Dihydrofolate reductase chemotherapy

The combined use of sulfonamides or sulfones with dihydrofolate reductase inhibitors, such as trimethoprim Bactrim, Septra) or pyrimethamine Fansidar), s, a good example of the synergistic possibilities that exist in multiple-drug chemotherapy. This type of impairment of the parasite s metabolism is termed sequential blockade. Using drugs that inhibit at two different points in the same biochemical pathway produces parasite lethality at lower drug concentrations than are possible when either drug is used alone. 

The principal mechanism of action is inhibition of dihydrofolate reductase, an enzyme important in the production of thymidine and purines. At the high doses used for chemotherapy, methotrexate inhibits cellular proliferation. However, at the low doses used in the treatment of inflammatory bowel disease (12-25 mg/wk), the antiproliferative effects may not be evident. Methotrexate may interfere with the inflammatory actions of interleukin-1. It may also stimulate increased release of adenosine, an endogenous anti-inflammatory autacoid. Methotrexate may also stimulate apoptosis and death of activated T lymphocytes. 

Correct answer = A. Methotrexate interferes with folate metabolism by acting as a competitive inhibitor of the enzyme dihydrofolate reductase. This starves cells for tetrahydrofolate, and makes them unable to synthesize purines and dTMP This is especially toxic to rapidly-growing cancer cells. Overproduction of dihydrofolate reductase, usually caused by amplification of its gene, can overcome the inhibition of the enzyme at the methotrexate concentrations used for chemotherapy, and can result in resistance of the tumor to treatment by this drug. 

Tetrahydrofolate cofactors participate in one-carbon transfer reactions. As described above in the section on vitamin B12, one of these essential reactions produces the dTMP needed for DNA synthesis. In this reaction, the enzyme thymidylate synthase catalyzes the transfer of the one-carbon unit of N 5,N 10-methylenetetrahydrofolate to deoxyuridine monophosphate (dUMP) to form dTMP (Figure 33-2, reaction 2). Unlike all of the other enzymatic reactions that utilize folate cofactors, in this reaction the cofactor is oxidized to dihydrofolate, and for each mole of dTMP produced, one mole of tetrahydrofolate is consumed. In rapidly proliferating tissues, considerable amounts of tetrahydrofolate can be consumed in this reaction, and continued DNA synthesis requires continued regeneration of tetrahydrofolate by reduction of dihydrofolate, catalyzed by the enzyme dihydrofolate reductase. The tetrahydrofolate thus produced can then reform the cofactor N 5,N 10-methylenetetrahydrofolate by the action of serine transhydroxy- methylase and thus allow for the continued synthesis of dTMP. The combined catalytic activities of dTMP synthase, dihydrofolate reductase, and serine transhydroxymethylase are often referred to as the dTMP synthesis cycle. Enzymes in the dTMP cycle are the targets of two anticancer drugs methotrexate inhibits dihydrofolate reductase, and a metabolite of 5-fluorouracil inhibits thymidylate synthase (see Chapter 55 Cancer Chemotherapy). 

Dihydrofolate reductase (DHFR), a classic target in antimicrobial and anticancer chemotherapy, has been shown to be a useful therapeutic target in plasmodium, toxoplasma, and eimeria species. Pyrimethamine is the prototypical DHFR inhibitor, exerting inhibitory effects in all three groups. However, pyrimethamine resistance in P falciparum has become widespread in recent years. This is largely attributable to specific point mutations in P falciparum DHFR that have rendered the enzyme less susceptible to the inhibitor. 

Both dihydrofolate reductase and thymidylate synthase reactions are targets for anticancer chemotherapy. Cancer is basically a disease of uncontrolled cell replication, and an essential part of cell replication is DNA synthesis. This means that a requirement exists for deoxynu-cleotide synthesis for growth. Inhibition of deoxynucleotide synthesis should inhibit the growth of cancer cells. 

Another way to reduce the supply of deoxynucleotides for cell replication is to target the reduction of dihydrofolate to tetrahydrofo-late. Folate antagonists are used in antimicrobial and anticancer chemotherapy. These compounds are competitive inhibitors of dihydrofolate reductase because they resemble the natural substrate. For example, methotrexate is used in antitumor therapy. 

During the 1980s and 1990s the role of folic acid analogues, especially methotrexate (MTX) (422), in cancer chemotherapy has been intensively studied enzyme dihydrofolate reductase (DHFR) has been the primary target of this effort. The introduction of 10-ethyl-10-deazaaminopterin (10-EDAM) (423), piritrexim (PTX) (424) and trimetrexate (TMX) (425) into clinical trials attests to the continued interest in this field <87MI 718-06). 

