DHFR-MTX

The crystal structure of the E. colt DHFR/MTX binary complex (9) reveals that the inhibitor binds in the active site in a kinked fashion (Figure 8). Asp-27 forms a salt-bridge to the bound pteridine ring while Phe-31 allows for the bend in the bound inhibitor. The remainder of the active site cavity surrounding the pterin ring is composed of amino acid residues that create a very hydrophobic environment. 

Conformation of Bound Inhibitor. Another area under investigation focuses on the conformations) of the substrate and an inhibitor (MTX) bound to DHFR. The form of the DHFR/MTX complex is known from the crystallographic studies of Kraut et al. (8-10). However, the orientation of the bound pterin ring in the reactive DHFR/DHF is known to differ dramatically from the MTX crystal structure (20). Basically, these differences arise because there are two possible orientations of the pterin ring in the active site one is flipped by 180° with respect to the other. Isotope labelling experiments on THF show that the reactive DHF must be bound in the conformation flipped from that observed by x-ray for MTX. In order to understand these differences, we ran simulation studies on altered forms of bound MTX and DHF to investigate the structural and energetic properties of these systems. 

One of the greatest problems in treatment with MTX and other anti-folates is the fact that the cancer cells develop immunity to the drugs. It has been found34 that this immunity is due mainly to DHFR mutations where some amino-acid residues are replaced by others which do not bind to anti-folates. The desire to better understand the mechanism of binding of anti-folates to DHFR, in order that this problem will be remediated, has led to numerous experimental studies. In addition, theoretical studies have complemented the attempts to elucidate the mechanism. 

The fact that N1 is preferentially protonated is in agreement with crystal data obtained for free triazines and enzyme-bound triazines in ternary complex with enzyme and enzyme cofactor (NADPH)45 and also with the difference spectroscopy evidence46 that the N1 of the DHFR-bound MTX is protonated. 

Fig. 14.1 Cellular pathway of methotrexate. ABCBl, ABCCl-4, ABC transporters ADA, adenosine deaminase ADP, adenosine diphosphate AICAR, aminoimidazole carboxamide ribonucleotide AMP, adenosine monophosphate ATIC, AICAR transformylase ATP, adenosine triphosphate SjlO-CH -THF, 5,10-methylene tetrahydrofolate 5-CHj-THF, 5-methyl tetrahydro-folate DHFR, dihydrofolate reductase dTMP, deoxythymidine monophosphate dUMP, deoxy-uridine monophosphate FAICAR, 10-formyl AICAR FH, dihydrofolate FPGS, folylpolyglutamyl synthase GGH, y-glutamyl hydrolase IMP, inosine monophosphate MTHFR, methylene tetrahydrofolate reductase MTR, methyl tetrahydrofolate reductase MTX-PG, methotrexate polyglutamate RFCl, reduced folate carrier 1 TYMS, thymidylate synthase. Italicized genes have been targets of pharmacogenetic analyses in studies published so far. (Reproduced from ref. 73 by permission of John Wiley and Sons Inc.)...

Interestingly, dihydroMTX and tetrahydroMTX were both less potent than MTX itself as inhibitors of DHFR, but proved to be more potent as inhibitors of TS as in the case of the above AP derivatives, dihydroMTX was more inhibitory than the tetrahydro derivative in every system examined [270]. It is unsurprising, in view of our current knowledge of the folate coenzyme pathways, that the unnatural D-enantiomer of 5,10-methylene-5,6,7,8-tetrahydroFA had no activity either as a substrate for or as an inhibitor of DHFR [271],... 

During the 1980s and 1990s the role of folic acid analogues, especially methotrexate (MTX) (422), in cancer chemotherapy has been intensively studied enzyme dihydrofolate reductase (DHFR) has been the primary target of this effort. The introduction of 10-ethyl-10-deazaaminopterin (10-EDAM) (423), piritrexim (PTX) (424) and trimetrexate (TMX) (425) into clinical trials attests to the continued interest in this field <87MI 718-06). 

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