Ribonucleotide reductase enzyme-activated inhibitors

Mimosine (9) is an agonist in folate metabolism and suggested to be an inhibitor toward the iron-containing ribonucleotide reductase, the activity of the transcription of the cytoplasmic serine hydroxymethyltransferase gene (shmtl ) and the copper enzymes... 

HU is an inhibitor of ribonucleotide reductase, a rate-limiting enzyme which catalyzes the conversion of ribonucleotides into deoxyribonucleotides. HU is thus a cytotoxic agent as it has the ability to inhibit DNA synthesis. Consequently, H U can affect only cells that are actively synthesizing DNA and, therefore, a drug of S-phase cell-cycle specific. Moreover, HU-mediated inhibition of ribonucleotide reductase is reversible, implying that the action of HU will exhibit a relatively straight forward concentration-time course dependence [2—4-]. 

The enzyme contains two catalytic sites, two regulatory sites and two specificity sites. The catalytic site binds the substrates, thioredoxin (reduced by NADPH + H+) and the nucleoside diphosphates. The allosteric regulatory site binds ATP as an activator in competition with dATP as an inhibitor. The specificity site binds dGTP, dTTP and dATP but not dCTP and modulates ribonucleotide reductase activity selectively for the four NDP substrates to balance the four dNTP pools. 

Peptide inhibitors or peptidomimetics (type 4) may represent a totally new type of drug with species-specific cytostatic activity 113). For HSVl ribonucleotide reductase, it was first shown that a short peptide (nine amino acid residues) with the same sequence as the C-terminal end in protein R2 inhibits the enzyme activity, probably by competitive... 

R1. It contains the active site. The two subunits make up the small subunit of the protein called R2, which contains the free radical. A clue to the mechanism of action of the enzyme (tyrosine free radical) is shown in Figure 22.14. Hydroxyurea, an inhibitor of ribonucleotide reductase, destroys the free radical. 

Hydroxyurea is an inhibitor of the enzyme ribonucleotide reductase. Hydroxurea s mechnism of inhibition involves destruction of the free radical in the enzyme that is essential for its activity (Figure 21.15). 

An ADA-resistant purine analog, cladribine (2-chlorodeoxyadenosine 2-CdA) has demonstrated potent activity in hairy cell leukemia, CLL, and low-grade lymphomas. After intracellular phosphorylation by deoxycytidine kinase and conversion to cladribine triphosphate, it is incorporated into DNA. It produces DNA strand breaks and depletion of NAD and adenosine triphosphate (ATP), as well as apoptosis, and is a potent inhibitor of ribonucleotide reductase. The drug does not require cell division to be cytotoxic. Resistance is associated with loss of the activating enzyme, deoxycytidine kinase, or escape of ribonucleotide reductase from inhibition. 

The topology of the radical site pocket of calf thymus and mouse fibroblast ribonucleotide reductase was recently probed with a series of hydroxamate inhibitors of increasing bulkiness and will be discussed in the following section. Other mammalian sources from which ribonucleotide reductases have been isolated and more or less purified include rat Novikoff hepatoma and regenerating rat liver" rabbit bone mar-row 5,66) Ejjj-iich ascites tumor cells of mice and cultured human lymphoblast cells Some of their properties are described in Table 2. Many more animal and human cells were assayed for enzyme activity, frequently in mutant cell lines, to test for cell cycle dependence, mechanisms of metabolic regulation, drug resistance, and correlation with tumor growth rates. Representative studies of this kind, which rapidly expand in number, are summarized in Table 3. 

In HeLa ceils hydroxyurea is an efficient inhibitor of histone synthesis. This action requires protein synthesis and leads to rapid disappearance of cytoplasmatic histone mRNA The effect is not specific for hydroxyurea since suppression of DNA synthesis by arabino-cytosine or temperature-sensitive mutations yields analogous results. Similarly, the synthesis of some enzymes necessary for DNA replication and active in S-phase is altered by hydroxyurea. Increased activity of ribonucleotide reductase in HeLa and in hamster cells and of the salvage enzyme thymidine kinase in HeLa cells and KB cells has been observed, probably as a consequence of the increased fraction of cells in S-phase. Repression occurs for thymidine kinase in human lymphocytes and for ornithine decarboxylase in Chinese hamster fibroblasts whereas no or only slight effects were seen on ribonucleotide reductase in hamster fibroblasts , on thymidylate synthase in extracts from synchronous mouse cells " , and on DNA polymerase in rabbit kidney cells or HeLa cells . ... 

The activity of the proteinaceous inhibitors isolated from animal cells is inversely correlated with the proliferation rate of their source. Only recently was such an inhibitor characterized as complex mixture of enzymes like phosphatases and kinases that act on the substrate CDP and on the effector ATP The assumption is justified that this class of substances merely interferes with the assay and does not define specific cellular inhibitors of ribonucleotide reductase. Nevertheless it must be realized that ribonucleotide reductase activity is well coordinated with cell proliferation and is strictly controlled within the cell cycle. Hence a regulatory role of inhibitors besides the control of gene expression and fine tuning by allosteric effectors is a priori conceivable. 

The initiation or arrest of enzyme synthesis responsible for the peak of ribonucleotide reductase is discussed below. Other factors which may modulate the activity in vivo or when measured in crude homogenates include allosteric control by the endogenous deoxyribonucleotides, the action of late S phase-specific inhibitors like the one found in Achlya, or the redox status of thioredoxin and glutaredoxin however the cell cycle dependence of these reactions is little known. It is therefore desirable to assay ribonucleotide reductase in preparations which have been subjected to at least one precipitation and dialysis step. While reliable cell cycle-dependent activity data are thus obtained, the absolute figures are frequently an order of magnitude too low to account for the cell s need of ribonucleotide reduction for DNA synthesis. This unsatisfactory condition is most likely a problem of quinary enzyme structure (see below) but is not felt to invalidate the accumulated evidence for tight correlation of ribonucleotide reduction and the cell cycle as a whole. 

Leishmania adenylosuccinate synthetase has a narrow substrate specificity but accepts several IMP analogs which include allopurinol ribonucleotide (34). The GMP reductase from L. donovani is quite different from the human GMP reductase (35) and IMP analogs are more potent inhibitors for it. Other leishmanial enzymes that have been investigated include IMP dehydrogenase (36), nucleoside hydrolase and phos-phorylase activities (37,38), adenosine kinase (39), nucleotidases (40) and the adenylosuccinate lyase (34). 

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