Enzymology of purine and pyrimidine antimetabolites used in the treatment of cancer.

Inhibition of cellular replication is one characteristic of cancer cells that has been effectively exploited in the past for the development of anticancer agents. Most of the drugs currently used to kill cancer cells inhibit the synthesis of DNA or interfere with its function in one manner or another. For a cell to divide into two cells, it must replicate all components including its genome, and unlike the synthesis of other major macromolecules (protein, RNA, lipid, etc.), the synthesis of DNA does not occur to a great degree in quiescent cells. Since most cells in an adult organism are quiescent and are not in the process of duplicating their genome, targeting DNA replication affords some level of selectivity. Of course, certain tissues (bone marrow, gastrointestinal, hair follicles, etc.) are in a replicative state, and all cells must continually repair their DNA. Therefore, inhibition of DNA replication in normal tissues results in considerable toxicity that limits the amount of drug that can be tolerated by the patient. In spite of this problem, very effective anticancer drugs have been developed that increase survival and, in some cases, cure the patient of his or her disease. Human cells have the capacity to salvage purines and pyrimidines for the synthesis of deoxyribonucleotides that are used for DNA synthesis, and analogues of these nucleotide precursors have proven to be an important class of anticancer agents. There are a total of 14 purine and pyrimidine antimetabolites that are approved by the FDA for the treatment of cancer (Table 1), which account for nearly 20% of all drugs that are used to treat cancer. Some of the first compounds approved by the FDA for the treatment of cancer were in this class of compounds. 6-Mercaptopurine was approved in 1953 for the treatment of childhood leukemia, where it is curative and is still the standard of treatment for this disease. Since 1991, nine nucleoside analogues have been approved by the FDA for the treatment of various malignancies. Four of these new agents were approved since 2004, and there are numerous agents that are currently being evaluated in clinical trials. These recent FDA approvals indicate that the design and synthesis of new nucleoside analogues is still a productive area for discovering new drugs for the treatment of cancer. In general, these compounds have been most useful in the treatment of hematologic malignancies, and even though there is still room for significant improvements in the treatment of these diseases, some of the newer agents are finding use in the treatment of solid tumors. Table 1 FDA Approved Purine and Pyrimidine Antimetabolites The basic mechanism of action of purine and pyrimidine antimetabolites is similar. These compounds diffuse into cells (usually with the aid of a membrane transporter1) and are converted to analogues of cellular nucleotides by enzymes of the purine or pyrimidine metabolic pathway. These metabolites then inhibit one or more enzymes that are critical for DNA synthesis, causing DNA damage and induction of apoptosis.2 Even though the compounds in this class are structurally similar and share many mechanistic details, it is clear that subtle quantitative and qualitative differences in the metabolism of these agents and their interactions with target enzymes can have a profound impact on their antitumor activity. As noted by Plunkett and Gandhi,3 “one of the remarkable features of purine and pyrimidine nucleoside analogues that remains unexplained is how drugs with such similar structural features, that share metabolic pathways, and elements of their mechanism of action show such diversity in their clinical activities”. Possibly the best example of this fact is the newly approved drug, clofarabine, which differs from cladribine by only one fluorine atom, because it has demonstrated excellent efficacy in the treatment of relapsed and refractory pediatric acute lymphoblastic leukemia, whereas cladribine is not effective against this disease. These clinical results indicate that the biochemical actions of clofarabine are sufficiently different from that of cladribine to impart unique clinical activities. This and other examples indicate that small structural modifications of nucleoside analogues can have profound effects on the chemical stability and biological activity of nucleoside analogues. 