DHFR

The 1- and 3-deaza analogues (463a, 463b) of the DHFR-inhibitory antitumour drug methotrexate have also been investigated (71JOC2818). 

Proguanil appears to have a dual activity. Part of it is metabolized to cycloguanil, which subsequently inhibits the protozaon dihydrofolate reduc-tase/thymidylate synthase (DHFR/TS) (Fig. 4). In addition, the native form, proguanil itself, exerts a potent antimalarial activity, especially in combination with other antimalarial drugs. The target of proguanil is unknown. 

The folate antagonists, pyrimethamine and sulfadiazine, inhibit the parasite s DHFR/TS synthase enzyme complex and the DHPS, respectively (Fig. 4) (see antimalarial drugs). To avoid deficiency of folic acid in patients treated with antifolate antagonists, folinic acid supplementation is recommended to reduce bone-marrow suppression. 

Overproduction of the chromosomal genes for the dihydrofolate reductase (DHFR) and the dihydroptero-ate synthase (DHPS) leads to a decreased susceptibility to trimethoprim and sulfamethoxazol, respectively. This is thought to be the effect of titrating out the antibiotics. However, clinically significant resistance is always associated with amino acid changes within the target enzymes leading to a decreased affinity of the antibiotics. 

Resistance to trimethoprim can be due to the acquisition of plasmid encoded non-allelic variants of the chromosomal DHFR enzyme that are antibiotic unsusceptible. The genes may be part of transposons that then insert into the chromosome. For instance, in gram-negative bacteria the most widespread gene is dhfrl on transposon Tn7. 

However, in the very end both projects failed with respect to drug design The Hb ligands do not permeate the erythrocyte membrane, and the trimethoprim analogs lost the high selectivity for bacterial DHFRs. 

Pneumocystis carinii DHFR inhibitor, Ki = 6.9 xM (about 30-foid seiectivity vs. human DHFR)... 

Plasmodium falciparum DHFR inhibitor, Ki = 0.9 jxM, Ki vs. different resistant strains = 0.6-2.1 xM... 

Streptococcus pneumoniae DHFR inhibitor, (R)-enantiomer, iCso = 9.8 niVI (S. pn. DHFR), iCgo = 2.8 nM (TMP-resistant S. pn. DHFR)... 

The plasmid vector pTEV-DHFR was used in this study. Internal ribosome entry sequence originated from tobacco etch virus (TEV) was locatai at upstream of dihydrofolate reductase (DHFR) gene. 

The catalytic activities of the fortified wheat germ cell-free systems supplemented with each fraction were investigated (Fig. 2). As shown in Fig. 2, only 0 - 40 % ammonium sulfate fraction showed an enhancement in DHFR protein synthesis. This enhancement of protein experimental results and the fact that the various eukaryotic initiation factors are contained in synthesis was also confirmed by SDS-PAGE and autoradiography (Fig. 3). From the above 0-40 % ammonium sulfate fraction [5, 6], it can be concluded that the amount of initiation factors in a conventionally prepared wheat germ cell-fi extract is deficient for the translation of DHFR with internal ribosome entry site. Therefore, it needs to supplement a wheat germ cell-free extract with the fraction containing the limited initiation factors for the efficient protein translation, and this fortified cell-free system can be easily made by simple... 

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.

Protein synthesis and selective inhibition 5.3 DHFR inhibitors... 

There is another fundamental difference between folate utilization in microbial and mammalian cells. Bacteria and protozoa are unable to take up exogenous folate and must synthesize it themselves. This is carried out in a series of reactions involving first the synthesis of dihydropteroic acid from one molecule each of pteridine and p-aminobenzoic acid (PABA). Glutamic acid is then added to form DHF which is reduced by DHFR to THF. Mammalian cells do not make their own DHF, instead they take it up firm dietary nutrients and convert it to THF using DHFR. 

Chromosomal mutations in E. coli result in overproduction of dihydrofolate reductase (DHFR). Higher concentrations of trimethoprim, which may not be therapeutically achievable, are therefore required to inhibit nucleotide metabolism. Other mutations lower the affinity of DHFR for trimethoprim. These two mechanisms of resistance may coexist in a single strain, effectively increasing the level of resistance to the antibiotic. 

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