Pyrimethamine induces phototoxicity in human keratinocytes via lysosomal and mitochondrial dependent signaling pathways under environmental UVA and UVB exposure.

Pyrimethamine (PYR) is used to treat parasitic infections including toxoplasmosis, pneumonia and cystoisosporiasis in HIV patients. Various oral medicines have shown phototoxicity therefore, we aimed to study the phototoxicity of PYR and its molecular mechanism involving stress responsive lysosomal protein Lamp2 and mitochondrial mediated signaling pathway under normal UVA/B exposure. We found that photodegradation and subsequent photoproduct formation was evident through LCMS/MS analysis. Photosensitized PYR produces ROS that cause damage to DNA, cell membrane and membrane bound organelles in human keratinocytes. PYR triggered cytotoxicity and phototoxicity that was evident through MTT and NRU assay respectively. Intracellular ROS generation caused phosphatidyl serine (PS) translocation in cell membrane, lysosome membrane permeabilization (LMP) and mitochondrial membrane potential (MMP) collapse that was further validated through caspase3 activation. DNA damage was measured as tail DNA formation and cell cycle arrest in G1 phase. Photosensitized PYR induces oxidative stress in the form of overexpression of Lamp2 that ultimately led to cellular apoptosis. Moreover, the effects of UVB were higher than UVA, probably due to its direct interaction with various macromolecules. We propose that photoexcited PYR may be harmful to human health even at normal sunlight exposure. Therefore, protective procedures should be practiced during PYR medication.

Discovery of novel STAT3 DNA binding domain inhibitors.

Background: STAT3 is a pro-oncogenic transcription factor. Pyrimethamine (PYM) is a STAT3 inhibitor that suppresses the proliferation of some cancer cells through downregulation of STAT3 target proteins. Methodology & Results: We have used structure-based tools to design novel PYM-based compounds. Intracellular target validation studies revealed that representative compounds 11b-d and 15a downregulate STAT3 downstream proteins and inhibit STAT3 DNA binding domain (DBD). Relative to PYM, a cohort of these compounds are >100-fold more cytotoxic to cancer cells with constitutively active (high pSTAT3) and basal (low pSTAT3) STAT3 signaling, suggesting that STAT3 DBD inhibition is deleterious to the proliferation of cancer cells with low and high pSTAT3 levels. Conclusion: These are promising leads for further preclinical evaluation as therapeutic agents for STAT3-dependent cancers.

Identification of Appropriate Endogenous Biomarker for Risk Assessment of Multidrug and Toxin Extrusion Protein-Mediated Drug-Drug Interactions in Healthy Volunteers.

Endogenous biomarkers are emerging to advance clinical drug-drug interaction (DDI) risk assessment in drug development. Twelve healthy subjects received a multidrug and toxin exclusion protein (MATE) inhibitor (pyrimethamine, 10, 25, and 75 mg) in a crossover fashion to identify an appropriate endogenous biomarker to assess MATE1/2-K-mediated DDI in the kidneys. Metformin (500 mg) was also given as reference probe drug for MATE1/2-K. In addition to the previously reported endogenous biomarker candidates (creatinine and N1 -methylnicotinamide (1-NMN)), N1 -methyladenosine (m1 A) was included as novel biomarkers. 1-NMN and m1 A presented as superior MATE1/2-K biomarkers since changes in their renal clearance (CLr ) along with pyrimethamine dose were well-correlated with metformin CLr changes. The CLr of creatinine was reduced by pyrimethamine, however, its changes poorly correlated with metformin CLr changes. Nonlinear regression analysis (CLr vs. mean total concentration of pyrimethamine in plasma) yielded an estimate of the inhibition constant (Ki ) of pyrimethamine and the fraction of the clearance pathway sensitive to pyrimethamine. The in vivo Ki value thus obtained was further converted to unbound Ki using plasma unbound fraction of pyrimethamine, which was comparable to the in vitro Ki for MATE1 (1-NMN) and MATE2-K (1-NMN and m1 A). It is concluded that 1-NMN and m1 A CLr can be leveraged as quantitative MATE1/2-K biomarkers for DDI risk assessment in healthy volunteers.

