Dihydrofolate purine biosynthesis

Trimethoprim acts in the body by interfering with the action of hydrofolate reductase, an enzyme that reduces dihydrofolic acid to tetrahydrofolic acid. This process is necessary for purine biosynthesis of live organisms and DNA, respectively. Reducing the dihydrofolic acid to tetrahydrofolic acid is also catalyzed in humans by dihydrofolate reductase. However, trimethoprim has thousands of more inhibitory effects with respect to bacterial enzymes than with respect of analogons enzymes of mammals, which is the main benefit of trimethoprim. 

Folate analogues, such as methotrexate (Figure 27-3), are folate antagonists. They block production of FH2 and FH4 by dihydrofolate reductase and lead to diminished purine biosynthesis (inhibition of reactions 3 and 9 in Figure 27-8). Methotrexate also affects metabolism of amino acids and pyrimidine (inhibition of thymidylate synthesis) and inhibits DNA, RNA, and protein synthesis. It is effective in the treatment of breast cancer, cancer of the head and neck, choriocarcinoma, osteogenic sarcoma, and acute forms of leukemia. High doses of methotrexate can be tolerated provided that the patient also receives folinic... 

Dihydrofolate reductase (DHFR) is by far the most extensively investigated enzyme. 3D structures of binary and ternary DHFR complexes from different bacteria and vertebrates have been published and an extremely large numter of QSAR equations have been derived, both for the isolated enzyme and for growth inhibition of whole cells [288, 396, 431, 432, 671, 677 — 691]. Due to the central role of DHFR in purine biosynthesis, DHFR inhibitors are therapeutically important as highly selective antibacterial (trimethoprim), antimalarial, and antitumor agents (methotrexate). 

I. Pharmacology. Leucovorin (folinic acid or citrovomm factor) Is a metabolically functional form of folic acid. Unlike folic acid, leucovorin does not require reduction by dihydrofolate reductase, and therefore it can participate directly in the one-carbon transfer reactions necessary for purine biosynthesis and cellular DNA and RNA production. In animal models of methanol intoxication, replace-... 

Because of the central role of dihydrofolate reductase (DHFR) in purine biosynthesis, DHFR inhibitors are important as antibacterial (trimethoprim), antimalarial, and antitumor agents (methotrexate). For a series of 5-(X-benzyl)-2,4-diaminopyrimidines 9 with several different groups X, QSAR equations were derived for the inhibition of Escherichia coli (equation 14 Xi app == experimental Inhibition constants) and of Lactobacillus casei (equation 15) in both equations the numbers 3, 4, and 5 refer to the X substituent positions at the benzyl group. " ... 

Dihydrofolate reductase (DHFR) catalyzes the reduction of 7,8-dihydrofolate (H2F) by nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH) to form 5,6,7,8-telrahydrofolate (H4F), a key step in furnishing the parental cofactor needed for de novo pyrimidine and purine biosynthesis. The enzyme has been the target of antitumor and antimicrobial drugs. A complete Idnelic scheme (Fig. 6) obtained primarily throngh transient kinetics has been described for the enzyme bosaEscherichia coli as well as other sources and provides a second case smdy as to how to define the catalytic process. 

The dihydrofolate reductase enzyme (DHFR) is involved in one-carbon metabolism and is required for the survival of prokaryotic and eukaryotic cells. The enzyme catalyzes the reduction of dihydrofolate to tetrahydrofolate, which is required for the biosynthesis of serine, methionine, purines, and thymidylate. The mouse dihydrofolate reductase (mDHFR) is a small (21 kD), monomeric enzyme that is highly homologous to the E. coli enzyme (29% identify) (Pelletier et al., 1998). The three-dimensional structure of DHFR indicates that it is comprised of three structural fragments F[l], F[2] andF[3] (Gegg etal., 1997). 

Inhibit Enzymes Many drugs are competitive inhibitors of key enzymes in pathways. The statin drugs (lovastatin, simvastatin), used to control blood cholesterol levels, competitively inhibit 3-hvdroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase in cholesterol biosynthesis. Methotrexate, an antineoplastic drug, competitively inhibits dihydrofolate reductase, depriving the cell of active folate needed for purine and deoxythymidine synthesis, thus interfering with DNA replication during S phase. 

In Fig. 1 various targets of some important cytostatic agents are depicted. Their main mechanisms of action can be briefly summarized as follows. Pentostatin blocks purine nucleotides by inhibiting adenosine deaminase. 6-Mercaptopurine and 6-thioguanine inhibit purine ring biosynthesis and they inhibit nucleotide interconversions. Methotrexate by inhibiting dihydrofolate reduction blocks thymidine monophosphate and purine synthesis. 5-Fluorouracil also blocks thymidine monophosphate synthesis. Dactinomycin, daunorubicin, doxorubicin and mitoxantrone intercalate with DNA and inhibit RNA synthesis. L-asparaginase deaminates... 

Folic Acid Antagonists. Folic acid antagonists block the biosynthesis of purine nucleotides. Methotrexate (7.76) is the prototypic fohc acid antagonist and functions by binding to the active catalytic site of dihydrofolate reductase, thereby interfering with the synthesis of the reduced form that accepts one-carbon units lack of this cofactor blocks the synthesis of purine nucleotides. As well as being used in the treatment of cancer, methotrexate has been used in the management of rheumatoid arthritis, psoriasis, and even asthma. 

