Folate synthesis inhibitors

The other major class of antimalarials are the folate synthesis antagonists. There is a considerable difference in the drug sensitivity and affinity of dihydrofolate reductase enzyme (DHFR) between humans and the Plasmodium parasite. The parasite can therefore be eliminated successfully without excessive toxic effects to the human host. DHFR inhibitors block the reaction that transforms deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) at the end of the pyrimidine-synthetic pathway. This reaction, a methylation, requires N °-methylene-tetrahydrofolate as a carbon carrier, which is oxidized to dihydrofolate. If the dihydrofolate cannot then be reduced back to tetrahydrofolate (THF), this essential step in DNA synthesis will come to a standstill. 

Combination of a sulfonamide with an inhibitor of dihydrofolate reductase (trimethoprim or pyrimethamine) provides synergistic activity because of sequential inhibition of folate synthesis (Figure 46-2). 

A number of antibiotics in addition to the folate antagonists and sulfonamides are modestly active antimalarials. The antibiotics that are bacterial protein synthesis inhibitors appear to act against malaria parasites by inhibiting protein synthesis in a plasmodial prokaryote-like organelle, the apicoplast. None of the antibiotics should be used as single agents in the treatment of malaria because their action is much slower than that of standard antimalarials. 

The active form of folate is the tetrahydro-derivative that is formed through reduction by dihydrofolate reductase. This enzymatic reaction (Figure 29.5) is inhibited by trimethoprim, leading to a decrease in the folate coenzymes for purine, pyrimidine, and amino acid synthesis. Bacterial reductase has a much stronger affinity for trimethoprim than does the mammalian enzyme, which accounts for the drug s selective toxicity. [Note Examples of other folate reductase inhibitors include pyrimethamine, which is used with sulfonamides in parasitic infections (see p. 353), and methotrexate, which is used in cancer chemotherapy (see p. 378).]... 

Folate analogs such as trimethoprim have potent antibacterial and antiprotozoal activity. Trimethoprim binds lO -fold less tightly to mammalian dihydrofolate reductase than it does to reductases of susceptible microorganisms. Small differences in the active-site clefts of these enzymes account for its highly selective antimicrobial action. The combination of trimethoprim and sulfamethoxazole (an inhibitor of folate synthesis) is widely used to treat infections. 

With respect to mechanism of action, the most extensive kinetic and equilibrium exchange studies have been carried out on monofunctional 10-formyl-H4-folate synthetase from Cl. cylindrosporum [84]. The data support a random sequential mechanism that does not involve the formation of freely dissociable intermediates. The most likely mechanism, however, is not concerted but probably involves the formation of a formyl phosphate intermediate, since the synthetase catalyzes phosphate transfer from carbamyl phosphate but not acetyl phosphate to ADP with H 4-folate serving as an activator. Carbamyl phosphate is an inhibitor of 10-formyl-H 4-folate synthesis - an inhibition that can be eliminated only when both ATP and formate are present in accord with the concept that it spans both sites [85]. It would be of considerable interest to attempt to demonstrate positional isotope exchange employing [, y- 0]ATP for this enzyme in order to further implicate an enzyme-bound formyl phosphate species [86]. 

Another frequently employed combination is that of a sulfonamide and an inhibitor of dihydrofolate reductase, such as trimethoprim this combination is synergistic because the drugs block sequential steps in microbial folate synthesis. 

Inhibitors of bacterial folate synthesis are considered here. Folate antagonists used as antineoplastic agents, i.e. methotrexate and lometrexol, are discussed in Section 3. 

Inhibit Enzymes Many drugs are competitive inhibitors of key enzymes in pathways. The statin drugs (lovastatin, simvastatin), used to control blood cholesterol levels, competitively inhibit 3-hvdroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase in cholesterol biosynthesis. Methotrexate, an antineoplastic drug, competitively inhibits dihydrofolate reductase, depriving the cell of active folate needed for purine and deoxythymidine synthesis, thus interfering with DNA replication during S phase. 

Pharmacology Trimetrexate, a 2.4-diaminoquinazoline, nonclassical folate antagonist, is a synthetic inhibitor of the enzyme dihydrofolate reductase. The end result is disruption of DNA, RNA, and protein synthesis, with consequent cell death. Pharmacokinetics Clearance was 38 15 ml /min/m and volume of distribution at steady state (Vdgs) was 20 8 L/m. The plasma concentration time profile declined... 

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. 

Other useful targets for pharmaceutical agents are thymidylate synthase and dihydrofolate reductase, enzymes that provide the only cellular pathway for thymine synthesis (Fig. 22-49). One inhibitor that acts on thymidylate synthase, fluorouracil, is an important chemotherapeutic agent. Fluorouracil itself is not the enzyme inhibitor. In the cell, salvage pathways convert it to the deoxynucleoside monophosphate FdUMP, which binds to and inactivates the enzyme. Inhibition by FdUMP (Fig. 22-50) is a classic example of mechanism-based enzyme inactivation. Another prominent chemotherapeutic agent, methotrexate, is an inhibitor of dihydrofolate reductase. This folate analog acts as a competitive inhibitor the enzyme binds methotrexate with about 100 times higher affinity than dihydrofolate. Aminopterin is a related compound that acts similarly. 

The dual alkylation/S Ar cyclization of A 1,A z-bis(2,4-dimethoxybenzyl)ethane-l,2-diamine (bis-DMB-ethylene-diamine) with the chloromethyl pyrimidine 102 provided the fused ring diazepine 103, a transformation that could not be accomplished by simply using unprotected ethylenediamine (Scheme 56). The product is an intermediate toward the synthesis of potential folate-related antitumor agents that act as glycinamide ribonucleotide formyltrans-ferase inhibitors <2004HC0405>. 

Methotrexate is a potent inhibitor of dihydrofolate reductase, with an affinity 1,000-fold greater than that of dUiydrofolate. Chemotherapy consists of alternating periods of administration of methotrexate and folate (normally as 5-formyl-tetrahydrofolate, leucovorin) to replete the normal tissues and avoid induction of folate deficiency- so-called leucovorin rescue. As well as depleting tissue pools of tetrahydrofolate, methotrexate leads to the accumulation of relatively large amounts of 10-formyl-dihydrofolate, which is apotentinhibitor of both thymidylate synthetase and glycinamide ribotide transformylase, an intermediate step in purine nucleotide synthesis. It is likely that this, rather than simple depletion of tetrahydrofolate, is the basis of the cytotoxic action of methotrexate (Barametal., 1988). 

---