Further studies of stereospecificity at carbon 6 for membrane transport of tetrahydrofolates: Diastereoisomers of 5-methyltetrahydrofolates as competitive inhibitors of transport of methotrexate in L1210 cells

The unnatural d diastereoisomer at carbon 6 of 5-methyltetrahydrofolate was only slightly less effective than the natural l diastereoisomer as a competitive inhibitor of the carrier-mediated membrane transport of [³H]methotrexate into L1210 murine leukemia cells. The apparent K_i for a mixture containing equal amounts of both natural and unnatural diastereoisomers was not significantly different from that found for the unnatural form. These results show that the reduced folate carrier system in these cells has a strong affinity for the unnatural stereoisomer, a finding in contrast to that obtained with the corresponding diastereoisomer of 5-formyltetrahydrofolate.     

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.     

A fluorescent analogue of methotrexate as a probe for folate antagonist molecular receptors

A dansyl-l-lysine analogue of methotrexate, $\text{N}{}^{\mathrm{\alpha }}\text{-(}\text{4-amino-4-deoxy-10-methylpteroyl}\text{)-N}{}^{\mathrm{\epsilon }}\text{-(5-[N,N-}\text{dimethylamino]-1-naphthalenesulfonyl]-1-naphthalenesulfonyl)-}\text{l}\text{-lysine}$, is a potent inhibitor of murine L1210 dihydrofolate reductase. The dansyl fluorescence emission was enhanced approximately 3-fold with a 10 nm blue shift upon binding to L1210 dihydrofolate reductase. The fluorescent analogue was only 10-fold less potent than methotrexate in inhibiting the growth of methotrexate-sensitive and -resistant L1210 cells and competes effectively for [³H]methotrexate transport with a K_i of 7.02 μM, a value virtually identical to the K_t for methotrexate in both cell lines. In addition, strong dansyl fluorescence was found to be associated with dihydrofolate reductase from methotrexate-resistant, dihydrofolate reductase-overproducing L1210 cells following incubation of viable cells with the fluorescent methotrexate analogue for 4 hr. The results demonstrate that the dansyl-l-lysine analogue of methotrexate was rapidly transported into L1210 cells where it formed a high-affinity, fluorescent complex with intracellular dihydrofolate reductase.     

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.     

Inhibitors of purine nucleoside phosphorylase, C(8) and C(5') substitutions

The C(8) and C(5') positions of base and nucleoside substrates of human erythrocytic purine nucleoside phosphorylase (PNP) are promising sites for the development of an inhibitor of this enzyme. The substrate analog, 8-aminoguanine (8-AG), has the lowest dissociation constant (K_i = 0.2–1.2 μM) of any compound reported to date and V_max = 16 per cent (relative to guanine). Other C(8) substituents decreased the affinity for PNP and, with the exception of the methyl and sulfhydryl groups, abolished substrate activity. Halogens or a thiomethyl group at C(5') of inosine resulted in unchanged or improved affinities (K_i = 10–30 μM) and greatly decreased substrate activity (V_max < 1 per cent relative to inosine). The K_i of formycin B was reduced from 100 μM to 10 μM or less by substitution of a halogen at C(5'). Phosphorolysis of purine nucleosides was inhibited significantly by 8-AG in intact human erythrocytes and murine Sarcoma 180, L1210 and L5178Y cells. Although a K_i value of 17 μM was determined for 8-aminoguanosine, it was equally effective in inhibiting PNP activity in intact cells. The nucleoside was cleaved to 8-AG which was not a substrate for guanase or hypoxanthine-guanine phosphoribosyltransferase. Despite its low substrate activity (V_max < 0.2%). 5′-deoxy-5′-iodoinosine was cleaved by intact L1210 and L5178Y cells.     