Metformin Transport in Native MDCK-Wt and MDCK-II Monolayers Unveils Functional Inter-Strains Differences Influencing Drug Permeability.

PURPOSE: MDCK cells are commonly used to assess drug permeability, but the existence of various strains merits a comparative functional study. Since metformin absorption is largely mediated by transporters and paracellular diffusion, we used it to functionally compare MDCK-wt and MDCK-II. METHODS: Uptake, bidirectional transport and efflux experiments were performed using different buffers, pH, and a panel of transporter inhibitors. Relative contributions to total transport in both strains were estimated. RESULTS: Metformin uptake into MDCK-wt was linear but saturable in MDCK-II. Uptake into MDCK-wt or -II was promoted at pH 5.4 or 8.4, respectively. Quinidine and cimetidine similarly inhibited uptake in both strains. Lopinavir (PMAT specific) at pH 5.4 or pyrimethamine (MATE specific) at pH 8.4 differentially inhibited MDCK-wt or -II, respectively. Transport at pH 7.4 was absorptive regardless of strains, but secretory (MDCK-II) or absorptive (MDCK-wt) at pH 5.4. Efflux was largely basolateral in both strains. While paracellular permeability was similar between strains, total transport was dominated by transporters in MDCK-II or paracellular diffusion in MDCK-wt. CONCLUSIONS: Metformin transport revealed functional differences between MDCK strains. Apical uptake was governed by MATE in MDCK-II or PMAT in MDCK-wt, such that metformin transport was either secretory or absorptive, respectively.

Cynomolgus Monkey as a Clinically Relevant Model to Study Transport Involving Renal Organic Cation Transporters: In Vitro and In Vivo Evaluation.

Organic cation transporter (OCT) 2, multidrug and toxin extrusion protein (MATE) 1, and MATE2K mediate the renal secretion of various cationic drugs and can serve as the loci of drug-drug interactions (DDI). To support the evaluation of cynomolgus monkey as a surrogate model for studying human organic cation transporters, monkey genes were cloned and shown to have a high degree of amino acid sequence identity versus their human counterparts (93.7, 94.7, and 95.4% for OCT2, MATE1, and MATE2K, respectively). Subsequently, the three transporters were individually stably expressed in human embryonic kidney (HEK) 293 cells and their properties (substrate selectivity, time course, pH dependence, and kinetics) were found to be comparable to the corresponding human form. For example, six known human cation transporter inhibitors, including pyrimethamine (PYR), showed generally similar IC50 values against the monkey transporters (within sixfold). Consistent with the in vitro inhibition of metformin (MFM) transport by PYR (IC50 for cynomolgus OCT2, MATE1, and MATE2K; 1.2 ± 0.38, 0.17 ± 0.04, and 0.25 ± 0.04 µM, respectively), intravenous pretreatment of monkeys with PYR (0.5 mg/kg) decreased the clearance (54 ± 9%) and increased in the area under the plasma concentration-time curve of MFM (AUC ratio versus control = 2.23; 90% confidence interval of 1.57 to 3.17). These findings suggest that the cynomolgus monkey may have some utility in support of in vitro-in vivo extrapolations (IVIVEs) involving the inhibition of renal OCT2 and MATEs. In turn, cynomolgus monkey-enabled IVIVEs may inform human DDI risk assessment.

Pyrimethamine Derivatives: Insight into Binding Mechanism and Improved Enhancement of Mutant ß-N-acetylhexosaminidase Activity.

