Inhibition of ribonucleotide reductase and growth of human colon carcinoma HT-29 cells and mouse leukemia L1210 cells by N-hydroxy-N'-aminoguanidine derivatives

A series of N-hydroxy-N'-aminoguanidine (HAG) derivatives were studied and compared for their effects on ribonucleotide reductase activity in cell-free extracts; on nucleic acid synthesis and the growth of human colon carcinoma HT-29 cells; and on mouse leukemia L1210 cells in culture. The HAG derivatives [RCH=NNHC(=NH)NHOH-tosylate] studied could be grouped as: (1) hydroxybenzylidines; (2) methoxybenzylidines; and (3) nitrobenzylidines substituted at the R position. 2'-Hydroxybenzylidine-HAG, the lead compound, was relatively active in both HT-29 cells and L1210 cells (20 +/- 5 and 13 +/- 4 microM for 50% inhibition of HT-29 and L1210 cell growth respectively). The monohydroxybenzylidene compounds were generally more active than the dihydroxy- and trihydroxybenzylidene-HAG derivatives. The methoxybenzylidene-HAGs were as active as the monohydroxybenzylidene-HAGs. 2'-Hydroxy-4'-methoxybenzylidene-HAG was much more active than 2',4'-dihydroxybenzylidene-HAG. The mononitrobenzylidene-HAGs were more active than the dinitrobenzylidene-HAG compound. In general, L1210 cells were more sensitive to the effects of the HAG compounds than were HT-29 cells. There was good agreement between the concentration of drug required to inhibit the growth of HT-29 cells and that required to inhibit the growth of L1210 cells. There was also good correlation between the ability of HAG derivatives to inhibit ribonucleotide reductase activity and to inhibit tumor cell growth. Some derivatives, such as 2',3',4'- and 3',4',5'-trihydroxybenzylidene-HAG inhibited L1210 cell growth by 50% at lower concentrations (7.8 and 11.9 microM respectively) than the concentrations needed for 50% inhibition of HT-29 cell growth (196 and 234 microM respectively) and ribonucleotide reductase activity (122 and 188 microM respectively). The studies of nucleic acid synthesis in L1210 cells using [3H]cytidine as a precursor showed that 2',3',4'-trihydroxybenzylidine-HAG inhibited DNA synthesis at a lower concentration (29 microM for 50% inhibition) than was needed for the inhibition of RNA synthesis and formation of [3H]deoxycytidine nucleotides in the acid-soluble fraction (320 and 820 microM for 50% inhibition respectively). These results indicate that 2',3',4'-trihydroxybenzylidine-HAG inhibits DNA synthesis in L1210 cells through other mechanisms rather than exclusively through the inhibition of ribonucleotide reductase activity.     

Properties of N-hydroxy-N'-aminoguanidine derivatives as inhibitors of mammalian ribonucleotide reductase

In previous studies, N-hydroxy-N'-aminoguanidine (HAG) derivatives were demonstrated to suppress growth and clonogenicity of tumor cells which correlated with the inhibition of ribonucleotide reductase and DNA synthesis. The present work has focused on the properties of five HAG derivatives as inhibitors of the ribonucleotide reductase from Ehrlich ascites tumor cells. HAG derivatives acted as non-competitive inhibitors of ribonucleotide reductase with respect to the substrates CDP and ADP. The apparent Ki values for the various HAG derivatives as inhibitors of CDP reductase ranged from 3.4 to 543 microM. However, the apparent Ki values for these inhibitors with respect to ADP reductase were 2- to 10-fold lower than the respective values for CDP reductase. After a preincubation of HAG derivatives and ribonucleotide reductase in the absence of substrates, an increased inhibition was observed. The activity of the inhibited enzyme could be restored by passage over a Sephadex G-25 column and subsequent incubation with dithioerythritol. The addition of either the non-heme iron subunit or the effector-binding subunit to the intact enzyme in the assay mixture resulted in a diminished inhibition of ADP reduction. Inhibition by HAG derivatives of ribonucleotide reductase activity in the test tube was not enhanced by iron chelators. However, a combination of HAG compounds and iron chelators synergistically inhibited the growth of L1210 cells.     

