E. coli dihydrofolate reductase

E. A. An affinity selection-mass spectrometry method for the identification of small molecule ligands from self-encoded combinatorial libraries, discovery of a novel antagonist of E. coli dihydrofolate reductase. Int. J. Mass. Spectrom. 2004, 238, 77-83. 

Figure 15-19 Drawings of the active site of E. coli dihydrofolate reductase showing the hound ligands NADP+ and tetrahydrofolate. Several key amino acid side chains are shown in the stereoscopic views on the right. The complete ribbon structures are on the left. (A) Closed form. (B) Open form into which substrates can enter and products can escape. From Sawaya and Kraut.381 Courtesy of Joseph Kraut. Molscript drawings (Kraulis, 1991).

Annis D.A., Athanasopoulos J., Curran P.J., Felsch J.S., Kalghatgi K., Lee W.H., Nash H.M., Orminati J.P.A., Rosner K.E., Shipps GW., Thaddupathy G.R.A., Tyler A.N., Vilenchik L., Wagner C.R., Wintner E.A., An affinity selection-mass spectrometry method for the identification of small molecule ligands from self-encoded combinatorial libraries. Discovery of a novel antagonist of E. coli dihydrofolate reductase Int. J. Mass Spectrom. 2004, 238, 77-83. 

Zimmermann, M., Tresch, A., Maass, A., Hofmann, M. Drilling into a HTS data set of E. coli dihydrofolate reductase. In Proceedings of the 15th European Symposium on Quantative Structure-Activity Relationships 2004, Aki E., Yalcin J. (eds) published by Computer Aided Drug Design Development Society, Turkey,... 

Fig. 15. Chain trace of E.coli dihydrofolate reductase. Bound inhibitor (meihoirexaie) is also shown, with nitrogen (black) and oxygen (shading) atoms indicated. Stereo drawing from the work of Kraut and colleagues [68]. 

Boehr DD, McElheny D, Dyson HJ, Wright PE. Substrate-specific us-ms time-scale motions in the E. coli dihydrofolate reductase NADPH complex. FASEB J. 2005 19 A846-A847. 

Sawaya, M. and Kraut, J. (1997). Loop and subdomain movements in the mechanism of E. coli dihydrofolate reductase crystallographic evidence. Biochemistry 36, 586-603... 

Figure 8. Probe map of E. coli dihydrofolate reductase-methotrexate (10) complex. The calculated minimum energy positions for an ammonium probe (blue) and carboxylate oxygen probe (yellow) closely match the experimental positions for the pteridine amino groups and the carboxyl of methotrexate (20,21). The molecular surface of the enzyme is purple, while all bonds are color-coded by atom type carbon = white, nitrogen = blue, oxygen = red, sulfur = yellow. 

Figure 21. Model of E. coli dihydrofolate reductase complex with 3 -carboxyalkoxytrimethoprim analog (lib), shown interacting with Arg-57 (208). The same orientation and color-coding are used as in Figure 8.

The second case used the binding of a series of quinazoline inhibitors to S. faecium dihydrofolate reductase (269). In this case, six site points were used to represent the binding site of the dihydrofolate reductase and the data for 22 inhibitors were correlated. Again the site points and ligand were compared with the only available crystal study at the time, a methotrexate complex of E. coli dihydrofolate reductase, and shown to be consistent. 

Huang, Z., Wagner, C. R., Benkovig, S. J. (1994) Nonadditivity of mutational effects at the folate binding site of E.coli dihydrofolate reductase, Biochemistry 33, 11576-11585. 

E. coli dihydrofolate reductase binary complex San Diego1 MWPC2a 93.2 73.6 P61 2.5 Matthews et al (1977)... 

To successfully perform Y3H screens, the choice of the anchor moiety of the hybrid ligand is important. MTX, as already indicated, shows much promise. It exhibits high affinity (low nanomolar to picomolar) for the monomeric form of E. coli dihydrofolate reductase (eDHFR), which is a small, compact molecule that can be easily expressed as a fusion protein in yeast cells [46], Furthermore, contrary to what is often observed with nonhybrid small molecules, MTX-hybrid molecules appear to generally permeate yeast cells quite readily. At GPC Biotech we have screened over 50 hybrid ligands in which MTX was coupled to various small molecule chemotypes. To date we have not encountered difficulties with cellular uptake of these molecules. Cellular uptake can readily be determined using appropriate competition experiments, as outlined in Fig. 18.2-5. 

The value of A, in turn, is exponentially related to the percentage of identical core residues so that a protein structure will conservatively provide a close general stmctural model for other proteins if the sequence similarity exceeds 50%. Consequently, there is an increasing use of this type of relationship to predict tertiary stmcture and secondary elements from primary sequences. Nevertheless, there are cases, such as the Lactobacillus casei and E. coli dihydrofolate reductases, where the primary sequence similarity is only 30% yet A is only 1.3 A (727). 

J. J. McDonald and C. L. Brooks ID,/. Am. Chem. Soc., 114, 2062 (1992). A Theoretical Approach to Drug Design, 3, Relative Thermodynamics of Inhibitor Binding by E. coli Dihydrofolate Reductase to Ethyl Derivatives of Trimethoprim Substituted at 3 -, 4 -, and 5 -Positions,... 

Reference (25) details a study in which time-resolved fluorescence intensity and anisotropy decay measurements were combined with stopped-flow mixing to monitor the local and global dynamics of E. coli dihydrofolate reductase during refolding. A general review of such (so-called) double kinetic experiments is given in (26). 

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