In order to identify structural features of pyrimethamine (5-(4-chlorophenyl)-6-ethylpyrimidine-2,4-diamine) that contribute to its inhibitory activity (IC50 value) and chaperoning efficacy toward ß-N-acetylhexosaminidase, derivatives of the compound were synthesized that differ at the positions bearing the amino, ethyl, and chloro groups. Whereas the amino groups proved to be critical to its inhibitory activity, a variety of substitutions at the chloro position only increased its IC50 by 2-3-fold. Replacing the ethyl group at the 6-position with butyl or methyl groups increased IC50 more than 10-fold. Surprisingly, despite its higher IC50, a derivative lacking the chlorine atom in the para-position was found to enhance enzyme activity in live patient cells a further 25% at concentrations >100 µM, while showing less toxicity. These findings demonstrate the importance of the phenyl group in modulating the chaperoning efficacy and toxicity profile of the derivatives.

Factors affecting uptake of optimal doses of sulphadoxine-pyrimethamine for intermittent preventive treatment of malaria in pregnancy in six districts of Tanzania.

BACKGROUND: Intermittent preventive treatment during pregnancy (IPTp) with optimal doses (two+) of sulphadoxine-pyrimethamine (SP) protects pregnant women from malaria-related adverse outcomes. This study assesses the extent and predictors of uptake of optimal doses of IPTp-SP in six districts of Tanzania. METHODS: The data come from a cross-sectional survey of random households conducted in six districts in Tanzania in 2012. A total of 1,267 women, with children aged less than two years and who had sought antenatal care (ANC) at least once during pregnancy, were selected for the current analysis. Data analysis involved the use of Chi-Square (χ2) for associations and multivariate analysis was performed using multinomial logistic regression. RESULTS: Overall, 43.6% and 28.5% of the women received optimal (two+) and partial (one) doses of IPTp-SP respectively during pregnancy. Having had been counseled on the dangers of malaria during pregnancy was the most pervasive determinant of both optimal (RRR = 6.47, 95% CI 4.66-8.97) and partial (RRR = 4.24, 95% CI 3.00-6.00) uptake of IPTp-SP doses. Early ANC initiation was associated with a higher likelihood of uptake of optimal doses of IPTp-SP (RRR = 2.05, 95% CI 1.18-3.57). Also, women with secondary or higher education were almost twice as likely as those who had never been to school to have received optimal SP doses during pregnancy (RRR = 1.93, 95% CI 1.04-3.56). Being married was associated with a 60% decline in the partial uptake of IPTp-SP (RRR = 0.40, 95% CI 0.17-0.96). Inter-district variations in the uptake of both optimal and partial IPTp-SP doses existed (P < 0.05). CONCLUSION: Counseling to pregnant women on the dangers of malaria in pregnancy and formal education beyond primary school is important to enhance uptake of optimal doses of SP for malaria control in pregnancy in Tanzania. ANC initiation in the first trimester should be promoted to enhance coverage of optimal doses of IPTp-SP. Programmes should aim to curb geographical barriers due to place of residence to enhance optimal coverage of IPTp-SP in Tanzania.

Antifolates inhibit Cryptococcus biofilms and enhance susceptibility of planktonic cells to amphotericin B.

The Cryptococcus neoformans species complex contains the most important agents of fungal meningoencephalitis. Therapeutic choices are limited and issues related to toxicity and resistance to antifungals have been described. The present study evaluated the inhibitory effect of the antifolate combinations sulfamethoxazole-trimethoprim (SMX/TMP) and sulfadiazine-pyrimethamine (SDZ/PYR) against planktonic cells and biofilms of C. neoformans and C. gattii. The influence of the antifolate combinations on the amphotericin minimum inhibitory concentration (MIC) of planktonic cells was also investigated. In addition, the effect of these combinations on the cellular ergosterol content of planktonic cells was studied. Strains of C. neoformans (n = 15) and C. gattii (n = 15) obtained from environmental or clinical sources were evaluated by the broth microdilution method. SMX/TMP and SDZ/PYR showed antifungal activity against free living cells and sessile cells of Cryptococcus spp. Moreover, planktonic cells showed increased susceptibility to amphotericin B after pre-incubation with sub-inhibitory concentrations of SMX/TMP or SDZ/PYR. The drug combinations SMX/TMP and SDZ/PYR were able to prevent the biofilm formation and showed inhibitory effect against mature biofilms of both species. Additionally, the study showed that antifolate drugs reduced the ergosterol content in C. neoformans and C. gattii planktonic cells. Our results highlight the antifungal potential of antifolate drugs.

