Mammalian dihydrofolate reductase

Enzymes that are addressed by major drugs have been studied in particular detail. Thus, well above one hundred stmctures have been reported for dihydrofolate reductases from a variety of organisms, including major pathogens such as Mycobacterium tuberculosis, which is the causative agent of tuberculosis, and of Plasmodium falciparum, which is the most important of the Plasmodium spp. that causes malaria. The interaction of mammalian dihydrofolate reductases with inhibitors that are used as cytostatic agents and/or immunosuppressants is also documented extensively by X-ray stmctures. 

Folate analogs such as trimethoprim have potent antibacterial and antiprotozoal activity. Trimethoprim binds lO -fold less tightly to mammalian dihydrofolate reductase than it does to reductases of susceptible microorganisms. Small differences in the active-site clefts of these enzymes account for its highly selective antimicrobial action. The combination of trimethoprim and sulfamethoxazole (an inhibitor of folate synthesis) is widely used to treat infections. 

Mammalian dihydrofolate reductases can also catalyze the transfer of a hydride ion to the C-7 position of folate in a reaction affording dihydrofolate, thus enabling the utilization of the fully oxidized vitamin from nutritional sources/ ... 

In order to counteract resistance development and to improve antibacterial activity, sulfonamides are typically combined with an inhibitor of dihydrofolate reductase (Figure 2, i). Dihydrofolate reductase is a core enzyme of human central intermediary metabolism. Although the bacterial and mammalian dihydrofolate reductases are orthologues with considerable sequence similarity, trimethoprim (35) has sufficient selectivity to enable the selective inhibition of the parasite enzyme. 

Trimethoprim is used as an antibiotic because it binds to bacterial dihydrofolate reductase much more tightly than to mammalian dihydrofolate reductase. 

It is, however, pertinent to mention here that the mammalian dihydrofolate reductase is approximately 1 10,000 to 1 50,000 as sensitive to it as the bacterial enzymes, so that there prevails almost little interference with folate metabolism in humans. 

Since trimethoprim binds to the mammalian dihydrofolate reductase much less tightly than to the enzyme of susceptible microorganisms, it causes fewer deleterious effects to humans than does methotrexate. 

Due to a mutation in the human genome, trimetoprim inhibits bacterial dihydrofolate reductase but not mammalian dihydrofolate reductase. Trimetoprim has therefore been used in combination with the sulphonamides to provide synergism in activity while reducing the toxidty potential of the individual compounds. A usefiil dinical example is co-trimoxazole, which is a compound formulation of trimetoprim and sulfamethoxazole. 

Product formation kinetics in mammalian cells has been studied extensively for hybridomas. Most monoclonal antibodies are produced at an enhanced rate during the Gq phase of the cell cycle (8—10). A model for antibody production based on this cell cycle dependence and traditional Monod kinetics for cell growth has been proposed (11). However, it is not clear if this cell cycle dependence carries over to recombinant CHO cells. In fact it has been reported that dihydrofolate reductase, the gene for which is co-amplified with the gene for the recombinant protein in CHO cells, synthesis is associated with the S phase of the cell cycle (12). Hence it is possible that the product formation kinetics in recombinant CHO cells is different from that of hybridomas. 

Folate metabolism Sulphonamides (also ) Trimethoprim Pyrimethamine Trimetrexate / Inhibit folate synthesis Inhibits dihydrofolate reductase Inhibits dihydrofolate reductase Inhibits dihydrofolate reductase Not present in mammalian cells Mammalian enzyme not inhibited Mammalian enzyme not inhibited Toxicity overcome with leucovorin... 

SV40 Promoter This is the promoter for expression of the DHFR gene. DHFR The enzyme dihydrofolate reductase (DHFR) is required for nucleotide (thymine) synthesis and cell growth and serves as a selection marker in mammalian cells. 

Trimethoprim, a trimethoxybenzylpyrimidine, selectively inhibits bacterial dihydrofolic acid reductase, which converts dihydrofolic acid to tetrahydrofolic acid, a step leading to the synthesis of purines and ultimately to DNA (Figure 46-2). Trimethoprim is about 50,000 times less efficient in inhibition of mammalian dihydrofolic acid reductase. Pyrimethamine, another benzylpyrimidine, selectively inhibits dihydrofolic acid reductase of protozoa compared with that of mammalian cells. As noted above, trimethoprim or pyrimethamine in combination with a sulfonamide blocks sequential steps in folate synthesis, resulting in marked enhancement (synergism) of the activity of both drugs. The combination often is bactericidal, compared with the bacteriostatic activity of a sulfonamide alone. 

