Mutations in the dihydrofolate reductase (dhfr) genes of Plasmodium falciparum and P. vivax are associated with resistance to the antifolate antimalarial drugs. P. vivax dhfr sequences were obtained from 55 P. vivax isolates (isolates Belem and Sal 1, which are established lines originating from Latin America, and isolates from patient samples from Thailand [n ‫؍‬ 44], India [n ‫؍‬ 5], Iran [n ‫؍‬ 2], and Madagascar [n ‫؍‬ 2]) by direct sequencing of both strands of the purified PCR product and were compared to the P. vivax dhfr sequence from a P. vivax parasite isolated in Pakistan (isolate ARI/Pakistan), considered to represent the wild-type sequence. In total, 144 P. vivax dhfr mutations were found at only 12 positions, of which 4 have not been described previously. An F3L mutation at residue 57 had been observed previously, but a novel codon (TTA) resulted in a mutation in seven of the nine mutated variant sequences. A new mutation at residue 117 resulted in S3T (S3N has been described previously). These two variants are the same as those observed in the P. falciparum dhfr gene at residue 108, where they are associated with different levels of antifolate resistance. Two novel mutations, I3L at residue 13 and T3M at residue 61, appear to be unique to P. vivax. The clinical, epidemiological, and sequence data suggest a sequential pathway for the acquisition of the P. vivax dhfr mutations. Mutations at residues 117 and 58 arise first when drug pressure is applied. Highly mutated genes carry the S3T rather than the S3N mutation at residue 117. Mutations at residues 57 and 61 then occur, followed by a fifth mutation at residue 13.With the global spread of chloroquine resistance in Plasmodium falciparum, the combination of sulfadoxine and pyrimethamine (S-P) is now used as first-line antimalarial treatment in an increasing number of countries. This is usually followed rapidly by the emergence of resistance. For example, in Thailand, high-level resistance to S-P arose within 5 years of its deployment in the late 1970s (14, 21). Similar patterns have been observed in other Asian countries, South America, and southern Africa. The combination of antifolate and sulfonamide drugs targets the folate metabolism of the malaria parasite. Molecular clinical and epidemiological studies have clearly shown that resistance to pyrimethamine and sulfadoxine results from specific point mutations in the parasite genes dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps), respectively. These cause alterations in crucial residues in the active sites of these enzymes, resulting in reduced drug affinity (5,11,13,(17)(18)(19)(20). Detection of these mutations in isolates in blood samples collected in the field has proved very valuable in the mapping of resistance and the monitoring of malaria control measures.In areas of malaria endemicity outside Africa, the prevalence of P. vivax equals and often exceeds that of P. falciparum. Chloroquine remains the first-line treatment for P. vivax infection, although in most areas wher...

Pyrimethamine (Pyr) targets dihydrofolate reductase of Plasmodium vivax (PvDHFR) as well as other malarial parasites, but its use as antimalarial is hampered by the widespread high resistance. Comparison of the crystal structures of PvDHFR from wild-type and the Pyr-resistant (SP21, Ser-58 3 Arg ؉ Ser-117 3 Asn) strain as complexes with NADPH and Pyr or its analog lacking p-Cl (Pyr20) clearly shows that the steric conflict arising from the side chain of Asn-117 in the mutant enzyme, accompanied by the loss of binding to Ser-120, is mainly responsible for the reduction in binding of Pyr. Pyr20 still effectively inhibits both the wild-type and SP21 proteins, and the x-ray structures of these complexes show how Pyr20 fits into both active sites without steric strain. These structural insights suggest a general approach for developing new generations of antimalarial DHFR inhibitors that, by only occupying substrate space of the active site, would retain binding affinity with the mutant enzymes.drug resistance ͉ malaria ͉ antifolates A major share of the global malaria burden, with an estimated 80 million cases annually, is caused by Plasmodium vivax (Pv) (1, 2). The problem has recently been worsened by emergence of resistance of the parasite to chloroquine (3-6). Pyrimethamine (Pyr) and other antifolates are generally not used against vivax malaria, because of the resistance of the parasite, which has commonly been considered to be inherent (7). It recently has been shown, however, that wild-type (WT) P. vivax dihydrofolate reductase (PvDHFR), the target of antifolates, is susceptible to inhibition by Pyr and other antifolates (8-11), whereas for the mutant parasites, of which the common one is the double mutant (SP21, Ser-58 3 Arg ϩ Ser-117 3 Asn), the affinities for binding with the antifolates are much reduced, rendering them ineffective. A similar, but less severe, situation was shown for Plasmodium falciparum (Pf), in which the homologous double mutant (K1, Cys-59 3 Arg ϩ Ser-108 3 Asn) PfDHFR has reduced affinities for the antifolates (12-14). In the case of P. falciparum, there was only a moderate reduction in affinity (50-to 90-fold) relative to the WT enzyme, and a moderate level of resistance ensued, which was augmented by further mutations at other sites (14-17). The crystal structures of the bifunctional enzyme dihydrofolate reductase (DHFR)-thymidylate synthase (TS) of P. falciparum (PfDHFR-TS) revealed basic structural features of Plasmodial DHFR-TS, including the insert regions and the junction region (18).Comparison of the structures of the WT and mutant forms of PfDHFR-TS demonstrates that Pyr is involved in steric conflict with the side chain of Asn-108, resulting in antifolate resistance (13,19,20). However, the steric conflict did not appear to result in major displacement of Pyr in the active site of P. falciparum. In contrast, analogous mutations in P. vivax resulted in relatively larger reduction in binding affinity of Pyr. Because the crystal structure of PvDHFR, either of the WT or the SP21...

Several of the enzymes related to the folate cycle are well-known for their role as clinically validated antimalarial targets. Nevertheless for serine hydroxymethyltransferase (SHMT), one of the key enzymes of this cycle, efficient inhibitors have not been described so far. On the basis of plant SHMT inhibitors from an herbicide optimization program, highly potent inhibitors of Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) SHMT with a pyrazolopyran core structure were identified. Cocrystal structures of potent inhibitors with PvSHMT were solved at 2.6 Å resolution. These ligands showed activity (IC50/EC50 values) in the nanomolar range against purified PfSHMT, blood-stage Pf, and liver-stage P. berghei (Pb) cells and a high selectivity when assayed against mammalian cell lines. Pharmacokinetic limitations are the most plausible explanation for lack of significant activity of the inhibitors in the in vivo Pb mouse malaria model.