEM cells in culture. In vitro growth inhibition can be reversed by addition of either hypoxanthine or AICA (5-aminoimidazole-4-carboxamide) to the culture medium. A cell line with impaired transport of reduced folates, L1210/CI920 [1], was resistant to AG2034 indicating that this compound can enter cells by utilizing the reduced folate carrier. AG2034 showed in vivo antitumor activity against the 6C3HED, C3HBA, and B-16 murine tumors and in the HxGC₃, KM20L2, LX-1, and H460 human xenograft models, and has been selected for preclinical development towards clinical trials.     

Inhibitors of purine nucleoside phosphorylase

Purine nucleoside phosphorylase (PNP) catalyses the reversible phosphorolysis of purine ribonucleosides and 2′-deoxyribonu-cleosides to the free base and ribose-1-phosphate or 2′-deoxyribose-1-phosphate. Mammalian PNP is specific for guanosine, inosine and certain analogues, although PNPs from other organisms show varying levels of specificity. Interest in PNP arises primarily from its critical role in purine nucleoside metabolism and in T-cell function, which indicates that inhibitors of this enzyme might be useful in the treatment of T-cell proliferative diseases, such as T-cell leukaemias and lymphomas, in the suppression of host-vs-graft response in organ transplant patients, and in the treatment of T-cell mediated autoimmune diseases. They may also be effective in the treatment of gout and certain parasitic diseases such as malaria. Most potent inhibitors of this enzyme are derivatives of guanine or 8-aminoguanine, including 9-arylmethyl derivatives. Of these, 9-benzylguanines substituted at position 2 or 3 of the phenyl ring by a side chain terminating in a phosphonate moiety are the most potent, with K_i in the 1 nM range, although these compounds have limited potential as drugs because of their very low cell permeability. As a result, inhibitors of this type that have been tested are effective in whole cells only at concentrations of 100 μM or higher. On the other hand, the three-dimensional structure of human PNP, determined by X-ray crystallography, has been used in designing novel inhibitors of this key enzyme, resulting in several families of membrane-permeable inhibitors with IC_50^S in the 6 to 30 nM range. The inhibition is competitive with respect to both inosine and phosphate. 9-(3-pyridylmethyl)-9-deazaguanine (BCX-34) was chosen from this group of inhibitors for further study. Its K_i for the inhibition of human erythrocytic PNP is 31 nM, and its IC₅₀ for inhibition of the proliferation of T-cells (CCRF-CEM) is 0.8 μM in the presence of 2′-deoxyguanosine, which alone has no effect on cells. It did not inhibit the proliferation of B-cells (MGL-8) up to 30 μM. Phase I/II clinical studies of a dermal formulation of BCX-34 have shown this drug to be efficacious and safe in the treatment of cutaneous T-cell lymphoma and psoriasis.     

Transport of 5-fluorouracil and uracil into human erythrocytes

The transport of 5-fluorouracil (5-FU) and uracil into human erythrocytes has been investigated under initial velocity conditions with an “inhibitor-stop” assay using a cold papaverine solution to terminate influx. At 37° and pH 7.3, 5-FU influx was nonconcentrative; was partially inhibited by adenine, hypoxanthine, thymine, and uracil; and was insensitive to inhibition by nucleosides or inhibitors of nucleoside transport. Inhibition of the influx of 5-FU or uracil by adenine (3.0 mM) did not increase when other pyrimidines or inhibitors of nucleoside transport were combined with adenine. 5-FU and uracil exhibited similar saturable (K_m 4 mM, V_max 500 pmol/sec/5μL cells) and nonsaturable (rate constant 80 pmol/sec/mM/5μL cells) components of influx. 5-FU, uracil, adenine, and hypoxanthine were competitive inhibitors of each other's influx with K_i, values matching their respective K_m values for influx. We conclude that 5-FU and uracil enter human erythrocytes at similar rates via both nonfacilitated diffusion and the same carrier that transports adenine and hypoxanthine.     

