Ribonucleoside diphosphate reductase inhibition

Inhibition of ribonucleoside diphosphate reductase by hydroxyurea. Cancer Res 1968 28 1559-1565. 

The primary action is inhibition of enzyme ribonucleoside diphosphate reductase. The drug is specific for S phase of the cell cycle and causes cell to arrest at the Gj-S interface. 

Hydroxyurea suppresses DNA synthesis by inhibiting ribonucleoside diphosphate reductase, which catalyzes the reduction of ribonucleotides to deoxyribonucleotides. Hydroxyurea is used in chronic cases of granulocytic leukemia that are unresponsive to busulfan. In addition, it is used for acute lymphoblastic leukemia. Hydroxyurea may cause bone marrow depression. 

The control of ribonucleotide reductase activity is affected in the classic feedback fashion by cellular nucleotide concentrations. dATP inhibits the reduction of all four ribonucleoside diphosphates. dTTP inhibits the reduction of only CDP and UDP. ATP is the positive effector for the reduction of these two nucleotides, and both dTTP and dGTP stimulate the reduction of GDP and ADP. Hydroxyurea, an antitumor agent, inhibits ribonucleotide reductase, and this depletes the deoxyribonucleotide supply required for tumor DNA biosynthesis. 

When the nitrate ester 277 was treated with BuaSnD and AIBN, the deuteriated alcohol 278 was formed, by 1,5-radical translocation. However, the 2 -chloro-2 -deoxy-system 279 gave the furanone 280 under the same conditions. These results suggest that the inhibition of ribonucleoside diphosphate reductase by 2 -chloro-2 -deoxynucleosides involves loss of Cl from a radical at C-3. Some related studies from the same team, also supporting this conclusion, were mentioned earlier. ... 

Two of the derivatives (i.e., those containing 5-methylamino and 5-ethyl-amino groups) demonstrated impressive antitumour activity against Sarcoma 180 ascites cells and several others were potent inhibitors of ribonucleoside diphosphate reductase, requiring concentrations in the range of 10" to 10" M for 50% inhibition [43], 5-Methylamino-l-formylisoquinoline thiosemicarbazone, which was the most effective of the newly synthesized compounds required a concentration of 3 10" M for 50% inhibition of reductase activity and increased the life span of tumour-bearing mice over untreated animals by a factor of 2.5 at the optimal daily dose. 

The a-(AO-heterocyclic carboxaldehyde thiosemicarbazones are primarily inhibitors of the synthesis of DNA in neoplastic cells [57,70] therefore, they exert their effect in the S phase of the cell cycle [71,72]. Inhibition of the biosynthesis of RNA and protein is also produced by agents of this class however, these metabolic processes are considerably less sensitive than is the replication of DNA [57,70,73-75]. Interference with the biosynthesis of DNA by these agents was shown to be due to inhibition of the enzyme ribonucleoside diphosphate reductase [70,73,74,76]. The a-(AO-heterocyclic carboxaldehyde thiosemicarbazones constitute, as a class, the most potent known inhibitors of ribonucleoside diphosphate reductase, being 80-5000 times more effective than the classical inhibitor of this enzyme, hydroxyurea [for appropriate references see 77]. 

The kinetic mechanism of inhibition of ribonucleoside diphosphate reductase by a-(AO-heterocyclic carboxaldehyde thiosemicarbazones is not clear. The concentrations of the nucleoside diphosphate substrate, the allosteric activator ATP, or magnesium ion do not influence the inhibition of the enzyme produced by all of the thiosemicarbazones tested to date. Interesting differences exist, however, between the ring hydroxylated and nonhydroxylated a-(AO-heterocyclic carbox-... 

The hydroxylated derivatives, 5-hydroxy-2-formylpyridine thiosemicarbazone and 3-hydroxy-2-formylpyridine thiosemicarbazone, show a different pattern of inhibition [74]. They appear competitive with iron and either non-competitive or uncompetitive with the dithiol substrate the imprecise nature of the assay does not allow a choice between these alternatives. The failure of the dithiol to reverse inhibition of ribonucleoside diphosphate reductase by the hydroxylated derivatives implies that the interaction of these inhibitors with the enzyme occurs at a site different from that involved in the action of (4) and (1). The impure nature of this complex enzyme system, however, makes it impossible to explain fully these differences and further advances will require the availability of a highly purified enzyme. 