Comparison of protein patterns between Plasmodium falciparum mutant clone T9/94-M1-1(b3) induced by pyrimethamine and the original parent clone T9/94.

OBJECTIVE: To compare the protein patterns from the extracts of the mutant clone T9/94-M1-1(b3) induced by pyrimethamine, and the original parent clone T9/94 following separation of parasite extracts by two-dimensional electrophoresis (2-DE). METHODS: Proteins were solubilized and separated according to their charges and sizes. The separated protein spots were then detected by silver staining and analyzed for protein density by the powerful image analysis software. RESULTS: Differentially expressed protein patterns (up- or down-regulation) were separated from the extracts from the two clones. A total of 223 and 134 protein spots were detected from the extracts of T9/94 and T9/94-M1-1(b3) clones, respectively. Marked reduction in density of protein expression was observed with the extract from the mutant (resistant) clone compared with the parent (sensitive) clone. A total of 25 protein spots showed at least two-fold difference in density, some of which exhibited as high as ten-fold difference. CONCLUSIONS: These proteins may be the molecular targets of resistance of Plasmodium falciparum to pyrimethamine. Further study to identify the chemical structures of these proteins by mass spectrometry is required.

Polysaccharide-based hydrogels: preparation, characterization, and drug interaction behaviour.

Oxidized alginate (ADA) and oxidized alginate blended with chitosan (ADA-Chit) were prepared in the presence of borax and CaCl 2, and their interactions with an antifolate drug, pyrimethamine (PYR), have been investigated. Tablets with a mean diameter of 1.2 +/- 0.06 cm were produced and drug interactions were performed in dimethyl sulfoxide (DMSO) using isothermal titration calorimetry (ITC). From ITC responses, the enthalpy changes of interaction PYR/materials, Delta int H, have been determined and were found to be -11.73 +/- 0.517 kJ mol (-1) for ADA and -4.86 +/- 0.156 kJ mol (-1) for ADA-Chit. The PYR encapsulation of approximately 75% was achieved for both materials, as measured by UV spectrometer.

In vitro assessment of the pharmacodynamic properties and the partitioning of OZ277/RBx-11160 in cultures of Plasmodium falciparum.

OBJECTIVES: Using synchronous cultures of Plasmodium falciparum malaria, the stage sensitivity of the parasite to OZ277 (RBx-11160), the first fully synthetic antimalarial peroxide that has entered Phase II clinical trials, was investigated in vitro over a concentration range of 1 x to 100 x the IC50. Secondly, partitioning of OZ277 into P. falciparum-infected red blood cells (RBCs) and uninfected RBCs was studied in vitro by measuring its distribution between RBCs and plasma (R/P). METHODS: The effects of timed in vitro exposure (1, 6, 12 or 24 h) to OZ277 were monitored by incorporation of [3H]hypoxanthine into parasite nucleic acids and by light-microscopic analysis of parasite morphology. Partitioning studies were performed with radiolabelled [14C]OZ277. RESULTS: After 1 h of exposure to OZ277 at the highest concentration (100 x the IC50) followed by removal of the compound, the hypoxanthine assay showed that growth of mature stages of P. falciparum was reduced to below 20%. Young ring forms were slightly less sensitive (43% growth). Similar stage-specific profiles were found for the antimalarial reference compounds artemether and chloroquine. Strong inhibition (< or = 6% growth) of all parasite stages was observed when the parasites were exposed to each of the three compounds for 6 h or longer. After removal of the compounds, the parasites did not recover, indicating that the observed growth inhibitions were cytotoxic rather than cytostatic. Pyrimethamine was confirmed to be active exclusively against young schizonts. Light-microscopic analysis also demonstrated the specificity of pyrimethamine against the schizont forms and showed that OZ277, artemether and chloroquine attenuated parasite growth more rapidly than did pyrimethamine. The R/P for OZ277 was 1.5 for uninfected RBCs and up to 270 for infected RBCs. CONCLUSIONS: The present study indicates similar stage-specific profiles for OZ277 and for the more well-established antimalarial agents artemether and chloroquine. Secondly, the study describes a significant accumulation of radiolabelled OZ277 in P. falciparum-infected RBCs.