Identification of efflux systems for large anions and anionic conjugates as the mediators of methotrexate efflux in L1210 cells

Two ATP-dependent efflux systems for methotrexate have been identified in inside-out vesicles from an L1210 mouse cell variant with a defective influx carrier for methotrexate. Transport at 40 μM [³H]methotrexate was separated by inhibitors into two components comprising 62 and 38% of total transport activity. The predominant route was inhibited by low concentrations of indoprofen (K_i = 2.5 μM), 4-biphenylacetic acid (K_i = 5.3 μM), and flurbiprofen (K_i = 5.2 μM), whereas the second component showed a high sensitivity to the glutathione conjugates of bromosulfophthalein (K_i = 0.08 μM), ethacrynic acid (K_i = 0.52 μM), and 1-chloro-2,4-dinitrobenzene (K_i = 0.77 μM). Bilirubin ditaurate was a potent inhibitor of both transport components (K_i = 1.5 and 0.17 μM, respectively). Separation of transport activities without interference from the other route was achieved by adding an excess (100 μM) of either the glutathione conjugate of ethacrynic acid or biphenylacetic acid. Double-reciprocal plots of transport at various substrate concentrations gave K_m values of 170 and 250 μM for methotrexate transport via the anion-sensitive and conjugate-sensitive routes, respectively. A comparison of inhibitor specificities indicated that the anion-sensitive transport activity in vesicles represents efflux system II for methotrexate in intact cells and is the same system identified previously in vesicles as an anion/anion conjugate pump. The conjugate-sensitive activity corresponds to efflux system I for methotrexate in intact cells and is the same system identified in vesicles as the high-affinity glutathione conjugate pump.     

Kinetic studies with 5-azacytidine-5'-triphosphate and DNA-dependent RNA polymerase.

In order to further understand the biochemical mode of action of 5-azacytidine, a potent antileukemic agent, kinetic studies were performed with 5-azacytidine-5'-triphosphate (5-aza-CTP) and purified DNA-dependent RNA polymerase from Escherichia coli and calf thymus. RNA polymerase could catalyze the incorporation of the fradulent nucleotide, 5-aza-CTP, into RNA. The apparent K_m value for 5-aza-CTP was estimated to be 350 and 390 for the E. coli and calf thymus enzymes respectively. The K_m value for 5-aza-CTP was about 18-fold greater than the K_m value for CTP (20 μM). The apparent V_max value for CTP was about 2-fold greater than the V_max value for 5-aza-CTP. 5-Aza-CTP was a weak competitive inhibitor with respect to CTP; the apparent K_i value for 5-aza-CTP was estimated to be 680 and 810 μM for the E. coli and calf thymus enzymes respectively. On the other hand, CTP was a potent competitive inhibitor with respect to 5-aza-CTP; the apparent K_i value of CTP was estimated to be 16 μM. 5-Aza-CTP did not appear to inhibit the incorporation of UTP into RNA in the reaction catalyzed by RNA polymerase. These data suggest that the inhibition of RNA synthesis in cells by 5-aza-cytidine is not produced by the inhibition of RNA polymerase by 5-aza-CTP.     

5-substituted 2′-deoxyuridines: Correlation between inhibition of tumor cell growth and inhibition of thymidine kinase and thymidylate synthetase

