Dihydrofolate synthase

Intracellular protozoa of the phylum Apicomplexa such as plasmodium, toxoplasma, and eimeria have long been known to respond to sulfonamides and sulfones. This has led to the assumption that Apicomplexa must synthesize their own folate in order to survive. The reaction of 2-amino-4-hydroxy-6-hydroxymethyl-dihydropteridine diphosphate with />aminobenzoate to form 7,8-dihydropteroate has been demonstrated in cell-free extracts of the human malaria parasite Plasmodium falciparum. 2-Amino-4-hydroxy-6-hydroxymethyl-dihydropteridine pyrophosphokinase and 7,8-dihydropteroate synthase have also been identified. Sulfathiazole, sulfaguanidine, and sulfanilamide act as competitive inhibitors of p-aminobenzoate. It has not been possible to demonstrate dihydrofolate synthase activity in the parasites, which raises the possibility that 7,8-dihydropteroate may have substituted for dihydrofolate in malaria parasites. Similar lack of recognition of folate as substrate was also observed in the dihydrofolate reductase of Eimeria tenella, a parasite of chickens. 

E) under the influence of dihydrofolate synthase (EC 6.3.2.12). Repetition of the same sequence of phosphorylation and amide formation serves to extend the amide side chain. Reduction (NADPH, dihydrofolate reductase, EC 1.5.1.3) leads to tet-rahydrofolate, which, itself, can undergo changes in the number of glutamate (Glu, E) side chains (tetrahydrofolate synthase, EC 6.3.2.17). 

Folic acid plays the central role in one-carbon metabolism and is directly or indirectly involved in the biosynthesis of nucleic acid bases and amino acids. In plants and microbes, the various folate derivatives involved in one-carbon metabolism originate from dihydropterin derivatives, via the enzymes 7,8-dihydropteroate and dihydrofolate synthase (Figure 5.18). 7,8-Dihydrofolate then undergoes reduction to 5,6,7,8-tetra-hydrofolate (THF), which can become substituted with formyl, methylene, or methyl groups for utilization in one-carbon donor reactions. ... 

In E. coli GTP cyclohydrolase catalyzes the conversion of GTP (33) into 7,8-dihydroneoptetin triphosphate (34) via a three-step sequence. Hydrolysis of the triphosphate group of (34) is achieved by a nonspecific pyrophosphatase to afford dihydroneopterin (35) (65). The free alcohol (36) is obtained by the removal of residual phosphate by an unknown phosphomonoesterase. The dihydroneoptetin undergoes a retro-aldol reaction with the elimination of a hydroxy acetaldehyde moiety. Addition of a pyrophosphate group affords hydroxymethyl-7,8-dihydroptetin pyrophosphate (37). Dihydropteroate synthase catalyzes the condensation of hydroxymethyl-7,8-dihydropteroate pyrophosphate with PABA to furnish 7,8-dihydropteroate (38). Finally, L-glutamic acid is condensed with 7,8-dihydropteroate in the presence of dihydrofolate synthetase. 

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. 

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. 

Toyoda T, Brobey RKB, Sano G, Horii T, Tomioka N, Itai Akiko. Lead discovery of inhibitors of the dihydrofolate reductase domain of Plasmodium falciparum dihydrofolate reductase-thymidylate synthase. Biochem Biophys Res Commun 1997 235 515-19. 

The methylation of deoxyuridine monophosphate (dUMP) to thymidine monophosphate (TMP), catalyzed by thymidylate synthase, is essential for the synthesis of DNA. The one-carbon fragment of methy-lene-tetrahydrofolate is reduced to a methyl group with release of dihydrofolate, which is then reduced back to tetrahydrofolate by dihydrofolate reductase. Thymidylate synthase and dihydrofolate reductase are especially active in tissues with a high rate of cell division. Methotrexate, an analog of 10-methyl-tetrahydrofolate, inhibits dihydrofolate reductase and has been exploited as an anticancer drug. The dihydrofolate reductases of some bacteria and parasites differ from the human enzyme inhibitors of these enzymes can be used as antibacterial drugs, eg, trimethoprim, and anti-malarial drugs, eg, pyrimethamine. 