Inhibition of human leukaemic thymidylate kinase and L1210 ribonucleotide reductase by dinucleotides of adenosine and thymidine and their phosphonate analogues

Dinucleotides of adenosine and thymidine in the ApnT series (n = 3,4,5 and 6) and their corresponding phosphonate analogues, where a methylene group replaces the oxygen between the alpha and beta phosphorus atoms adjacent to thymidine, have been evaluated as inhibitors of human leukaemic thymidylate kinase (dTMP kinase, EC 2.7.4.9) and ribonucleotide reductase (EC 1.17.4.1) from L1210 cells. Ap3T, Ap4T, Ap2cpT and Ap3cpT were poor inhibitors of both enzymes. Ap5T, Ap6T and their phosphonate analogues were potent inhibitors of dTMP kinase, possibly acting as bisubstrate analogues (IC50 values: Ap5T, 7.9 microM; Ap4cpT, 5.8 microM; Ap6T, 5.4 microM; Ap4cpT, 4.0 microM). For CDP reductase, where these compounds may bridge activity/effector sites on the M1 subunit of the enzyme, Ap5T and Ap6T were inhibitors with IC50 values of 14.4 microM and 20.3 microM respectively. The phosphonate series of compounds was far less active. The thymidine moiety of the compounds was essential for inhibition since Ap5A was inactive against both enzymes. dTTP, although a poor inhibitor of thymidylate kinase was a potent negative effector of CDP reductase (IC50, 19.3 microM). Significantly, Ap5T was not hydrolysed to release dTTP under the conditions of the assay. These studies show that the activities of both enzymes may be modulated by nucleotide analogues.     

Dopamine inhibits cell growth and cell cycle by blocking ribonucleotide reductase

Dopamine (DA) is a classical neurotransmitter modulating various brain functions by acting on its specific receptors. In addition, DA is a reactive molecule that has been implicated in neurodegeneration, especially in Parkinson's disease. Here we show that DA inhibited cell growth of dopamine transporter transfected cells by intracellularly blocking cell cycle progression. To pinpoint the site of this effect, we measured DNA distribution and 5-bromo-2'-deoxyuridine (BrdU) incorporation, as well as the levels of the key cell cycle proteins. DA increased number of cells with a G1 DNA content, decreased BrdU incorporation and simultaneously increased cyclin A but had no effect on cyclin D2, D3, E, nor on cdk4 and p21. These results narrowed down the DA effect to the beginning of S phase, suggesting inhibition of the ribonucleotide reductase, an enzyme essential for DNA synthesis. Indeed, measurement of enzyme activity in situ revealed that DA, within 1h of addition to cells labelled with [3H]cytidine, strongly reduced the cell content of [3H]2'-deoxycytidine 5'-triphophate. The time course of this DA effect preceded the cell cycle progression. This novel molecular mechanism of intracellular DA action independent of plasmamembrane receptors may be involved in processes controlling the development and survival of brain dopaminergic neurons.     

In vivo growth of mouse leukemia L1210 cells with metabolic alterations in the subunits of ribonucleotide reductase

Mouse leukemia L1210 cells selected for resistance to drugs targeted specifically at each of the protein subunits of ribonucleotide reductase were studied for their ability to grow in vivo. The life-span of the mice injected with hydroxyurea-resistant L1210 cells, which have elevated levels of the mRNA and protein for the non-heme iron (NHI, R2) subunit of ribonucleotide reductase, was approximately twice that of the mice injected with equal numbers of the parental wild-type L1210 leukemia cells. The life-span of mice injected with the L1210 cells that had alterations in the effector-binding subunit (EB, R1) was considerably shorter than the mice injected with the parental wild-type L1210 cells. These results provide direct evidence that tumor cells with alterations in the properties of ribonucleotide reductase grow differently in vivo, with defined effects on the host mouse that cause either an increased survival time or a decreased survival time compared to the effects of wild-type L1210 leukemia cells on tumor-bearing mice.     

