Protein dihydrofolate reductase

Figure 2.34. Ligand, methotrexate bound to the protein dihydrofolate reductase visualized in MarvinSpace. Detailed view of the binding pocket. The surface is colored by residue type and it is transparent. Behind it the ball and stick representation of the protein is seen. Lengths of some hydrogen bonds are monitored. Two pharmacophore regions are also defined an acceptor (red sphere) and a hydrophobic region (large yellow sphere), these are also semi-transparent. Image courtesy of ChemAxon...

An extensively studied enzyme-inhibitor system involves the protein dihydrofolate reductase/ and demonstrates an important feature of drug-enzyme interactions - the fact that an inhibitor drug may not... 

An extensively studied enzyme-inhibitor system involves the protein dihydrofolate reductase [88]. Crystallographic results demonstrate an important feature of drug-enzyme interactions an inhibitor drug may not bind in the same way as substrate even though both have similar chemical formulae. This enzyme catalyzes the reduction of 7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate, an essential... 

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. 

Gschwend DA, Sirawaraporn W, Santi DV, Kuntz ID. Specificity in structure-based drug design identification of a novel, selective inhibitor of Pneumocystis carinii dihydrofolate reductase. Proteins Struct Funct Genet 1997 29 59-67. 

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 %. 

Fig. 3. Autoradiograph of SDS-PAGE of in vitro translated dihydrofolate reductase (DHFR) in the wheat germ cell-free protein synthesis systems with (n) 4 pi of ribosome fiaction, (III) 4 pi of 0 -40 % ammonium sulfate fraction, or (IV) 4 pi of 40 - 60% ammonium sulfate fraction, respectively. Lane I is control dihydrofolate reductase produced in the normal wheat germ cell-free protein synthesis system.

The author assumed that the Born radii of atoms can be estimated from the solvent exposure factors for sampling spheres around the atoms. Two spheres were used in a five-parameter equahon to calculate the Born radii. The parameters of the equahon were eshmated using numerical calculahons from X-ray protein structures for dihydrofolate reductase. In addition to AGol the author also considered the AGJ term accounting for cavity formahon and dispersion of the solute-solvent interactions as ... 

Folic acid antagonist inhibits dihydrofolate reductase (DHFR) blocks reduction of folate to tetrahydrofolate inhibits de novo purine synthesis results in arrest of DNA, RNA, and protein synthesis... 

Protein fragment complementation using dihydrofolate reductase... 

Gegg, C. V., Bowers, K. E., and Matthews, C. R. (1997). Probing minimal independent folding units in dihydrofolate reductase by molecular dissection. Protein Sci. 6, 1885-1892. 

Bajorath, J., D. H. Kitson, G. Fitzgerald, J. Andzelm, J. Kraut, and A. T. Hagler. 1991. Local Density Functional Calculations on a Protein System Folate and Escherichia Coli dihydrofolate reductase. Electron Redistribution on Binding of a Substrate to an Enzyme. PROTEINS 9, 217. 

Hagler, Structure and energetics of ligand binding to proteins Escherichia coli dihydrofolate reductase-trimethoprim, a drug-receptor system, Proteins 4 31 (1988). 

Arabinose-binding protein domains 1 and 2 Dihydrofolate reductase Adenylate kinase Rhodanese domains 1 and 2 Glutathione reductase domains 1 and 2 Phosphoglycerate mutase Phosphoglycerate kinase domain 2 Pyruvate kinase domain 3 Hexokinase domains 1 and 2 Catalase domain 3 Aspartate aminotransferase... 

These three compounds exert many similar effects in nucleotide metabolism of chicks and rats [167]. They cause an increase of the liver RNA content and of the nucleotide content of the acid-soluble fraction in chicks [168], as well as an increase in rate of turnover of these polynucleotide structures [169,170]. Further experiments in chicks indicate that orotic acid, vitamin B12 and methionine exert a certain action on the activity of liver deoxyribonuclease, but have no effect on ribonuclease. Their effect is believed to be on the biosynthetic process rather than on catabolism [171]. Both orotic acid and vitamin Bu increase the levels of dihydrofolate reductase (EC 1.5.1.4), formyltetrahydrofolate synthetase and serine hydroxymethyl transferase in the chicken liver when added in diet. It is believed that orotic acid may act directly on the enzymes involved in the synthesis and interconversion of one-carbon folic acid derivatives [172]. The protein incorporation of serine, but not of leucine or methionine, is increased in the presence of either orotic acid or vitamin B12 [173]. In addition, these two compounds also exert a similar effect on the increased formate incorporation into the RNA of liver cell fractions in chicks [174—176]. It is therefore postulated that there may be a common role of orotic acid and vitamin Bj2 at the level of the transcription process in m-RNA biosynthesis [174—176]. 

Timelines can also be portrayed in charts or figures, as illustrated in excerpt 14D. In fact, charts and figures represent excellent ways to illustrate how smaller, individual projects contribute to larger research goals and how smaller projects complement one another and overlap in time. (Note In excerpt 14D, Kohen uses the following abbreviations in his chart, each defined previously in the proposal hydrogen (H), tritium (T), deuterium (D), kinetic isotope effect (KIE), dihydrofolate reductase (a relatively small protein) (DHFR), and wild type (WT).)... 

Pharmacology Trimetrexate, a 2.4-diaminoquinazoline, nonclassical folate antagonist, is a synthetic inhibitor of the enzyme dihydrofolate reductase. The end result is disruption of DNA, RNA, and protein synthesis, with consequent cell death. Pharmacokinetics Clearance was 38 15 ml /min/m and volume of distribution at steady state (Vdgs) was 20 8 L/m. The plasma concentration time profile declined... 

Fig. 2. Mechanisms causing resistance to antitumor treatment. ATM. ataxia telangiectasia gene, (Westphal et al., 1998 Xu and Baltimore, 1996), bcl-2/bax (Farrow and Brown, 1996, Zunino et al., 1997 Haq and Zanke, 1998), bcr/abl (McGahon et al., 1994), BCRP, breast cancer resistance protein (Doyle et d., 1998 Ross et al, 1999) bleomycin hydrolase (El-Deiry, 1997), BRCAl (Husain et al., 1998 Chen et al., 1998), BRCA2 (Chen et al., 1998 Chen et 1999), c-abl (White and Prives, 1999), c-jun (Sanchez-Perez and Perona, 1999), cytidine deaminase (El-Deiry, 1997), DNA poip, DNA polymerase p (Ochs et al., 1999), dihydrofolate reductase (Schimke, 1986), DT-diaphorase (Riley and Workman, 1992 Fitzsimmons et al., 1996 El-Deiry, 1997), EGR-1 (Ahmed et al., 1996), fos (Niimi et al., 1991), glucosylceramide synthase...

Fig. 6 Transition temperature of protein unfolding measured by CE and CD. Proteins lysozyme from chicken egg (LYS), a-lactalbumin from human milk (LAL-h), a-lactalbumin type III from bovine milk (LAL-III), cytochrome c from bovine heart (CYT), recombinant dihydrofolate reductase (DHFR).

Trimethoprim (TMP)-Sulfamethmazole (SMX) [Co-Trimoxazole] (Bactrim, Septra) [Antibiotic/Folate Antagonist] Uses un Rx prophylaxis, otitis media, sinusitis, bronchitis Action SMX T synth of dihydro-folic acid TMP T dihydrofolate reductase to impair protein synth Dose Adul. 1 DS tab PO bid or 5-20 mg/kg/24 h (based on TMP) IV in 3-4 doses P. jiroveci ... 

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