Studies using free energy calculations for the design and analysis of potential drug candidates are reviewed in section five. The chapters in this section cover drug discovery programs targeting fructose 1,6-bisphosphatase (diabetes), COX-2 (inflammation), SRC SH2 domain (osteoporosis and cancer), HIV reverse transcriptase (AIDS), HIV-1 protease (AIDS), thymidylate synthase (cancer), dihydrofolate reductase (cancer) and adenosine deaminase (immunosuppression, myocardial ischemia). 

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.)...

The DNA component deoxythymidine triphosphate (dTTP) is synthesized from UDP in several steps. The base thymine, which only occurs in DNA (see p. 80), is formed by meth-ylation of dUMP at the nucleoside monophosphate level. Thymidylate synthase and its helper enzyme dihydrofolate reductase are important target enzymes for cytostatic drugs (see p. 402). 

Dihydrofolate reductase acts as an auxiliary enzyme for thymidylate synthase. It is involved in the regeneration of the coenzyme N, N -methylene-THF, initially reducing DHF to THF with NADPH as the reductant (see p. 418). The folic acid analogue methotrexate, a frequently used cytostatic agent, is an extremely effective competitive inhibitor of dihydrofolate reductase. It leads to the depletion of N, N -methylene-THF in the cells and thus to cessation of DNA synthesis. 

Thymidylate synthase [EC 2.1.1.45] reductively methylates 2 -deoxyuridine-5 -monophosphate to form 2 -deoxythymidine-5 -monophosphate in the following folate-dependent reaction dUMP + A, A -methylene-tetrahydrofolate dTMP + dihydrofolate. 

CARNOSINE SYNTHETASE CHAPERONES CHOLINE KINASE CHOLOYL-CoA SYNTHETASE COBALAMIN ADENOSYLTRANSFERASE 4-COUMAROYL-CoA SYNTHETASE CREATINE KINASE CTP SYNTHETASE CYTIDYLATE KINASE 2-DEHYDRO-3-DEOXYGLUCONOKINASE DEHYDROGLUCONOKINASE DEOXYADENOSINE KINASE DEOXYADENYLATE KINASE DEOXYCYTIDINE KINASE (DEOXYjNUCLEOSIDE MONOPHOSPHATE KINASE DEOXYTHYMIDINE KINASE DEPHOSPHO-CoA KINASE DETHIOBIOTIN SYNTHASE DIACYLGLYCEROL KINASE DIHYDROFOLATE SYNTHETASE DNA GYRASES DNA REVERSE GYRASE ETHANOLAMINE KINASE EXONUCLEASE V... 

DEOXYOCTULOSONATE 8-PHOS-PHATE SYNTHASE DETHIOBIOTIN SYNTHASE DIHYDROFOLATE SYNTHETASE... 

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...

The answer is c. (Hardman, pp 1058-1059. Katzung, pp 793-795.) Trimethoprim inhibits dihydrofolic acid reductase. Sulfamethoxazole inhibits p-aminobenzoic acid (PABA) from being incorporated into folic acid by competitive inhibition of dihydropteroate synthase. Either action inhibits the synthesis of tetrahydrofolic acid. 

Pyrimethamine and proguanil selectively inhibit plasmodial dihydrofolate reductase, a key enzyme in the pathway for synthesis of folate. Sulfonamides and sulfones inhibit another enzyme in the folate pathway, dihydropteroate synthase. As described in Chapter 46 and shown in Figure 46-2, combinations of inhibitors of these two enzymes provide synergistic activity. 

In many areas, resistance to folate antagonists and sulfonamides is common for P falciparum and less common for P vlvax. Resistance is due primarily to mutations in dihydrofolate reductase and dihydropteroate synthase, with increasing numbers of mutations leading to increasing levels of resistance. At present, resistance seriously limits the efficacy of sulfadoxine-pyrimethamine (Fansidar) for the treatment of malaria in most areas, but in Africa most parasites exhibit only moderate resistance, such that antifolates appear to continue to offer preventive efficacy against malaria. Because different mutations may mediate resistance to different agents, cross-resistance is not uniformly seen. 

---