Inhibitory activity of diarylamidine derivatives on murine leukemia L1210 cell growth

Summary A series of 96 diarylamidine (and diarylimidazoline) derivatives were evaluated for their inhibitory effects on the growth and DNA synthesis of murine leukemia L1210 cells. The amidino- and imidazolino-substituted aryl moieties of the compounds consisted of phenyl, indole, indene, benzofuran, benzo[b]thiophene or benzimidazole. Several of these compounds were found to inhibit L1210 cell proliferation with an ID₅₀ (50% inhibitory dose) of 1 g/ml or lower. Structure-function analysis revealed that the antitumor cell activity of the diarylamidines depended on the planarity of the molecule, the presence of amidino- (or, preferably, imidazolino-) groups on both aryl moieties, the nature of the bridge connecting the two aryl moieties (preferably no bridge at all, phenoxy or ethene) and, finally, the nature of the aryl moieties (preferably, benzofuran or benzo[b]thiophene). Hence, compound 20 (6-(2-imidazolin-2-yl)-2-[4-(2-imidazolin-2-yl)phenyl] benzo[b]thiophene) emerged as the most potent inhibitor of L1210 cell growth (ID₅₀: 0.21 g/ml). Its inhibitory potency was similar to that of the well-known trypanocidal drug ethidium bromide (compound 98). For all diarylamidine derivatives taken together, some correlation (r = 0.612) was noted between the log ID₅₀ for L1210 cell proliferation and the log ID₅₀ for L1210 cell DNA synthesis (as monitored by [methyl ³H]dThd incorporation). These findings suggest that the inhibitory effects of the diarylamidines on L1210 cell proliferation may at least partially reside in an inhibition of DNA synthesis. Compound 41 (2,2-vinylenedi-1-benzofuran-5-carboxamidine), that exhibited a potent antitumor activity in vitro (ID₅₀: 1.5 g/ml), was further evaluated for its antitumor efficacy in vivo and found to increase the median survival time of L1210 cell-inoculated BDF₁ mice up to 204%, if administered at a dose of 200 mg/kg.     

Inhibition of ribonucleotide reductase by antitumor agents related to levodopa and dopamine

Using partially purified enzyme from L1210 cells, dihydroxybenzene derivatives related structurally to dopamine were shown to reversibly inactivate ribonucleotide reductase. A structure-activity analysis revealed that derivatives with side-chains, which contain a negatively-charged group, had significantly reduced inhibitory activity. The ability of these compounds to inhibit ribonucleotide reductase was dependent on the hydroxyl groups being in the ortho position and did not correlate with free radical inhibitory activity. A kinetic analysis by the method of Lineweaver-Burk indicated that the inhibition of ribonucleotide reductase by the derivative 3,4-dihydroxybenzylamine was competitive with the reducing substrate dithioerythritol. This analog, in combination with hydroxyurea, gave synergistic inhibition or ribonucleotide reductase, suggesting different sites of action. Using Tween 80-treated L1210 cells, it was found that these drugs had an immediate inhibitory effect on ribonucleotide reductase activity in intact, reversibly permeabilized cells. Furthermore, although these drugs had no immediate effect on DNA polymerase, in permeabilized L1210 cells (when the cells were preincubated with the dihydroxybenzene derivatives for 1 hr prior to permeabilization), there was significant inhibition of DNA polymerase activity. The two key enzymes for DNA synthesis appear to be sequentially inhibited by these analogs, with the reduced form (quinol) inhibiting ribonucleotide reductase and the oxidized form (quinone) inhibiting DNA polymerase.     

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.     

Inhibition of dihydrofolate reductase, methotrexate transport, and growth of methotrexate-sensitive and -resistant L1210 leukemia cells in vitro by 5-substituted 2,4-diaminoquinazolines