Cytotoxicities of the l and d isomers of phenylalanine mustard in L1210 cells

The cytotoxicity of d-phenylalanine mustard (medphalan) to murine L1210 leukemia cells in culture was reduced by both the d and l isomers of leucine. l-Leucine only partially protected L1210 cells from medphalan cytotoxicity at drug concentration's above 10 μM, suggesting that medphalan uptake occured via both an amino acid carrier and an as yet undetermined agency, possibly passive diffusion. At equitoxic concentrations of l-phenylalanine mustard (melphalan) and medphalan, l-leucine reduced medphalan cytotoxicity by only one-sixth that obtained with melphalan. Analysis of melphalan and medphalan inhibition of the initial rate of l-leucine transport indicated a melphalan K_i of 0.085 mM, a value one-seventh that of melphalan (K_i, 0.635 mM).     

Transport and interaction of drugs in leukocytes

The accumulation of selected CNS drugs by rat leukocytes was previously reported. This paper presents evidence for the transport into leukocytes of additional drugs. Also studied was the inhibition of the latter processes by various structurally related compounds. The markedly rapid and sodium-independent uptakes into rat leukocytes of amphetamine, codeine, methadone and naloxone fulfilled the basic criteria for active transport. The uptake of morphine was apparently accomplished by more than one process. The affinities of the high capacity transport systems (approximate V_max: 100 nmoles/g cells/5sec) varied considerably as reflected by the two extreme K_m values obtained for methadone (20 μM) and morphine (1.8 mM). A variety of amines inhibited the cellular transport of the drugs. Most potent inhibitors were quinacrine (K_i: 0.5 to 3 μM), desipramine (K_i: 6–20 μM) and methadone (K_i: 18–25 μM). Morphine and tryptamine exhibited inhibition constants higher than 1 mM. The cellular transport processes newly described in rat leukocytes apparently represent a novel addition to the heterogenous biological transport of basic amines. The structural specificity of amine transport in various tissues is discussed.     

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.     

Growth inhibitory,transport and biochemical properties of the γ-glutamyl and γ-aspartyl peptides of methotrexate inL1210 leukemia cells in vitro

Both methotrexate-γ-glutamate and methotrexate-γ-aspartate are equivalent to metho-trexate as inhibitors of L1210 cell dihydrofolate reductase. However, the initial influx of both peptides into L1210 cells during transport studies is substantially lower than that of methotrexate. The apparent K_m tor influx of methotrexate-γ-glutamate and methotrexate-γ-aspartate is 15-fold and 100-fold greater than methotrexate respectively. Efflux measurements, which were possible only for methotrexate-γ-glutamate, showed a similar rate for this peptide and methotrexate. The intracelluiar accumulation and subsequent metabolism to methotrexate of methotrexate-γ-glutamate, but not of methotrexate-γ-aspartate, were confirmed by bioautographic analysis of cell extracts. After correction for extracellular cleavage of both peptides mediated by enzymes in calf serum supplementing the culture medium, the relative growth (L1210 cell)-inhibitory potency for the three agents was 1:18:210 for methotrexate, methotrexate-γ-glutamate and methotrexate-γ-aspartate respectively. Both the relative inhibitory potency and the difference in absolute inhibitory concentration among the three agents were predictable solely from the data on the influx of each measured during transport studies. Methotrexate-γ-aspartate is apparently more resistant to enzymic cleavage than is methotrexate-γ-glutamate.     

Inhibition of CYP3A by Antimalarial Piperaquine and Its Metabolites in Human Liver Microsomes With IVIV Extrapolation

The potential of the antimalarial piperaquine and its metabolites to inhibit CYP3A was investigated in pooled human liver microsomes. CYP3A activity was measured by liquid chromatography-tandem mass spectrometry as the rate of 1′-hydroxymidazolam formation. Piperaquine was found to be a reversible, potent inhibitor of CYP3A with the following parameter estimates (%CV): IC₅₀ = 0.76 μM (29), K_i = 0.68 μM (29). In addition, piperaquine acted as a time-dependent inhibitor with IC₅₀ declining to 0.32 μM (28) during 30-min pre-incubation. Time-dependent inhibitor estimates were k_inact = 0.024 min^?1 (30) and K_I = 1.63 μM (17). Metabolite M2 was a highly potent reversible inhibitor with estimated IC₅₀ and K_i values of 0.057 μM (17) and 0.043 μM (3), respectively. M1 and M5 metabolites did not show any inhibitory properties within the limits of assay used. Average (95th percentile) simulated in vivo areas under the curve of midazolam increased 2.2-fold (3.7-fold) on the third which is the last day of piperaquine dosing, whereas for its metabolite M2, areas under the curve of midazolam increased 7.7-fold (13-fold).     