A considerable number of enzymes occupy a central and crucial role in the activity of drugs. Dihydrofolate reductase, an enzyme involved in purine and amino acid biosynthesis, is the target of antibacterial sulfanilamides, which act both as bacteriostatics and antimalarials. These drugs act on the enzyme in different ways, some being so-called antimetabolites (i.e., reversible enzyme inhibitors). Some diuretics act on carbonic... 

Methotrexate acts by inhibition of dihydrofolate reductase, the enzyme requisite for the reduction of dihydrofolic acid (3) to 5,6,7,8-tetrahydrofolic acid (4). In turn, (4) is a precursor to a series of enzyme cofactors (5-7) essential for the transfer of one carbon unit necessary for the biosynthesis of purines and pyrimidines and hence, ultimately, DNA. As an inhibitor of dihydrofolate reductase, methotrexate kills cells during the S phase of the cell cycle, when the cells are in the log phase of growth. Unfortunately, this cytotoxicity is non-selective, and rapidly proliferating normal cells, e.g., gastrointestinal epithelium cells and bone marrow, are dramatically affected as well. In addition, recent use of high dose methotrexate therapy with leucovorin rescue has led to additional clinical problems arising from a dose-related nephrotoxic metabolite, 7-hydroxy methotrexate (8). Finally, the very polar nature of methotrexate renders it virtually impenetrable to the blood-brain barrier, which can necessitate direct intrathecal injection in order to achieve therapeutic doses for the treatment of CNS tumours. 

Methotrexate is a folic acid antagonist, binding reversibly to dihydrofolate reductase. This prevents purine/pyrimidine biosynthesis and DNA synthesis.3... 

Tetrahydrofolic acid (THF) is a coenzyme in the synthesis of purine bases and thymidine. These are constituents of DNA and RNA and are required for cell growth and replication. Lack of THF leads to inhibition of cell proliferation. Formation of THF from dihydrofolate (DHF) is catalyzed by the enzyme dihydrofolate reductase. DHF is made from folic acid, a vitamin that cannot be synthesized in the body but must be taken up from exogenous sources. Most bacteria do not have a requirement for folate, because they are capable of synthesizing it-more precisely DHF-ffom precursors. Selective interference with bacterial biosynthesis of THF can be achieved with sulfonamides and trimethoprim. 

Folic acid or the folate coenzyme [6] is a nutritional factor both for the parasites and the hosts. It exists in two forms, viz. dihydro- and tetrahydrofolic acids [4,5] which act as cofactors involved in the transfer of one carbon units like methyl, hydroxymethyl and formyl. The transfer of a one carbon unit is associated with de novo synthesis of purines, pyrimidines and amino acids. Mammals can not synthesize folate and, therefore, depend on preformed dietary folates, which are converted into dihydrofolate by folate reductase. Contrary to this, a number of protozoal parasites like plasmodia, trypanosomes and leishmania can not utilize exogenous folate. Consequently, they carry out a de novo biosynthesis of their necessary folate coenzymes [12]. The synthesis of various folates follows a sequence of reactions starting from 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine (1), which is described in Chart 4 [13,14]. 

Dihydrofolate reductase (DHFR, EC 1.5.1.3) is an essential enzyme required for normal folate metabolism in prokaryotes and eukaryotes. Its role is to maintain necessary levels of tetrahydrofolate to support the biosynthesis of purines, pyrimidines and amino acids. Many compounds of pharmacological value, notably methotrexate and trimethoprim, vork by inhibition of DHFR. Their clinical importance justified the study of DHFR in the rapidly evolving field of enzymology. Today, there is a vast amount of published literature (ca. 1000 original research articles) on the broad subject of dihydrofolate reductase contributed by scientists from diverse disciplines. We have selected kinetic, structural, and computational studies that have advanced our understanding of the DHFR catalytic mechanism with special emphasis on the role of the enzyme-substrate complexes and protein motion in the catalytic efficiency achieved by this enzyme. 

What are called antifolate drugs pertain in general to blocking the biosynthesis of purines and pyrimidines, the heterocyclic bases used in the further synthesis of DNA and RNA, where folic acid is required as a coenzyme (or vitamin) for the enzyme dihydrofolate reductase. The previously mentioned compound called methotrexate or amethopterin (4-amino-A °-methyl folic acid), being a structural analog of folate or folic acid, locks up the enzyme dihydrofolate reductase, which in turn blocks the synthesis of a thymidine nucleotide necessary for cell division. 

See also De Novo Biosynthesis of Purine Nucleotides, DHF, THF, 10-FormyItetrahydrofolate, Adenosylmethionine and Biological Methylation, Metabolism of Serine, Glycine, and Threonine, Dihydrofolate Reductase, FdUMP, Vitamin B12 Coenzymes... 

See also Coenzymes in Nitrogen Metabolism, De Novo Biosynthesis of Purine Nucleotides, N-10-Formyltetrahydrofolate, 5-Methyltetrahydrofolate, 7,8-Dihydrofolate (DHF)... 

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