Dihydrofolate reductase DHFR /trimethoprim

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. 

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. 

Manganese dioxide can also be used for oxidations of activated alkyl groups, as demonstrated by the oxidation of the clinical dihydrofolate reductase (DHFR) inhibitor trimethoprim 586, to give the aryl ketone 587, without any need for protection of the two amino groups <2006BML4366>. 

The action of trimethoprim (43) as an antibacterial depends on its inhibition of the bacterial enzyme dihydrofolate reductase (DHFR). The essential coenzyme tetrahydrofolate (47) operates in a cyclic process where the nucleotide thymidylate (48) is synthesized whilst... 

Plasmid- and transposon-mediated resistance to trimethoprim involves a by-pass of the sensitive step by duplication of the chromosomally-encoded dihydrofolate reductase (DHFR) target enzyme [203]. Several trimethoprim-resistant bacterial DHFRs have been identified, resistance ensuing because of altered enzyme target sites [204], Low-level resistance to tetracyclines arises in E. coli as a result of chromosomal mutations leading to loss of the outer membrane porin OmpF through which these drugs normally pass [6, 193],... 

A classic example of a drug that works by species-specific protein inhibition is trimethoprim (TMP). Because this drug binds to bacterial dihydrofolate reductase (DHFR) 10 moie tightly than to the mammalian enzyme, there is a therapeutic concentration in which the drug can be used as an antibacterial with little deleterious consequences for a mammalian host. 

Dihydrofolate Reductase. The reduced form of folate (tetrahydrofolate) acts as a one-carbon donor in a wide variety of biosynthetic transformations. This includes essential steps in the synthesis of purine nucleotides and of thymidylate, essential precursors to EHA and I A. For this reason, folate-dependent enzymes have been useful targets for the development of anticancer and anti-inflammatory drugs Ce.g., methotrexate) and anti-infectives (trimethoprim, pyrimethamine). During the reaction catalyzed by thymidylate synthase (TS), tetrahydrofolate also acts as a reducltant and is converted stoichiometrically to dihydrofolate. The regeneration of tetrahydrofolate, required for the continuous fimc-tioning of this cofactor, is catalyzed by dihydrofolate reductase (DHFR). 

The recommendations for the treatment of EPM using pyrimethamine, trimethoprim and sulfadiazine were originally based on the use of these drugs for the treatment of malaria and toxoplasmosis in humans. Either pyrimethanune or trimethoprim in combination with sulfadiazine or sulfamethoxazole have been used with some success and have gained widespread acceptance as the treatment of choice for EPM. Pyrimethamine and trimethoprim are diaminopyrimidine antimicrobial agents that inhibit dihydrofolate reductase (DHFR see Ch. 2). These agents interfere with... 

Figure 28-24 Dihydrofolate reductase (DHFR) has been a popular target for drug design. Methotrexate (MTX). 12. and trimethoprim (TMP). 13. resemble folic acid. 14. the natural substrate.

Trimethoprim competitively inhibits dihydrofolate reductase (DHFR) and resistance can be caused by overproduction of host DHFR, mutation in the structural gene for DHFR and acquisition of the dfr gene encoding a resistant form. There are at least 15 DHFR enzyme types based on sequence homology and acquisition of dfr genes encoding alternative DHFR of type I, II or V is the most common mechanism of trimethoprim resistance among the Enterobacteriaceae. 

Dihydrofolate reductase (DHFR, EC 1.5.1.3) is an essential enzyme required for normal folate metabolism in prokaryotes and eukaryotes. Its role is to maintain necessary levels of tetrahydrofolate to support the biosynthesis of purines, pyrimidines and amino acids. Many compounds of pharmacological value, notably methotrexate and trimethoprim, vork by inhibition of DHFR. Their clinical importance justified the study of DHFR in the rapidly evolving field of enzymology. Today, there is a vast amount of published literature (ca. 1000 original research articles) on the broad subject of dihydrofolate reductase contributed by scientists from diverse disciplines. We have selected kinetic, structural, and computational studies that have advanced our understanding of the DHFR catalytic mechanism with special emphasis on the role of the enzyme-substrate complexes and protein motion in the catalytic efficiency achieved by this enzyme. 

Pyrimethamine and trimethoprim are inhibitors of both bacterial and protozoal dihydrofolate reductase (DHFR) enzymes but do not affect the mammalian enzyme. Further specificity is achieved by the use of PABA antagonists, since they are competitive inhibitors of the protozoal dihydropteroate (DHP) synthase reaction, which condenses PABA with hydroxymethyldihydropteridine to form DHP, an intermediate in the tetrahydrofolate (THF) biosynthetic pathway. Protozoa synthesize THF de novo whereas humans require dietary folate. For this reason sulfur drugs are selective and virtually non-toxic to humans. 

Dihydrofolate reductase (DHFR) is by far the most extensively investigated enzyme. 3D structures of binary and ternary DHFR complexes from different bacteria and vertebrates have been published and an extremely large numter of QSAR equations have been derived, both for the isolated enzyme and for growth inhibition of whole cells [288, 396, 431, 432, 671, 677 — 691]. Due to the central role of DHFR in purine biosynthesis, DHFR inhibitors are therapeutically important as highly selective antibacterial (trimethoprim), antimalarial, and antitumor agents (methotrexate). 

Because of the central role of dihydrofolate reductase (DHFR) in purine biosynthesis, DHFR inhibitors are important as antibacterial (trimethoprim), antimalarial, and antitumor agents (methotrexate). For a series of 5-(X-benzyl)-2,4-diaminopyrimidines 9 with several different groups X, QSAR equations were derived for the inhibition of Escherichia coli (equation 14 Xi app == experimental Inhibition constants) and of Lactobacillus casei (equation 15) in both equations the numbers 3, 4, and 5 refer to the X substituent positions at the benzyl group. " ... 

Pathway compensation Trimethoprim Overproduction of DHFR (dihydrofolate reductase)... 

A most interesting and useful development concerning DHR inhibitors was the selectivity of inhibition observed between different classes of compounds against dihydrofolate reductases from mammals, protozoa and bacteria, which was found to be due to marked differences in binding affinity to the enzyme methotrexate binds very tightly to all reductases tested and is lethal to any cell it can enter, while trimethoprim and pyrimethamine have selectively strong affinity for bacterial and plasmodial reductases, respectively. This helped to rationalise the clinical use of DHFR inhibitors alone or in combination with sulphonamides and sulphones while trimethoprim is used mainly for bacterial infections, pyrimethamine is used for protozoal infections [58a]. 

---