Structural basis for inhibition of histamine N-methyltransferase by diverse drugs.

In mammals, histamine action is terminated through metabolic inactivation by histamine N-methyltransferase (HNMT) and diamine oxidase. In addition to three well-studied pharmacological functions, smooth muscle contraction, increased vascular permeability, and stimulation of gastric acid secretion, histamine plays important roles in neurotransmission, immunomodulation, and regulation of cell proliferation. The histamine receptor H1 antagonist diphenhydramine, the antimalarial drug amodiaquine, the antifolate drug metoprine, and the anticholinesterase drug tacrine (an early drug for Alzheimer's disease) are surprisingly all potent HNMT inhibitors, having inhibition constants in the range of 10-100nM. We have determined the structural mode of interaction of these four inhibitors with HNMT. Despite their structural diversity, they all occupy the histamine-binding site, thus blocking access to the enzyme's active site. Near the N terminus of HNMT, several aromatic residues (Phe9, Tyr15, and Phe19) adopt different rotamer conformations or become disordered in the enzyme-inhibitor complexes, accommodating the diverse, rigid hydrophobic groups of the inhibitors. The maximized shape complementarity between the protein aromatic side-chains and aromatic ring(s) of the inhibitors are responsible for the tight binding of these varied inhibitors.

Crystal structure of dihydrofolate reductase from Plasmodium vivax: pyrimethamine displacement linked with mutation-induced resistance.

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 --> Arg + Ser-117 --> 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.

Linkage group selection: rapid gene discovery in malaria parasites.

The identification of parasite genes controlling phenotypes such as drug resistance, virulence, immunogenicity, and transmission is vital to malaria research. Classical genetic methods have achieved these goals only rarely and with difficulty. We describe here a novel genetic method, Linkage Group Selection (LGS), which achieves rapid de novo location of genes encoding selectable phenotypes of malaria parasites. A phenotype-specific selection pressure is applied to the uncloned progeny of a genetic cross between two malaria parasites that differ in the relevant phenotype. Selected and unselected progeny are analyzed using genome-wide quantitative genetic markers. Markers of the "sensitive" parent, which are reduced after selection, are sequenced and located in genomic databases. They are expected to be closely linked to gene(s) determining the phenotype under selection. We have validated LGS with the rodent malaria parasite Plasmodium chabaudi chabaudi using a phenotype, pyrimethamine resistance, whose controlling gene, that encoding dihydrofolate reductase (dhfr), is known. We show that molecular markers closely linked to dhfr, and only those linked to this gene, were reduced or removed by pyrimethamine treatment in accordance with the expectations of LGS.

Transfection studies to explore essential folate metabolism and antifolate drug synergy in the human malaria parasite Plasmodium falciparum.

Folate metabolism in Plasmodium falciparum is the target of important antimalarial agents. The biosynthetic pathway converts GTP to polyglutamated derivatives of tetrahydrofolate (THF), essential cofactors for DNA synthesis. Tetrahydrofolate can also be acquired by salvage mechanisms. Using a transfection system adapted to studying this pathway, we investigated modulation of dihydropteroate synthase (DHPS) activity on parasite phenotypes. Dihydropteroate synthase incorporates p-aminobenzoate (pABA) into dihydropteroate, the precursor of dihydrofolate. We were unable to obtain viable parasites where the dhps gene had been truncated. However, parasites where the protein was full-length but mutated at two key residues and having < 10% of normal activity were viable in folate-supplemented medium. Metabolic labelling showed that these parasites could still convert pABA to polyglutamated folates, albeit at a very low level, but they could not survive on pABA supplementation alone. This degree of disablement in DHPS also abolished the synergy of the antifolate combination pyrimethamine/sulfadoxine. These data indicate that DHPS activity above a low but critical level is essential regardless of the availability of salvageable folate and formally prove the role of this enzyme in antifolate drug synergy and folate biosynthesis in vivo. However, we found no evidence of a significant role for DHPS in folate salvage. Moreover, when biosynthesis was compromised by the absence of a fully functional DHPS, the parasite was able to compensate by increasing flux through the salvage pathway.

