p-Alkoxyphenols, a new class of inhibitors of mammalian R2 ribonucleotide reductase: possible candidates for antimelanotic drugs.

The inhibition by different p-alkoxyphenol derivatives of the growth-regulating enzyme ribonucleotide reductase (RR) in purified Escherichia coli and mouse R2 protein preparations was studied by EPR spectroscopy. The inhibitor-induced inactivation of the catalytic subunit protein R2 was measured at 77 degrees K by observing the decrease of the typical EPR signal from the functionally essential protein-linked tyrosyl free radical. p-Methoxy-, p-ethoxy-, p-propoxy-, and p-allyloxyphenol were about 2 orders of magnitude more effective in inhibiting mouse R2, compared with E. coli R2. Among the p-alkoxyphenols studied, p-propoxyphenol was the most effective inhibitor of mouse R2 (IC50, 0.7 microM) and p-methoxyphenol was the least effective (IC50, 11 microM); p-ethoxy- and p-allyloxyphenol were intermediate. The observed half-maximal inhibition values characterized p-alkoxyphenols as a new class of strong inhibitors of the R2 protein of mammalian RR. p-Propoxy-, p-ethoxy-, and p-allyloxyphenol could be considered as new candidates for anticancer drugs. A special cellular inhibition assay of RR in proliferating tumor cells, in which the tyrosyl radical of R2 at natural concentration was monitored by EPR, showed that the four para-substituted alkoxyphenols also inhibited the enzyme with high efficiency in tumor cells (IC50, between 0.5 microM and 5 microM). Our results with inactivation of protein R2 of RR imply that the cytostatic effect of p-alkoxyphenols on melanoma cells, which has been hitherto explained by inhibition of tyrosinase [Melanoma Res. 2:295-304 (1992)], may be caused at least partly by inhibition of RR. Protein R2 of RR may be considered as an additional target that could be used for future cancer chemotherapy.

ESR studies on reactivity of protein-derived tyrosyl radicals formed by prostaglandin H synthase and ribonucleotide reductase.

Using ESR spectroscopy, the ability of enzyme inhibitors to quench protein-derived tyrosyl radicals was studied in two different enzymes, prostaglandin H synthase and ribonucleotide reductase. The prostaglandin H synthase inhibitors indomethacin, eugenol, and MK-410 effectively prevent the formation of tyrosyl radicals during the oxidation of arachidonic acid by prostaglandin H synthase from ram seminal vesicles. A direct reaction with preformed tyrosyl radicals was observed only with eugenol. The other prostaglandin H synthase inhibitors were ineffective. The ribonucleotide reductase inhibitors hydroxyurea and 4-hydroxyanisole, which effectively inactivate the tyrosyl radical in the active site of ribonucleotide reductase present in tumor cells, exhibit a different reactivity with tyrosyl radicals formed by prostaglandin H synthase. Hydroxyurea quenches preformed tyrosyl radicals in prostaglandin H synthase weakly, whereas 4-hydroxyanisole does not quench tyrosyl radicals in prostaglandin H synthase at all. Eugenol, which quenches preformed prostaglandin H synthase-derived tyrosyl radicals, also quenches the tyrosyl radical in ribonucleotide reductase. The results suggest that the reactivity of protein-linked tyrosyl radicals in ribonucleotide reductase and those formed during prostaglandin H synthase catalysis are very different and have unrelated roles in enzyme catalysis.

Ribonucleotide reductase in melanoma tissue. EPR detection in human amelanotic melanoma and quenching of the tyrosine radical by 4-hydroxyanisole.

The characteristic EPR doublet of tyrosine radicals of the growth-regulating enzyme ribonucleotide reductase was detected in human melanoma tissue grown in nude mice. This was possible through the use of an amelanotic melanoma that does not exhibit disturbing EPR signals from melanin. The content of tyrosine radicals is higher in young tumor tissues than in older ones. The clinically applied antimelanotic drug, 4-hydroxyanisole, inhibits ribonucleotide reductase in Ehrlich ascites tumor cells as demonstrated by a pronounced quenching of tyrosine radicals (IC50 = 5 microM). In amelanotic melanoma tissue tyrosine radicals of the enzyme are also quenched by 4-hydroxyanisole in concentrations down to 50 microM. Thus, the inactivation of ribonucleotide reductase, which provides deoxyribonucleotides for DNA synthesis, may be a hitherto unexpected mechanism for the antitumor action of 4-hydroxyanisole.

Quenching of tyrosine radicals of M2 subunit from ribonucleotide reductase in tumor cells by different antitumor agents: an EPR study.