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).]... 

Pyrimethamine and trimethoprim reversibly inhibit the second step in the synthesis of folic acid by inhibiting the enzyme dihydrofolate reductase, which catalyzes the reduction of dihydrofolic acid to tetrahydrofolic acid. The trimethoprim-binding affinity is much stronger for the bacterial enzyme than the corresponding mammalian enzyme, which produces selective toxicity. A powerful synergism exists between either pyrimethamine or trimethoprim and sulfonamides (e g., sulfemethoxazole and trimethoprim) because of sequential blockage of the same biosynthetic pathway. 

A classic example of a drug that works by species-specific protein inhibition is trimethoprim (TMP). Because this drug binds to bacterial dihydrofolate reductase (DHFR) 10 moie tightly than to the mammalian enzyme, there is a therapeutic concentration in which the drug can be used as an antibacterial with little deleterious consequences for a mammalian host. 

Urlaub G, Kas E, Carothers AM Chasin LA (1983) Detection of the diploid dihydrofolate reductase locus from cultured mammalian cells. Cell 33 405-412. 

Fig. 12.7 Pathways of folate metabolism and use in microbial cells (upper) and mammalian cells (lower). Bacterial and protozoal cells must synthesize dihydrofolic acid (DHF) from p-aminobenzoic acid (PABA). DHF is converted to tetrahydrofolic acid (THF) by the enzyme dihydrofolate reductase (DHFR). THF supplies single carbon units for various pathways including DNA, RNA and methionine synthesis. Mammalian cells do not make DHF, it is supplied from the diet, conversion to THF occurs via a DHFR enzyme as in microbial cells.

Methotrexate is used as an oral anticancer agent, but resistance may develop in tumour cells, and there are a number of unwanted side-effects. It has a high affinity for the mammalian form of dihydrofolate reductase and cannot be used as an antibacterial or antimalarial drug. After use at high doses in humans, the extent of depletion of folic acid may be such, that rescue with administration of folinic acid (a form of tetrahydrofolate) is necessary. 

Inhibition of folic acid synthesis in susceptible microorganisms and ultimately the synthesis of nucleic acids. By competing with para-aminobenzoic acid (PABA) for the enzyme dihydropteroate synthetase, sulphonamides prevent the incorporation of PABA into dihydrofolate, while trimethoprin, by selectively inhibiting dihydrofolate reductase, prevents the reduction of dihydrofolate to tetrahydrofolate (folic acid). Animal cells, unlike bacteria, utilize exogenous sources of folic acid. Pyrimethamine inhibits protozoal dihydrofolate reductase, but is less selective for the microbial enzyme and therefore more toxic than trimethoprim to mammalian species. 

Dihydrofolate reductase is present in mammalian cells as well as bacterial cells, so we might wonder why trimethoprim does not affect our own cells. The answer is that trimethoprim is able to distinguish between the enzymes in either cell. Although this enzyme is present in both types of cell and carries out the same reaction, mutations over millions of years have resulted in a significant difference in structure between the two enzymes such that trimethoprim recognizes and inhibits the bacterial enzyme, but does not recognize the mammalian enzyme. 

Inhibitors of dihydrofolate reductase. Methotrexate, a structural analogue of dihydrofolate, is effective against intact mammalian cells but ineffective against protozoa and some bacteria owing to permeability barriers. Trimethoprim and pyrimethamine (2,4-diaminopyrimidines) are effective against microorganisms. The former is antibacterial and antimalarial the latter is primarily antimalarial. 

To date, commercial antibody expression exclusively uses mammalian cell line expression. These mammalian cell lines have expression cassettes for the antibody heavy and light chain stably integrated into the host cell chromosome. The most commonly used cell lines are derived from Chinese hamster ovary (CHO) cells. About half the approved antibody therapeutics are made in CHO cell lines. The dihydrofolate reductase (dhfr) gene is used as a selectable marker owing to the development of CHO cell lines that are deficient for dhfr genes such as CHO DG44 and CHO... 

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