Placental transfer of pyrimethamine studied in an ex vivo placental perfusion model.

Pyrimethamine is used as and anti-infectious agent because of its antifolate properties. Its action is synergistic with that of dapsone and sulfamides on Toxoplasma gondii. The goal of the present study was to evaluate the placental transfer of pyrimethamine in an ex vivo model of perfused human placental cotyledon at term. Human placentas were perfused according to the slightly modified method of Schneider. The pyrimethamine fetal transfer rate was approximately 30%, while cotyledon clearance was about 1.8 ml/min. The placental transfer of pyrimethamine seems to be independent of the maternal concentrations of pyrimethamine, suggesting passive diffusion mechanisms or a nonsaturable active transport at the tested concentrations.

Multiple mechanisms of resistance to polyglutamatable and lipophilic antifolates in mammalian cells: role of increased folylpolyglutamylation, expanded folate pools, and intralysosomal drug sequestration.

Chinese hamster ovary PyrR100 cells display more than 1000-fold resistance to pyrimethamine (Pyr), a lipophilic antifolate inhibitor of dihydrofolate reductase. PyrR100 cells had wild-type DHFR activity, lost folate exporter activity, and had a 4-fold increased activity of a low pH folic acid transporter. Here we report on the marked alterations identified in PyrR100 cells compared with parental cells: 1) approximately 100-fold decreased folic acid growth requirement; 2) a 25-fold higher glucose growth requirement in Pyr-containing medium; 3) a 2.5- to 4.1-fold increase in folylpolyglutamate synthetase activity; 4) a 3-fold increase in the accumulation of [3H]folic acid and a 3-fold expansion of the intracellular folate pools; 5) a 4-fold increase in the activity of the lysosomal marker beta-hexoseaminidase, suggesting an increased lysosome number/PyrR100 cell; and 6) a small reduction in the steady-state accumulation of [3H]Pyr and no evidence of catabolism or modification of cellular [3H]Pyr. Consequently, PyrR100 cells were markedly resistant to the lipophilic antifolates trimetrexate (40-fold) and AG377 (30-fold) and to the polyglutamatable antifolates 5,10-Dideaza-5,6,7,8-tetrahydrofolic acid (DDATHF) (26-fold) and AG2034 (14-fold). Resistance to these drugs was reversed in PyrR100 cells transferred into folate-depleted medium. In conclusion, these multiple resistance factors collectively result in a prominent increase in folate accumulation, an expansion of the intracellular folylpolyglutamate pool, and abolishment of the cytotoxic activity of polyglutamatable and lipophilic antifolates. The role of increased lysosome number per cell in sequestration of hydrophobic weak base drugs such as Pyr is also discussed as a novel mechanism of drug resistance.

[Effect of nitroquine on the membrane phospholipid of intraerythrocytic Plasmodium yoelii in vitro].

AIM: To study the mechanism of antimalarial action of nitroquine. METHODS: Intraerythrocytic P. yoelii was cultured by the method of Trager and Jensen. The amount of [3H]-ethanolamine incorporation was measured as an index of the phospholipid synthesis. DPH was used as a probe to measure the plasmodial fluorescent polarization. RESULTS: The incorporation of [3H]-ethanolamine into the P. yoelii infected erthrocytes was markedly inhibited by nitroquine. The plasmodial membrane polarization and viscosity were significantly increased by nitroquine. CONCLUSION: Nitroquine could inhibit the phospholipid synthesis and decrease the membrane fluidity of P. yoelii.

Sulfadoxine resistance in the human malaria parasite Plasmodium falciparum is determined by mutations in dihydropteroate synthetase and an additional factor associated with folate utilization.

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.