A series of eighteen 2,4-diaminoquinazoline analogues of folic, isofolic, pteroic and isopteroic acids having various substituents at position 5 was studied. Each compound was evaluated as an inhibitor of L1210 dihydrofolate reductase, methotrexate influx into L1210 leukemia cells, and growth of methotrexate-sensitive and -resistant L1210 cells in vitro. Bridge reversal at positions 9 and 10 reduced the effectiveness of the classical analogues only with regard to the inhibition of the drug-sensitive cells as compared to methotrexate (MTX). Absence of the glutamate moiety adversely affected the potency of the compounds, particularly when coupled with reversal of the 9,10-bridge. However, the presence of -Cl at position 5 restored significantly the potency of these compounds. The pteroate and isopteroate analogue ethyl esters were generally more effective inhibitors of cell growth than their non-esterified counterparts. Regarding the effects of substituents at position 5, the data suggest that -Cl greater than -CH3 greater than -H for inhibition of methotrexate transport and growth of methotrexate-sensitive L1210 cells. The 5-Cl pteroate analogue and its corresponding ethyl ester were highly effective as growth inhibitors of methotrexate-resistant, transport-defective, L1210 cells in vitro.     

Inhibition of cellular thioredoxin reductase by diaziquone and doxorubicin. Relationship to the inhibition of cell proliferation and decreased ribonucleotide reductase activity.

The flavoenzyme thioredoxin reductase (TR) is an important enzyme for many aspects of cellular function. The antitumor quinones diaziquone and doxorubicin have been shown to produce a time- and concentration-dependent inhibition of TR when incubated for up to 24 hr with intact A204 human rhabdomyosarcoma cells. There was a positive correlation between the inhibition of TR and the inhibition of cell colony formation measured 7 days later for diaziquone (r = 0.84, P less than 0.01), and for doxorubicin (r = 0.87, P less than 0.01). 2,6-Dichloroindophenol, which in previous studies was shown to be a good inhibitor of TR in vitro, was a poor inhibitor of TR in intact A204 cells and there was no significant correlation with inhibition of colony formation. The activity of ribonucleotide reductase, which catalyzes the first unique step of DNA synthesis and which obtains its reducing equivalents from TR through thioredoxin, was decreased in diaziquone- and doxorubicin treated A204 cells. We suggest that the inhibition of TR by some antitumor quinones leading to a decreased activity of TR and, consequently, a decreased activity of thioredoxin-dependent enzymes including ribonucleotide reductase may contribute to the growth inhibitory activity of these quinones.     

Inhibition of ribonucleotide reductase in thymocytes of rats treated by ditiocarb sodium.

The i.p. administration of ditiocarb sodium (diethyldithiocarbamate, DDC) at doses of 500 and 1000 mg/kg body wt. decreased within 1 h and in a competitive manner the ribonucleotide reductase (RNR) activity of rat thymocytes by about 25 and 40%, respectively. Under the same conditions, scheduled DNA synthesis was inhibited by almost 30 and 75%, whereas RNA synthesis remained unchanged. 1000 mg DDC/kg body wt. resulted in a long-lasting diminution in thymus weight; the spleen revealed no significant drug effects. It is suggested that the decrease of RNR activity in thymocytes of DDC-treated rats is a major determinant in the (reversible) inhibition of DNA synthesis which, in turn, may be implicated in the radio- and chemoprotective effects of the drug.     

Inhibition of initiation of translation in L1210 cells by 8-azaguanine

The inhibition of protein synthesis by 8-azaguanine (azaG) in L1210 cells in culture was investigated. AzaG selectively inhibited protein synthesis at concentrations where viability was decreased significantly. AzaG altered the polyribosome sedimentation profile, increasing the numbers of monosomes and smaller polysemes and decreasing the number of larger polysomes. The reversal by cycloheximide of the alterations in the polysome profile suggested that azaG inhibited the initiation of translation. This was confirmed by the demonstration of inhibition of the formation of the 43S and 80S initiation complexes.     