The inhibition of ribonucleotide reductase (RR) of intact Ehrlich ascites tumor cells by different antitumor agents was compared using EPR spectroscopy. The inactivation of M2 subunit was measured via quenching of the functionally essential tyrosine radical. Inhibitors of different classes, for example, hydroxyurea, pyrogallol, and thiosemicarbazones, differ in their efficiency by three orders of magnitude. Most effective inhibition was found for isoquinoline-1-aldehyde-thiosemicarbazone (IQ-1) with an IC50 value of 0.18 microM. Inhibition of RR inside tumor cells is comparable with that reported for isolated enzymes.

Stability and reactivation of tyrosine radicals from ribonucleotide reductase in tumor cells studied by ESR.

Naturally occurring tyrosine radicals from the M2 subunit of ribonucleotide reductase (RR) have been recorded by ESR in proliferating ordinary Ehrlich-ascites (EA) tumor cells of mice. Tyrosine radicals are stable in EA cells at room temperature for 2 h. Up to 500 mW no microwave saturation occurs. The relatively high stability and non-saturation of tyrosine radicals in EA cells suggests a suitable protein conformation in the M2 subunit enabling a close contact between the tyrosine radical and the antiferromagnetic iron complex. This facilitates an ESR study of functionally essential tyrosine radicals of RR in EA cells at low temperature and recommends this cellular system for studying such processes as inhibition and activation, which change the content of tyrosine radicals of the proliferation-linked RR. Oxygen treatment of non-proliferating (quiescent) EA cells reactivates tyrosine radicals 2-3 fold as found in strongly proliferating cells. We conclude that in quiescent cells, suffering from a lack of oxygen due to their high density in the peritoneal cavity, a reactivation of tyrosine radicals occurs by oxidation of non-radical tyrosine residues of inactive M2 subunits.

ESR studies of structure and kinetics of radicals from hydroxyurea. An antitumor drug directed against ribonucleotide reductase.

Hydroxyurea (HU) is a clinically applied antineoplastic drug, which quenches tyrosine radicals in the active site of ribonucleotide reductase (RR) and inhibits DNA synthesis in proliferating cells. Under oxidizing conditions (Cu2+ or H2O2) long-lived radicals from HU have been found by ESR. The structure of HU radicals was established to be: (formula; see text). The kinetics of formation and decay of HU radicals after reaction of HU with H2O2 is complex; it exhibits a lag-phase, a maximum, and a decay, all depending on the concentration of HU. Biological consequences of HU radicals for the inhibition of RR as well as their role in cytotoxic events during chemotherapy of cancer are discussed.

Ribonucleotide reductase in ascites tumour cells detected by electron paramagnetic resonance spectroscopy.

Tyrosine radicals localized in the M2 subunits of ribonucleotide reductase have been detected by electron paramagnetic resonance (EPR) in ordinary ascites tumour cells. The intensity of its doublet EPR spectrum is higher in rapidly proliferating cells. Hydroxyurea, a specific inhibitor of this enzyme, decreases the concentration of the tyrosine radical. Whereas in different ascites tumours the doublet EPR spectrum dominates at g = 2.004, in solid tumours another more intense EPR spectrum from nitrosyl-hemoproteins appears. In conclusion, EPR spectroscopy can be used to monitor the content and variations of active M2 subunits of ribonucleotide reductase in intact ascites tumour cells.

Human tissues degrade uridine much less than thymidine. Possible consequence for 5-fluorouracil therapy.

In view of clinical trials to improve FUra chemotherapy of cancer by combined application with Urd and dThd, we investigated the capacity of human tissues to split these nucleosides. All human normal and neoplastic tissues gave a uridine-splitting activity which can be inhibited by beta-L-pTdR and behaves in this respect as uridine-deoxyuridine phosphorylase (EC 2.4.2.3). dThd splitting, however, which is 2-9-fold higher than that of Urd, is insensitive towards beta-L-pTdR, confirming earlier results that it is due to thymidine phosphorylase (EC 2.4.2.4). On the other hand, tissues, e.g., spleen of rats and mice, in which dThd and Urd are split by uridine-deoxyuridine phosphorylase, degrade 2-5-fold more Urd than dThd. Thus, free pyrimidine base competing with FUra for degradation and thus prolonging the life time of the drug in the body, will be formed mainly from dThd in the human body but more so from Urd in the rat or mouse.

(E)-5-(2-Bromovinyl)-2'-deoxyuridine: a good substrate for mammalian pyrimidine nucleoside phosphorylases.

Specific antiherpetic thymidine analogues are split by mammalian pyrimidine nucleoside phosphorylases with the following order of activity: VUdR greater than BVUdR greater than thymidine greater than EtUdR.