1.1. Primary Enzymes Involved in the Metabolism and Activity of Purine and Pyrimidine Analogues To adequately understand the mechanism of action of this class of compounds it is necessary to be familiar with the enzymes that are involved in the metabolism of natural purines and pyrimidines. Human cells have all the enzymes needed for de novo synthesis of purine and pyrimidine nucleotides; however, other than orotate phosphoribosyl transferase with fluorouracil, these enzymes are not involved in the activation of the purine and pyrimidine antimetabolites and are only secondary targets responsible for antitumor activity of these compounds. Although salvage of purines and pyrimidines is not required for growth, human cells express many enzymes that can utilize purines and pyrimidines as substrates, and it is these enzymes (shown in Figures 1 and ​and2)2) that are most important to the anabolism and catabolism of the purine and pyrimidine antimetabolites that are used in the treatment of cancer. The catabolic enzymes are important because they are often responsible for detoxifying the nucleoside analogues, and these enzymes are expressed thoughout the body. Dihydropyrimidine dehydrogenase and xanthine oxidase are the initial enzymes in the degradation pathways of pyrimidines and purines. Adenosine deaminase and purine nucleoside phosphorylase are two important enzymes in the inactivation of purine nucleoside analogues but have also been successful targets of two agents, pentostatin and forodesine. Figure 1 Primary enzymes involved in the metabolism of pyrimidine analogues. Figure 2 Primary enzymes involved in the metabolism of purine analogues. Phosphoribosyl transferases are responsible for activating the 3 base analogues (mercaptopurine, thioguanine, and fluorouracil), and there are five enzymes in human cells that can phosphorylate deoxynucleoside analogues4–6 (deoxycytidine kinase, thymidine kinase 1, thymidine kinase 2, deoxyguanosine kinase, and 5′-nucleotidase). The primary rate-limiting enzyme for activation of most of the approved nucleoside analogues is deoxycytidine kinase. Although deoxycytidine is the preferred natural substrate for this enzyme, it also recognizes deoxyadenosine and deoxyguanosine as substrates. The purine analogues are also substrates for deoxyguanosine kinase expressed in mitochondria, and this enzyme can contribute to the activation of these agents. Once formed, the monophosphate metabolites are phosphorylated by the appropriate monophosphate kinases7 to the diphosphate metabolite, which is phosphorylated by nucleoside diphosphate kinase. The first step in the formation of the 5′-triphosphates is typically the rate-limiting step and is, therefore, the most important step in activation of deoxynucleoside analogues. The X-ray crystal structure of deoxycytidine kinase has recently been solved,8 and given its importance in the activation of deoxynucleoside analogues, its structure is used for design of new agents. The primary target of the deoxynucleoside analogues are the DNA polymerases involved in DNA replication. There are at least 14 eukaryotic DNA polymerases expressed in human cells,9 three of which are primarily involved in chromosomal replication (DNA polymerases α, δ, and e) and are the primary targets for the anticancer nucleoside analogues. The other major cellular polymerases are DNA polymerase β, which is involved in DNA repair; DNA polymerase γ, which is the polymerase responsible for mitochondrial DNA replication; and telomerase, which is responsible for the replication of DNA telomeres, but these enzymes are not primary targets for the anticancer antimetabolites. Inhibition of DNA polymerase γ or telomerase activity does not result in the immediate inhibition of cell growth. A deoxynucleotide triphosphate analogue could theoretically interact with a DNA polymerase in one of three ways: (i) it could compete with the natural substrate, but not be used as a substrate; (ii) it could substitute for the natural substrate with little effect on subsequent DNA synthesis; or (iii) it could substitute for the natural substrate and interfere with subsequent DNA synthesis, causing chain termination. The second two possibilities are the primary manners in which the anticancer nucleotide analogues interact with DNA polymerases, and all of these analogues have been shown to be good substrates for the replicative DNA polymerases. The primary differences in these compounds are (i) how easily the DNA chain is elongated after the incorporation of the analogue and (ii) how easily they can be removed from the DNA by the proof-reading exonucleases. The incorporation of these agents into DNA is one of the most important aspects of their mechanism of action resulting in antitumor activity, because the incorporation is difficult to repair and causes a lasting inhibition of DNA synthesis or disruption of DNA function. The inhibition of DNA synthesis by agents, such as aphidicolin, that only inhibit DNA polymerase activity without being incorporated into the DNA chain have not made good anticancer agents, because the DNA is not damaged by these agents and DNA synthesis resumes after the removal of the agent. Indeed, aphidicolin is used to synchronize cell populations,10 because of its ability to temporarily inhibit DNA synthesis without inducing cell death.