Effects of bis(6-mercaptopurine-9-beta-D-ribofuranoside)-5',5"-phosphate and its butyryl derivative on mouse leukaemia L1210 and a 6-mercaptopurine-resistant subline in culture

Bis(6-mercaptppurine-9-β-d-ribofuranoside)-5′-5″′-monophosphate (bis(MPR)P) and its butyryl derivative, bis(O²,$\text{O}{}^3\text{-}\text{dibutyryl}\text{-6-}\text{mercaptopurine}\text{-9-β-}\text{d}\text{-}\text{ribofuranoside}$)-5′,5″′-monophosphate (bis(dibutyrylMPR)P) were synthesized from 6-mercaptopurine-9-β-d-ribofuranoside (MPR). Bis(MPR)P (ec₅₀ = 0.014 μM) and MPR (ec₅₀ = 0.022 μM) were essentially equivalent in their growth inhibitory activities against L1210/0 cell cultures, whilst bis(dibutyrylMPR)P (ec₅₀ = 1.1 μM) was considerably less effective. L1210/MPR cells grew normally in the presence of 1 mM MPR but were inhibited by bis(MPR)P (ec₅₀ = 580 μM) and (bis(dibutyrylMPR)P (ec₅₀ = 42 μM). Bis(dibutyrylMPR)P was less readily broken down to MPR by enzymes in the serum component of the culture medium than was bis(MPR)P, and leukaemia cells did not contribute to the extracellular degradation of the acylated derivative. The delayed cytotoxic effects of bis(MPR)P and bis(dibutyrylMPR)P on L1210/0 cells were those of the MPR breakdown product. Exposure to bis(MPR)P resulted in delayed cytotoxicity in L1210/MPR cultures whilst bis(dibutyrylMPR)P produced only acute growth inhibition and no delayed effect on the MPR-resistant subline. MPR was incorporated into DNA of L1210/0 cells as 6-thioguanine deoxyribonucleotide whilst bis(MPR)P was not incorporated into L1210/MPR cell DNA. These results suggested that the ultimate mechanisms of action of bis(MPR)P and bis(dibutyrylMPR)P in L1210/ MPR cells may have been different from that of MPR in sensitive L1210/0 cells and therefore might not represent true circumvention of resistance to MPR.     

Calcium binding properties of rat heart plasma membrane and inhibition by structural analogues of dl-propranolol

The isolation and characterization of a plasma membrane preparation from rat heart is described. Enzymatic, chemical, and electron microscopic analysis revealed a relative lack of contamination with nuclear, mitochondrial, ribosomal, and sarcoplasmic reticulum membrane. One calcium binding site (K_d) = 265 μM, B_max = 65 $\text{nmoles}\text{mg}$ protein) was detected by equilibrium dialysis. Monovalent metal ions exhibited 10–100-fold less inhibition potency than divalent metal ions when analyzed by competitive inhibition of calcium binding. The range of K_i values found for divalent metal ions was similar to the K_d value for calcium. La⁺³ produced a potent non-competitive inhibition. A large variety of structural analogues of d,l-propranolol, many of which have been shown to lack β-adrenergic blocking activity, were competitive inhibitors of the calcium binding activity, with K_i values ranging from 40–900 μM. Electrophilic, hydrophobic, and diamino substituents greatly increased the inhibitory activity. There was no significant difference between related tertiary and quaternary amines. The experimental antiarrhythmic agent UM 272 had the least ability to inhibit calcium binding to the cardiac plasma membrane preparation (K_i = 795 μM). However, UM 424, another experimental antiarrhythmic agent, had an inhibitory activity similar to dl-propranolol (k_i = 115 μM and 108 μM, respectively).     

