Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Sulphur-based antimalarial drugs targeted at dihydropteroate synthetase (DHPS) are frequently used in synergistic combination with inhibitors of dihydrofolate reductase (DHFR) to combat chloroquine-resistant malaria. We have previously shown that lines of Plasmodium falciparum resistant to the most commonly used sulpha drug, sulphadoxine, carry point mutations in the DHPS coding region, relative to the sequence of sensitive strains (Brooks et al., Eur. J. Biochem. 224 (1994) 397-405). We have now developed PCR diagnostic assays based on allele-specific amplification that are able to detect such mutations. The four tests described can reliably discriminate all of the mutations observed to alter codons 436, 581 and 613, yielding allele-specific amplification products of different sizes in each case. Moreover, by careful adjustment of primer length and the degree of mismatch to target and non-target alleles, we were able to standardise all four tests to a single set of PCR conditions, allowing all possible mutations to be monitored simultaneously on one thermocycler. These assays should prove invaluable in further assessing the contribution of specific base changes in the DHPS gene of the parasite to the sulphadoxine resistance phenotype and to the clinical failure of the sulphadoxine/pyrimethamine combination Fansidar.
Mol Biochem Parasitol 1995 Apr
PMID:A mutation-specific PCR system to detect sequence variation in the dihydropteroate synthetase gene of Plasmodium falciparum. 763 Mar 75

Sulfadoxine/pyrimethamine (Fansidar) is widely used in Africa for treating chloroquine-resistant falciparum malaria. To clarify how parasite resistance to this combination arises, various lines of Plasmodium falciparum were used to investigate the role of naturally occurring mutations in the target enzyme, dihydropteroate synthetase (DHPS), in the parasite response to sulfadoxine inhibition. An improved drug assay was employed to identify a clear correlation between sulfadoxine-resistance levels and the number of DHPS mutations. Moreover, tight linkage was observed between DHPS mutations and high-level resistance in the 16 progeny of a genetic cross between sulfadoxine-sensitive (HB3) and sulfadoxine-resistant (Dd2) parents. However, we also demonstrate a profound influence of exogenous folate on IC50 values, which, under physiological conditions, may have a major role in determining resistance levels. Importantly, this phenotype does not segregate with dhps genotypes in the cross, but shows complete linkage to the two alleles of the dihydrofolate reductase (dhfr) gene inherited from the parental lines. However, in unrelated lines, this folate effect correlates less well with DHFR sequence, indicating that the gene responsible may be closely linked to dhfr, rather than dhfr itself. These results have major implications for the acquisition of Fansidar resistance by malaria parasites.
Mol Microbiol 1997 Mar
PMID:Sulfadoxine resistance in the human malaria parasite Plasmodium falciparum is determined by mutations in dihydropteroate synthetase and an additional factor associated with folate utilization. 907 34

Resistance of Plasmodium falciparum to antifolate chemotherapy is a significant problem where combinations such as Fansidar (pyrimethamine-sulfadoxine; PYR-SDX) are used in the treatment of chloroquine-resistant malaria. Antifolate resistance has been associated with variant sequences of dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS), the targets of PYR and SDX respectively. However, while the nature and distribution of mutations in the dhfr gene are well established, this is not yet the case for dhps. We have thus examined by DNA sequence analysis 141 field samples from several geographical regions with differing Fansidar usage (West and East Africa, the Middle East and Viet Nam) to establish a database of the frequency and repertoire of dhps mutations, which were found in 60% of the samples. We have also simultaneously determined from all samples their dhfr sequences, to better understand the relationship of both types of mutation to Fansidar resistance. Whilst the distribution of mutations was quite different across the regions surveyed, it broadly mirrored our understanding of relative Fansidar usage. In samples taken from individual patients before and after drug treatment, we found an association between the more highly mutated forms of dhps and/or dhfr and parasites that were not cleared by antifolate therapy. We also report a novel mutation in a Pakistani sample at position 16 of DHFR (A16S) that is combined with the familiar C59R mutation, but is wild-type at position 108. This is the first observation in a field sample of a mutant dhfr allele where the 108 codon is unchanged.
Mol Biochem Parasitol 1997 Nov
PMID:Resistance to antifolates in Plasmodium falciparum monitored by sequence analysis of dihydropteroate synthetase and dihydrofolate reductase alleles in a large number of field samples of diverse origins. 936 63

The antifolate combination pyrimethamine/sulphadoxine (PYR/SDX; Fansidar) is frequently used to combat chloroquine-resistant malaria. Its success depends upon pronounced synergy between the two components, which target dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) in the folate pathway. This synergy permits clearance of parasites resistant to either drug alone, but its molecular basis is still unexplained. Plasmodium falciparum can use exogenous folate, which is normally present in vivo, bypassing SDX inhibition of DHPS and, apparently, precluding synergy under these conditions. However, we have measured parasite inhibition by SDX/PYR combinations in assays in which folate levels are strictly controlled. In parasites that use exogenous folate efficiently, SDX inhibition can be restored by levels of PYR significantly lower than those required to inhibit DHFR. Isobolograms show that the degree of synergy between PYR and SDX is highly dependent upon prevailing folate concentrations and are indicative of PYR acting to block folate uptake and/or utilization. No significant synergy was observed at physiological drug levels when PYR/SDX acted on purified DHFR, whether wild type or mutant. We conclude that the primary basis for antifolate synergy in these organisms arises from PYR targeting a site (or sites) in addition to DHFR, which restores DHPS as a relevant target for SDX.
Mol Microbiol 1999 Jun
PMID:Utilization of exogenous folate in the human malaria parasite Plasmodium falciparum and its critical role in antifolate drug synergy. 1038 65

We have expressed dhfr alleles of Plasmodium falciparum in the budding yeast, Saccharomyces cerevisiae, and used this yeast model to identify single amino acid substitutions that confer high level pyrimethamine resistance on the background of the triple mutant dhfr (I51+R59+N108). Mutations in three clusters were identified: codons 50-57, 187-193, and 213-214. Several mutations previously identified in field samples were also isolated, including codons 50 and 164. The I164L mutation is of particular interest, because the quadruple mutant genotype (N51I+C59R+S108N+I164L) encodes an enzyme that is no longer inhibited by pyrimethamine, rendering sulfadoxine/pyrimethamine (SP; Fansidar) clinically ineffective. Thirty-six novel alleles were tested to determine their sensitivity to chlorcycloguanil and WR99210, two DHFR inhibitors that are in clinical and pre-clinical development, respectively. Chlorcycloguanil is effective against parasites that carry the triple mutant allele, but in vitro analysis has suggested that chlorcycloguanil will be clinically ineffective against parasites that carry the quadruple mutant allele of dhfr. In our screen, 23 of 36 novel strains were as resistant to chlorcycloguanil as the quadruple mutant, and one strain was 10-fold more resistant. WR99210 is still effective in the nM range against parasites that carry the quadruple mutant allele. In the preliminary screen, 31 of 36 novel alleles were as sensitive to WR99210 as the quadruple mutant. Detailed analysis of the remaining five showed that four of the five had IC(50) values in the same range as the quadruple mutant, and one, N51I+C59R+S108N+E192G, had an IC(50) value about fivefold higher. This result suggests that WR99210 and related compounds will be clinically effective against quadruple mutants currently found in Southeast Asia and South America and against most novel alleles that could be selected on the background of the triple mutant genotype now prevalent in East Africa.
Mol Biochem Parasitol 2001 Sep 28
PMID:Novel alleles of the Plasmodium falciparum dhfr highly resistant to pyrimethamine and chlorcycloguanil, but not WR99210. 1155 35