The sulfas, including co-trimoxazole (sulfamethoxazole plus trimethoprim, see p. 293), are bacteriostatic. These drugs are active against selected enterobacteria, chlamydia, Pneumocystis, and nocardia. Typical clinical applications are shown in Figure 29.3. In addition, sulfadiazine [sul fa DYE a zeen] in combination with the dihydrofolate reductase inhibitor pyrimethamine [py ri METH a meen] is the only effective form of chemotherapy for toxoplasmosis (p. 353). 

The active form of folate is the tetrahydro-derivative that is formed through reduction by dihydrofolate reductase. This enzymatic reaction (Figure 29.5) is inhibited by trimethoprim, leading to a decrease in the folate coenzymes for purine, pyrimidine, and amino acid synthesis. Bacterial reductase has a much stronger affinity for trimethoprim than does the mammalian enzyme, which accounts for the drug s selective toxicity. [Note Examples of other folate reductase inhibitors include pyrimethamine, which is used with sulfonamides in parasitic infections (see p. 353), and methotrexate, which is used in cancer chemotherapy (see p. 378).]... 

Methotrexate is a potent inhibitor of dihydrofolate reductase, with an affinity 1,000-fold greater than that of dUiydrofolate. Chemotherapy consists of alternating periods of administration of methotrexate and folate (normally as 5-formyl-tetrahydrofolate, leucovorin) to replete the normal tissues and avoid induction of folate deficiency- so-called leucovorin rescue. As well as depleting tissue pools of tetrahydrofolate, methotrexate leads to the accumulation of relatively large amounts of 10-formyl-dihydrofolate, which is apotentinhibitor of both thymidylate synthetase and glycinamide ribotide transformylase, an intermediate step in purine nucleotide synthesis. It is likely that this, rather than simple depletion of tetrahydrofolate, is the basis of the cytotoxic action of methotrexate (Barametal., 1988). 

Methotrexate is a potent inhibitor of dihydrofolate reductase, with an affinity 1,000-fold greater than that of dihydrofolate. Chemotherapy consists of alternating periods of administration of methotrexate and folate (normally as... 

In addition to the examples discussed above, the structures of many clinically and biologically important proteins have been determined. Some of these are amenable to immediate use in drug discovery efforts. These include enzymes such as dihydrofolate reductase (Jansy, 1988), and thymidilate synthase (Appelt et al, 1991) both of which are involved in the synthesis of DNA precursors. Their inhibition is a target in anticancer chemotherapies. Structures of other proteins such as cAMP-dependent protein kinase, acetylcholinesterase, and the glucocorticoid receptor add to the knowledge base that will open new avenues... 

Covalent bonds are not as important in drug-receptor binding as noncovalent interactions. Alkylating agents in chemotherapy tend to react and form an immonium ion, which then alkylates proteins, preventing their normal participation in cell divisions. Baker s concept of active site directed irreversible inhibitors was well established by covalent formation of Baker s antifolate and dihydrofolate reductase (46). 

Rapidly dividing cells require an abundant supply of thymidylate for the synthesis of DNA. The vulnerability of these cells to the inhibition of TMP synthesis has been exploited in cancer chemotherapy. Thymidylate synthase and dihydrofolate reductase are choice targets of chemotherapy (Figure 25.14). 

Figure 25.14. Anti cancer Drug Targets. Thymidylate synthase and dihydrofolate reductase are choice targets in cancer chemotherapy because the generation of large quantities of precursors for DNA synthesis is required for rapidly dividing cancer cells.

Fohc acid analogues containing amino acids other than glutamate, and also folate covalendy bound to a protein for the purposes of antibody production, have been prepared (58—60). Methotrexate is an analogue of fohc acid that is widely used in cancer chemotherapy (61) (see ChemotHERAPEUTICS, anticancer). Other analogues such as trimethoprim and pyrimethamine are used in the treatment of malaria and protozoal diseases (62). These analogues bind extremely tightly to dihydrofolate reductase. 

However, in view of its apparent success, this work is bound to advance further our understanding of the modes of action and structure-activity relationships of non-classical antimetabolites, and to produce new leads for the rational design of future drugs for chemotherapy. It should be remembered that it was the rational approach , based on the concepts of antimetabolites and of dihydrofolate reductase inhibitors as chemotherapeutic agents, that has... 

Folic Acid Antagonists - Methotrexate (MTX), folinic acid and ara-C were used sequentially with positive results in patients who had become resistant to conventional chemotherapy.18 The AT-3000 MTX resistant line of S-180 cells were found to have at least 150 times more dihydrofolate reductase than MTX sensitive cells.28 Similar data were obtained with MTX resistant hamster cells.21... 

A number of compounds derived from biguanides, triazines and diami-nopyrimidines have been shown to be potent inhibitors of dihydrofolate reductase (DHFR) and, therefore, occupy an important position in the chemotherapy of human malaria and bacterial infections. 

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