Antiviral, antitumor, and thymidylate synthetase inhibition studies of 5-substituted styryl derivatives of 2'-deoxyuridine and their 5'-phosphates

2′-Deoxyuridine derivatives containing styryl, 3-nitrostyryl, 4-nitrostyryl, and phenylethyl groups substituted at the 5-position of the pyrimidine ring have been evaluated for their effects on vaccinia and herpes simplex virus replication (in primary rabbit kidney cell cultures) and mouse leukemia L-1210 cell culture growth. 5-Phenylethyl-2′-deoxyuridine inhibited herpes simplex (type 1 and 2) virus-induced cytopathogenicity by 50 per cent at a dose (id₅₀) of 10–30 μg/ml. It was inactive against tumor cell growth. The corresponding styryl derivative showed an id₅₀ of 30–70 μg/ml for herpes simplex virus, 20 μg/ml for vaccinia virus, and 280 μg/ml for L-1210 cell growth. 5(E)-(3-Azidostyryl)-2′-deoxyuridine 5′-phosphate inhibited vaccinia replication with an IC₅₀ of 20 μg/ml and L-1210 cell culture growth with an id₅₀ of 80 μg/ml. The nucleotides of these compounds were all potent reversible inhibitors of thymidylate synthetase (Lactobacillus casei) with the following $\text{K}{}_{\mathrm{i}}\text{K}{}_{\mathrm{m}}$ ratios: 3-nitrostyryl, 0.035; 4-nitrostyryl, 0.05; 3-azidostyryl, 0.06; styryl, 0.08; and phenylethyl, 0.31. The photodecomposition of the azidostyryl derivative, a photoaffinity labeling reagent for thymidylate synthetase, was examined at two wavelengths.     

Inhibition of gastric K+ ATPase by phenylbutazone and indomethacin.

Inhibition of gastric H⁺ secretion by phenylbutazone and indomethacin was investigated by examining the effects of these agents on a putative H⁺ transport enzyme, a K⁺ stimulated ATPase. unique to gastric mucosa. Phenylbutazone and indomethacin were found to inhibit both the K⁺ ATPase and the K⁺ pNPPase. K_is were 430 μM and 710 μM for the K⁺ ATPase and 330 μM and 670 μM for the K⁺ pNPPase for phenylbutazone and indomethacin respectively. Inhibition was not reversed by Mg²⁺, ATP, pNPP, or KCl and obeyed non-competitive kinetics. Inhibition of the pNPPase suggested that the mechanism of inhibition involved the K⁺ sensitive dephosphorylation of the phosphoenzyme. In the presence of 500 μM phenylbutazone dephosphorylation was significantly less at 3, 5, 7.5, 10 and 15 sec following KCl addition. These studies provide an alternate mechanism for inhibition of gastric H⁺ secretion by phenylbutazone and indomethacin.     

Inhibition of cyclic AMP phosphodiesterase by 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylic acid 3-[2-(N-benzyl-N-methylamino)] ethyl ester 5-methyl ester hydrochloride (YC-93), a potent vasodilator.

A new, potent vasodilator (YC-93), 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylic acid 3-[2-(N-benzyl-N-methyl amino)] ethyl ester 5-methyl ester hydrochloride, competitively inhibited cyclic adenosine 3′,5′-monophosphate (cyclic AMP) phosphodiesterase in the 105,000 g supernatant solutions from canine basilar, carotid, coronary and femoral arteries. The inhibition constant (K_i) of YC-93 for these enzyme preparations was in the range of 2.0–4.3 μM at substrate concentrations near the low K_m (about 1 μM for each enzyme preparation), and was 4.0–12 μM at substrate concentrations near the high K_m (50–70 μM). The potency of YC-93 for inhibition of coronary phosphodiesterase at 1 μM cyclic AMP and 50 μM cyclic AMP was much greater than that of papaverine and 3-isobutyl-l-methyl xanthine (IBMX). Commercially available cyclic AMP phosphodiesterase purified from beef heart was also strongly inhibited by YC-93 in a competitive manner and its K_i value was 2.0 μM in the wide range of substrate concentrations tested. Studies on the structure-activity relationship using low K_m phosphodiesterase from canine coronary artery and high K_m phosphodiesterase from beef heart, demonstrated that 3,5-diethoxycarbonyl-1,4-dihydro-2, 4,6-collidine, the simplest 1,4-dihydropyridine derivative (tested in the present studies) resulted in slightly less inhibition than papaverine, and the inhibitory potency of the former compound was greatly increased mainly by two structural modifications. Firstly, addition of a nitrophenyl group at position 4 of the 1,4-dihydropyridine ring, secondly, the replacement of ethylester at position 3 of the 1,4-dihydro-pyridine ring by N-benzyl-N-methylaminoethyl ester. A few dihydropyridine derivatives together with YC-93 were the most potent inhibitors of cyclic AMP phosphodiesterase among the compounds tested. The finding that the level of cyclic AMP in canine arterial strips was increased by 64 per cent (P < 0.01) even after 1 min exposure to 1 μM YC-93 supports the possibility of at least a partial involvement of phosphodiesterase inhibition in vasodilation by the drug.     

