Dihydrofolate reductase, enhancement

The sulfa dmgs are stiH important as antimicrobials, although they have been replaced in many systemic infections by the natural and semisynthetic antibiotics. They are of great value in third world countries where problems of storage and lack of medical personnel make appropriate use of antibiotics difficult. They are especially useful in urinary tract infections, particularly the combination of sulfamethoxazole with trimethoprim. Their effectiveness has been enhanced by co-adniinistration with dihydrofolate reductase inhibitors, and the combination of sulfamethoxazole with trimethoprim is of value in treatment of a number of specific microbial infections. The introduction of this combination (cotrimoxazole) in the late 1960s (1973 in the United States) resulted in increased use of sulfonamides. 

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. 

A wheat germ, cell-free, translation extract was fractionated into three concentrated parts using ammonium sulfate the 0 - 40 % saturated fraction, the 40 - 60 % saturated fraction, and the ribosome fraction. These fractions were tested for their ability to enhance the translational activity of the wheat germ, cell-free extract for dihydrofolate reductase. The fortified cell-free system supplemented with the 0 - 40 % ammonium sulfate fraction enhanced the efficiency of protein synthesis by 50 %. 

Since there are strict stereochemical requirements for the relative positions and orientations of the two participating cysteine residues,11 addition of new disulfides to existing proteins by site-directed mutagenesis has not always produced the desired increase in stability. Introduction of disulfide bonds has been attempted for phage T4 lysozyme,4-71 phage A repressor,81 dihydrofolate reductase,91 and subtilisins.10-131 Among them the most extensive study has been performed on T4 lysozyme, and enhancement of protein stability has been successful. 

Proguardl (t) 17 h) inhibits dihydrofolate reductase which converts folic to folinic acid, deficiency of which inhibits plasmodial cell division. Plasmodia, like most bacteria and unlike humans, cannot make use of preformed foUc acid. Pyrimethamine and trimethoprim, which share this mode of action, are collectively known as the antifols. Their plasmod-icidal action is markedly enhanced by combination with sulphonamides or sulphones because there is inhibition of sequential steps in folate synthesis (see Sulphonamide combinations, p. 231). 

The combination of molecular modeling with genetic engineering to enhance protein stability has been successful in certain cases. For instance, introducing carefully sited novel disulfide bonds increased protein stability in T4 lysozyme (11-13) and in X-repressor (14). However, the results in other proteins, for instance, in subtilisin (15,16) and in dihydrofolate reductase (17) have been less predictable. 

In a validation study, we successfuUy applied LUDI to the design of inhibitors of dihydrofolate reductase and HIV-protease [56]. Pisabarro et al. [62] used a combination of GRID and LUDI to successfully design novel inhibitors of human synovial fluid phospholipase A2 with enhanced activity. A calculation with GRID pointed to a lipophilic binding pocket not occupied by the lead compound. LUDI was then used to search for suitable substituents to fill this pocket. One suggestion from LUDI was synthesized and found to yield a ten-fold improvement in binding affinity. 

Rajagopalan, P., Lutz, S. and Benkovic, S. (2002). Coupling interactions of distal residues enhance dihydrofolate reductase catalysis mutational effects on hydride ransfer rates. Biochemistry 41, 12618-12628... 

Scheme I. The preferred pathway is represented by the closed loop (heavier arrows), which bypasses free enzyme. Following the release of NADP+, the release of H4F from the E-H4F complex is too slow to account for turnover, but it is enhanced 10-fold by the binding of NADPH at the neighboring site. Studies on dihydrofolate reductase have provided a complete analysis of the effects of point mutations on the rate and equilibrium constants for each step in the reaction sequence. The active site structure of dihydrofolate reductase is shown schematically in Fig. 5, illustrating some of the amino acids interacting with the substrates that have been mutated.

Sulfamethoxazole inhibits bacterial synthesis of dihydrofohc acid, and trimethoprim blocks the production of tetrahydrofolic acid by inhibiting the enzyme dihydrofolate reductase. Thus two consecutive steps ate blocked in the biosynthesis of nucleic acids and proteins essential to many bacteria. In vitro serial dilution tests have shown that the combination of sulfamethoxazole and trimethoprim [738-70-5] inhibits the growth of common urinary tract pathogens with the exception of Pseudomonas aeruginosa. Table 3 illustrates the enhanced effect of the combination over that of either agent alone. 

I. Pharmacology. Folic acid is a B-complex vitamin that is essential for protein synthesis and erythropoiesis. In addition, the administration of folate to patients with methanol poisoning may enhance the elimination of the toxic metabolite formic acid, based on studies in folate-deficient primates. Note Folic acid requires metabolic activation and is not effective for treatment of poisoning by dihydrofolate reductase inhibitors (eg, methotrexate and trimethoprim). Leucovorin (see p 460) is the proper agent in these situations. 

Rice GC, Hoy C, Schimke RT (1986) Transient hypoxia enhances the frequency of dihydrofolate reductase gene amplification in Chinese hamster ovary cells. Proc Natl Acad Sci USA 83 5978-5982... 

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. 

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