Biosynthesis and Degradation of Nucleotides:- Many Chemotherapeutic Agents Target Enzymes in the Nucleotide Biosynthetic Pathways
The growth of cancer cells is not controlled in the same way as cell growth in most normal tissues. Cancer cells have greater requirements for nucleotides as precursors of DNA and RNA, and consequently are generally more sensitive than normal cells to inhibitors of nucleotide biosynthesis. A growing array of important chemotherapeutic agents—for cancer and other dis eases—act by inhibiting one or more enzymes in these pathways. We describe here several well-studied examples that illustrate productive approaches to treatment and help us understand how these enzymes work. The first set of agents includes compounds that inhibit glutamine amidotransferases. Recall that glutamine is a nitrogen donor in at least half a dozen separate re actions in nucleotide biosynthesis. The binding sites for glutamine and the mechanism by which NH4 is extracted are quite similar in many of these enzymes. Most are strongly inhibited by glutamine analogs such as azaserine and acivicin (Fig. 1). Azaserine, characterized by John Buchanan in the 1950s, was one of the first examples of a mechanism-based enzyme inactivator . Acivicin shows promise as a cancer chemotherapeutic agent. Other useful targets for pharmaceutical agents are thymidylate synthase and dihydrofolate reductase, en zymes that provide the only cellular pathway for thymine synthesis (Fig. 2). One inhibitor that acts on thymidylate synthase, fluorouracil, is an important chemotherapeutic agent. Fluorouracil itself is not the enzyme inhibitor. In the cell, salvage pathways convert it to the deoxynucleoside monophosphate FdUMP, which binds to and inactivates the enzyme. Inhibition by FdUMP (Fig. 3) is a classic example of mechanism-based enzyme inactivation. Another prominent chemotherapeutic agent, methotrexate, is an inhibitor of dihydrofolate reductase. This folate analog acts as a competitive inhibitor; the enzyme binds methotrexate with about 100 times higher affinity than dihydrofolate. Aminopterin is a related compound that acts similarly.
FIGURE 1 Azaserine and acivicin, inhibitors of glutamine amidotransferases. These analogs of glutamine interfere in a number of amino acid and nucleotide biosynthetic pathways.
FIGURE 2 Thymidylate synthesis and folate metabolism as targets of chemotherapy. (a)During thymidylate synthesis, N5, N10-methylenetetrahydrofolate is converted to 7,8-dihydrofolate; the N5, N10-methylenetetrahydrofolate is regenerated in two steps . This cycle is a major target of several chemotherapeutic agents. (b)Fluorouracil and methotrexate are important chemothera peutic agents. In cells, fluorouracil is converted to FdUMP, which inhibits thymidylate synthase. Methotrexate, a structural analog of tetrahydrofolate, inhibits dihydrofolate reductase; the shaded amino and methyl groups replace a carbonyl oxygen and a proton, respectively, in folate (see Fig. 22–44). Another important folate analog, aminopterin, is identical to methotrexate except that it lacks the shaded methyl group. Trimethoprim, a tight-binding inhibitor of bacterial dihydrofolate reductase, was developed as an antibiotic.
MECHANISM FIGURE 3 Conversion of dUMP to dTMP and its inhibition by FdUMP. The top row is the normal reaction mechanism of thymidylate synthase. The nucleophilic sulfhydryl group contributed by the enzyme in step 1 and the ring atoms of dUMP taking part in the reaction are shown in red; :B denotes an amino acid side chain that acts as a base to abstract a proton in step 3. The hydrogens de rived from the methylene group of N5,N10-methylenetetrahydrofolate are shaded in gray. A novel feature of this reaction mechanism is a 1,3-hydride shift (step 3), which moves a hydride ion (shaded pink) from C-6 of H4 folate to the methyl group of thymidine, resulting in the oxidation of tetrahydrofolate to dihydrofolate. It is this hydride shift that apparently does not occur when FdUMP is the substrate (bottom). Steps 1 and 2 proceed normally, but result in a stable complex— with FdUMP linked covalently to the enzyme and to tetrahydrofolate— that inactivates the enzyme.
The medical potential of inhibitors of nucleotide biosynthesis is not limited to cancer treatment. All fastgrowing cells (including bacteria and protists) are potential targets. Trimethoprim, an antibiotic developed by Hitchings and Elion, binds to bacterial dihydrofolate reductase nearly 100,000 times better than to the mammalian enzyme. It is used to treat certain urinary and middle ear bacterial infections. Parasitic protists, such as the trypanosomes that cause African sleeping sickness (African trypanosomiasis), lack pathways for de novo nucleotide biosynthesis and are particularly sensitive to agents that interfere with their scavenging ofnucleo tides from the surrounding environment using salvage pathways. Allopurinol and a number of related purine analogs have shown promise for the treat ment of African trypanosomiasis and related afflictions. See Box 22–2 for another approach to combating African trypanosomiasis, made possible by advances in our understanding of metabolism and enzyme mechanism.