A large variety of 5-substituted 2′-deoxyuridines (dUrds) and 2′-deoxyuridylates (dUMPs) have been evaluated for their inhibitory effects on the thymidine (dThd) kinase or thymidylate (dTMP) synthetase isolated from mouse leukemia L1210 cells. The most potent inhibitors of dThd kinase were 5-chloro-, 5-bromo- and 5-iodo-dUrd. Their K_i/K_m values ranged from 0.57 to 0.82. All dUrd analogs tested showed competitive kinetics with respect to dThd. However, there was little, if any, correlation between the inhibitory effects of the compounds on L1210 cell growth and their inhibitory activities against dThd kinase (r = 0.16). The most potent inhibitors of dTMP synthetase were (in order of decreasing activity): 5-nitro-dUMP > 5-formyl-dUMP > 5-fluro-dUMP > 5-oxime of 5-formyl-dUMP > 5-azidomethyl-dUMP > (E)-5-(2-bromovinyl)-dUMP. The k_i/K_m values for these compounds ranged from 0.001 to 0.665. All dUMP analogs tested showed competitive kinetics with respect to dUMP (if not preincubated with the enzyme at 37°). There was a strong correlation (r = 0.833) between the inhibitory effects of these compounds on L1210 cell growth and their inhibitory activities against dTMP synthetase. Thus, the suppressive action of 5-substituted dUrd derivatives on tumor cell growth would involve prior conversion of the nucleoside analogs to the corresponding 5′-monophosphates followed by an inhibition of dTMP synthetase.     

Inhibition of rat hepatic guanylate kinase by 6-thioguanosine-5'-phosphate and 6-selenoguanosine-5'-phosphate.

Guanylate kinase has been partially purified from rat liver and subjected to isoelectric focusing. Three peaks of enzymic activity at isoelectric points of 4.7, 4.9 and 5.1 were obtained. The greatest amount of activity occurred at pH 4.9 with an over-all purification of about 250.fold. GMP, dGMP and 8-azaGMP act as substrates and no essential differences were detected in the K_m and V_max values of the three isoelectric focusing peaks. Initial velocity studies with the principal activity peak (pI 4.9), with either GMP or ATP as the changing-fixed substrate, confirmed earlier results with guanylate kinase preparations from hog brain and human erythrocytes and were consistent with either a “random” or “ordered” reaction mechanism. 6-Thioguanosine-5′-phosphate (6-thioGMP) and 6-selenoguanosine-5′-phosphate (6-SeGMP) are good competitive inhibitors of the main activity peak (pI 4.9) with inhibition constants (K_i) of 6.8 × 10^?5 M and 9.5 × 10^?5 M for 6-thioGMP and 6-SeGMP respectively. No substrate activity was detected for 6-thioGMP or 6-SeGMP with any of the three isoelectric focusing enzyme peaks in assays capable of detecting reaction velocities greater than 1 per cent of that seen with GMP as the substrate.     

Potent mechanism-based inhibition of CYP3A4 by imatinib explains its liability to interact with CYP3A4 substrates

BACKGROUND AND PURPOSE

Imatinib, a cytochrome P450 2C8 (CYP2C8) and CYP3A4 substrate, markedly increases plasma concentrations of the CYP3A4/5 substrate simvastatin and reduces hepatic CYP3A4/5 activity in humans. Because competitive inhibition of CYP3A4/5 does not explain these in vivo interactions, we investigated the reversible and time-dependent inhibitory effects of imatinib and its main metabolite N-desmethylimatinib on CYP2C8 and CYP3A4/5 in vitro.

EXPERIMENTAL APPROACH

Amodiaquine N-deethylation and midazolam 1′-hydroxylation were used as marker reactions for CYP2C8 and CYP3A4/5 activity. Direct, IC₅₀-shift, and time-dependent inhibition were assessed with human liver microsomes.

KEY RESULTS

Inhibition of CYP3A4 activity by imatinib was pre-incubation time-, concentration- and NADPH-dependent, and the time-dependent inactivation variables K_I and k_inact were 14.3 µM and 0.072 min⁻¹ respectively. In direct inhibition experiments, imatinib and N-desmethylimatinib inhibited amodiaquine N-deethylation with a K_i of 8.4 and 12.8 µM, respectively, and midazolam 1′-hydroxylation with a K_i of 23.3 and 18.1 µM respectively. The time-dependent inhibition effect of imatinib was predicted to cause up to 90% inhibition of hepatic CYP3A4 activity with clinically relevant imatinib concentrations, whereas the direct inhibition was predicted to be negligible in vivo.