Some of the structural features required for inhibition of ribonucleoside diphosphate reductase have been determined [30]. These studies suggested that position 6 of (4) and position 3 of compound (1) are equivalent with respect to orientation of the inhibitor at the enzymatic binding site and that little or no tolerance exists for modification at this position. In addition, substitution of the terminal amino group of the thiosemicarbazone side chain decreased enzyme inhibition, suggesting the presence of a low bulk tolerance zone in this position [23]. The results also indicated that the isoquinoline derivative (1), which can be visualized as a pyridine derivative with a benzene ring fused across the 3- and 4-positions, is about 6-fold more potent as an inhibitor of the enzyme than is (4). Likewise, introduction of a Me group on the pyridine ring of (4) at either the 3-, 4-, or 5-positions resulted in derivatives that were better inhibitors of ribo-... 

The primary lesion created in cells by the heterocyclic carboxaldehyde thiosemicarbazones is interference with the biosynthesis of DNA, and this action is primarily due to the potent inhibition of ri-bonucleoside diphosphate reductase activity. 5-Hydroxypicolinaldehyde thiosemicarbazone (NSC-107392) inhibits ribonucleoside diphosphate reductase by the chelation of iron. Krakoff et al. (1974) tried the drug by intravenous injection to 30 patients with leukaemia or various solid tumours. Marked haemolysis, iron chelation, and urinary excretion of iron occurred. 

Earlier data and those derived from these inhibition studies can be summarized in a model of ribonucleoside diphosphate reductase in which the active site is formed both from B1 and B2. It contains active dithiols contributed by Bl and a free radical contributed by B2. The active dithiols donate the electrons required for ribonucleotide reduction while participating in catalysis the function and nature of the free radical remain unknown. 

Studies conducted on the inhibition of ribonucleoside diphosphate reductase by the preformed iron chelate of 1-formylisoquinoline thiosemicarbazone (1) [(Fe)IQ-1 ] have shown that under appropriate conditions (Fe)IQ-l was essentially equal to (1) as an inhibitor of enzymatic activity [78]. At a concentration of (Fe)IQ-l which inhibited enzymatic activity by 73% in the absence of added Fe, only 30% inhibition was observed when Fe was added to the preparation. In contrast, (1) decreased enzyme activity 65% in the presence of Fe but only 15% without Fe. The findings imply that Fe(IQ-l) is the active form of the inhibitor and stress further complexities in the mode of inhibition. 

Anticancer examples, Thiosemicarbazones have been mentioned above in connexion with their antiviral and antitubercular properties. They also have anticancer activity, as already mentioned for 5-hydroxypicolinic aldehyde thiosemicarbazone (4.18) in Section 4.0. Such substances powerfully inhibit ribonucleoside diphosphate reductase, the enzyme which converts ribo- to deoxyribo- nucleotides, leading to inhibition of DNA synthesis. Interaction with the enzymatically-required iron by these drugs in thought to lie at the root of their action (Agrawal, et al,y 1972). 

Inhibition of DNA synthesis is brought about by the action of dTTP as an allosteric inhibitor of ribonucleotide reductase (Reichard et al., 1961 Moore and Hurlbert, 1966 Brown and Reichard, 1969 Rummer et al., 1978). This enzyme is responsible for reducing all four ribonucleoside diphosphates (NDP) to the corresponding de-oxyribonucleoside diphosphates (dNDP). It is subject to a complex allosteric control which has been most studied with the bacterial enzyme. Most studies with the mammalian enzyme show it to be similar to the bacterial enzyme (Fig.11.7). 

The partly purified reductase from the Novikoflf tumor is specific for ribonucleoside diphosphates, and a single enzyme reduces CDP, UDP, ADP, and GDP. In analogy with the E. coli enzyme, the tumor reductase appears to require nonheme iron, as indicated by stimulation of reductase activity with iron salts and inhibition with iron-chelating agents. The purified reductase requires either a reductant such as dihydrolipoate or dithiothreitol, or an NADP-linked hydrogen transport system the E. coli thioredoxin-thioredoxin reductase couple will link the tumor reductase with NADPH. There appears to be a thioredoxin-like hydrogen transport system in the tumor and in rat liver 32) that links nucleotide reduction with NADPH. 

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