Potent ribonucleotide reductase inhibitors: Thiazole‐containing thiosemicarbazone derivatives

The antioxidant, antimalarial, antibacterial, and antitumor activities of thiosemicarbazones have made this class of compounds important for medicinal chemists. In addition, thiosemicarbazones are among the most potent and well‐known ribonucleotide reductase inhibitors. In this study, 24 new thiosemicarbazone derivatives were synthesized, and the structures and purity of the compounds were determined by IR, ¹H NMR, ¹³C NMR, mass spectroscopy, and elemental analysis. The IC₅₀ values of these 24 compounds were determined with an assay for ribonucleotide reductase inhibition. Compounds 19, 20 , and 24 inhibited ribonucleotide reductase enzyme activity at a higher level than metisazone as standard. The cytotoxic effects of these compounds were measured on the MCF7 (human breast adenocarcinoma) and HEK293 (human embryonic kidney) cell lines. Similarly, compounds 19, 20 , and 24 had a selective effect on the MCF7 and HEK293 cell lines, killing more cancer cells than cisplatin as standard. The compounds (especially 19, 20 , and 24 as the most active ones) were then subjected to docking experiments to identify the probable interactions between the ligands and the enzyme active site. The complex formation was shown qualitatively. The ADME (absorption, distribution, metabolism, and excretion) properties of the compounds were analyzed using in‐silico techniques.     

Structure-activity relationships of sparsomycin and its analogues. Inhibition of peptide bond formation in cell-free systems and of L1210 and bacterial cell growth

The biological activity of 14 analogues of sparsomycin (1) was studied in cell-free systems of Escherichia coli, Saccharomyces cerevisiae, and Sulfolobus solfataricus by measuring the inhibition of protein synthesis. The inhibition of L1210 colony formation in soft agar and bacterial cell growth in solid as well as in liquid medium was also examined. Each analogue possesses not more than two structural modifications of the sparsomycin molecule. This enabled us to determine unambiguously several structural and stereochemical features that are required for an optimal biological activity in these assays. Sparsomycin, having the SCRS chirality, is the most potent of the four possible stereoisomers. The results obtained with compounds 5-7 indicate that the presence of an oxygen atom on the S (alpha) atom is essential. Substitution of the bivalent sulfur atom by a CH2 group (10) or of the SCH3 moiety by a Cl atom (12) affects the activity of the molecule partially. Compound 12 is surprisingly active against intact cells. Substitution of the C(6)-CH3 group by a H(14) reduces the activity of the molecule. Isomerization of the trans double bond into the cis double bond yields cis-sparsomycin (15), which is inactive. The hydrophobic derivatives 8, 9, and 11 are considerably more active than sparsomycin; thus the ribosomal binding site for sparsomycin may have a hydrophobic character.     

Inhibition of HCV replicon cell growth by 2-arylbenzofuran derivatives isolated from Mori Cortex Radicis

Medicinal herbs are increasingly used in the search for safe and efficient drug candidates for hepatitis C virus infection. In this study, we have investigated the anti-HCV effect of compounds from Mori Cortex Radicis. During a screening for extracts with anti-HCV affinity from medicinal plants (173 species), the methanol extract of Mori Cortex Radicis was selected. Fractionation of the extract by monitoring antiviral activity with a replicon cell-based assay resulted in the isolation of five compounds, mulberroside C ( 1), moracin P ( 2), moracin O ( 3), moracin M ( 4) and mulberrofuran K ( 5) from the ethyl acetate-soluble fraction. Compounds 1 approximately 4 showed significant inhibitory activities. Compounds 2 and 3 showed potent inhibitory activity (IC (50) 35.6 microM, 80.8 microM, respectively) in the replicon cell assay, which was confirmed by NS3 helicase inhibitory activity (IC (50) 42.9 microM, 27.0 microM, respectively).     

Human ribonucleotide reductase. Activation and inhibition by analogs of ATP

Sixteen ATP analogs were studied as activators of CDP reduction catalyzed by human ribonucleotide reductase. Activation constants were determined. Three analogs, 3-deazaATP, 5'-adenylimidodiphosphate, and 3'-dATP, activated approximately as efficiently as ATP. Four analogs were partial activators. These seven activators were also accessory activators of GDP reduction. Furthermore, two other analogs, adenosine-5'-O-(1-thiotriphosphate) and 8-bromoATP, selectively stimulated GDP reduction. Ten analogs, at equal molar concentrations with ATP, inhibited ATP-dependent activation of CDP reduction and/or accessory activation of GDP reduction by greater than 45%. No analog inhibited as potently as 2'-dATP, which had an IC50 of 30-50 microM versus the stimulation of CDP and GDP reduction by 2.0 mM ATP.