Characteristics of membrane transport of methotrexate by cultured human breast cancer cells

Methotrexate, a folic acid analogue, enters cells using a high-affinity carrier system that is shared with naturally occurring reduced folates. Methotrexate transport by MCF-7 cells, a hormonally responsive line of human breast cancer cells, exhibited a high-affinity carrier system that displayed Michaelis-Menten kinetics [K_m = 8.22 ± 0.62 μM; V_max = 12.22 ± 2.8 nmoles · min^?1·(g cell protein)^?1] was competitively inhibited by leucovorin and aminopterin, but not by folic acid; and was temperature-sensitive (Q₁₀ = 2.25 ± 0.32). Initial uptake rates were not affected by ouabain or sodium azide, but efflux of intracellular drug was inhibited markedly by sodium azide, suggesting the presence of an energy-dependent active efflux mechanism. At extracellular methotrexate concentrations in excess of 10 μM, a second, low-affinity uptake component could be identified that may represent a lower affinity membrane carrier or passive diffusion. Examination of hormonal influences on methotrexate transport revealed that growth of MCF-7 cells in serum-free medium induced a significant increase in the transport K_m value (15.93 ± 1.6 μM) compared to the K_m of 8.22 μM for cells grown in fetal calf serum. This change in affinity of the transport carrier could be reversed by the additon of insulin, but not of estradiol, to the culture medium. Methotrexate transport by human breast-cancer cells displayed characteristics that were similar to those of transport reported for human leukemia cells but that have not been documented previously for cells derived from a human solid tumor. In addition, the drug transport carrier was subject to modulation by insulin but not by estrogen.     

Inhibition of buttermilk xanthine oxidase by folate analogues and derivatives

Folic acid has been reported recently to be an effective agent for the treatment of hyperuricernia, although conflicting data exist. The relative inhibitory activities of this compound and its breakdown products, pterin aldehyde and 6-hydroxymethylpterin, for the enzyme xanthine oxidase have not been clear. In this study, folic acid purified from these two compounds competitively inhibited buttermilk xanthine oxidase under aerobic conditions by a mechanism kinetically distinct from that of pterin aldehyde, with an inhibition constant (K_i) of 0.12 μM. Methotrexate, leucovorin and N⁵-methylH₄folate were competitive inhibitors of the enzyme with K_i values ranging from 12 to 53 μM. MethyleneH⁴folate, H₂folate and H₄folate did not inhibit xanthine oxidase. N⁵-MethylH₄folate could not be evaluated by the reduction of cytochrome c because of the nonenzymatic oxidation of this folate derivative by cytochrome c to a compound, shown to be N⁵-methylH₂folate. Unless high intracellular concentrations of unchanged folic acid, pterin aldehyde or hydroxymethylpterin can be achieved or folic acid proves to be a more effective inhibitor of reduced than of oxidized enzyme, it is unlikely that this compound will be an effective clinical agent for the inhibition of xanthine oxidase.     

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.     

Inhibition of mushroom tyrosinases by lamprene and thiambutosine

The inhibition by lamprene of the oxidation of l-dopa, catalysed by tyrosinase from the wild mushroom, Xerocomus badius, was of the mixed type with a K_i, of 30 μM. The inhibition by thiambutosine of the tyrosinase from the cultivated mushroom, Agaricus campestris, was competitive with a K_i of 15 μM.     

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.