CONCLUSIONS AND IMPLICATIONS

Imatinib is a potent mechanism-based inhibitor of CYP3A4 in vitro and this finding explains the imatinib–simvastatin interaction and suggests that imatinib could markedly increase plasma concentrations of other CYP3A4 substrates. Our results also suggest a possibility of autoinhibition of CYP3A4-mediated imatinib metabolism leading to a less significant role for CYP3A4 in imatinib biotransformation in vivo than previously proposed.     

Synthesis and biologic studies of arabinosylcytosine 5'-methylphosphonate

Arabinosylcytosine (ara-C), a clinically useful antitumor agent, is ineffective against cells that have deleted deoxycytidine kinase, the enzyme necessary for conversion of ara-C to its active nucleotide form. To circumvent this resistance, arabinosylcytosine-5'-methylphosphonate (ara-CMeP) was synthesized as an analogue of ara-CMP that would be membrane-permeable, resistant to serum phosphatase attack, and resistant to nucleoside deaminase inactivation. Ara-CMP was inhibitory to leukemia P388 in vitro but required concentrations 90-fold greater than that of ara-C for comparable cell inhibition. Both ara-CMeP and ara-CMP were competitive inhibitors of dCMP kinase from leukemia L1210 with K_i values of 4.0 × 10^?3 and 4.4 × 10^?3 M respectively. However, ara-CMP is a substrate for dCMP kinase, whereas ara-CMeP was not. Thus, the inability of ara-CMeP to be phosphorylated precludes its usefulness as a functional analogue of ara-CMP.     

Kinetics of in vitro nitro reduction of 2,4-dinitrophenol gy rat liver homogenates

The in vitro metabolism of [¹⁴C]-2,4-dinitrophenol (DNP) was examined in rat liver homogenates (300–500 g males). DNP and the metabolites, 4-amino-2-nitrophenol (4A2NP) and 2-amino-4-nitrophenol (2A4NP) were identified and quantified. The Michaelis-Menten constant, K_m, and the maximum velocity, V_max, were determined for the reduction reaction using a Hofstee plot. The K_m and V_max for DNP reduction were, respectively: 1.8 × 10^?4m and 1.13 nmol/mg protein/min. p-Nitrobenzoic acid (PNBA), o-nitrophenol (ONP), p-nitrophenol (PNP) and 2,4-dinitro-6-sec-butylphenol (DNBP) were examined as inhibitors of DNP nitro reduction. PNBA had no effect, but PNP, ONP and DNBP significantly inhibited DNP nitro reduction. ONP was a competitive inhibitor of DNP reduction while PNP and DNBP were noncompetitive. The K_t values calculated from the regression lines, for ONP, PNP and DNBP respectively, were 5.1 × 10^?4m, 7.1 × 10^?4m.     

Inosine 5′-monophosphate dehydrogenase from Sarcoma 180 cells—Substrate and inhibitor specificity

The substrate and inhibitor specificity of IMP dehydrogenase from Sarcoma 180 ascites tumor cells has been studied with twenty purine nucleotide analogs. Several were found to be substrates with the following efficiencies (V_max/K_m):IMP (4000), 8-azaIMP (1360), 6-thioIMP (250), araIMP (250) and dIMP (240). While substrate activity was not detected with the 5′-phosphates of 6-methylmercaptopurine riboside or 1-ribosylallopurinol (rates less than 1/10,000th that of IMP), they were competitive inhibitors with respect to IMP (both with K_i, values of 0.43 mM). Seven XMP analogs and six GMP analogs were also found to be competitive inhibitors with respect to IMP. Methods for the synthesis of the 5′-phosphates of arabinosylhypoxanthine. arabinosylxanthine. arabinosylguanine and 2′-deoxyxanthosine are described.