Mutant Dihydrofolate Reductase Research Articles

Immobilization of Malarial (Plasmodium falciparum) Dihydrofolate Reductase for the Selection of Tight-Binding Inhibitors from Combinatorial Library

A simple procedure for selection of tight-binding inhibitors of mutant dihydrofolate reductases from Plasmodium falciparum (PfDHFRs) based on preferential binding to the enzyme immobilized on a Sepharose column has been described. PfDHFRs with a cysteine residue at the C-terminal have been prepared in order to immobilize to a thiopropyl-Sepharose gel via S-S linkage. The amount of immobilized DHFRs was estimated to be 4-5 mg/g of dried gel, and the activities of bound DHFRs were comparable to that of free enzymes. The prepared immobilized enzyme has been used for the selection of tight-binding inhibitors from combinatorial libraries, based on the affinities of each ligand with the enzyme. Free ligands were then identified and analyzed quantitatively by high-performance liquid chromatography-mass spectrometry, and the components with high binding affinity of the library could thus be realized. Results could be confirmed by quantitative analysis of the bound ligands released from the enzyme by guanidine hydrochloride treatment.

The Catalytic Effect of Dihydrofolate Reductase and Its Mutants Is Determined by Reorganization Energies

The effect of distant mutations on the catalytic reaction of dihydrofolate reductase (DHFR) is reexamined by empirical valence bond simulations. The simulations reproduce for the first time the changes in the observed rate constants (without the use of adjustable parameters for this purpose) and show that the changes in activation barriers are strongly correlated with the corresponding changes in the reorganization energy. The preorganization of the polar groups of enzymes is the key catalytic factor, and anticatalytic mutations destroy this preorganization. Some anticatalytic mutations in DHFR also increase the distance between the donor and acceptor, but this effect is not directly related to catalysis since the native enzyme and the uncatalyzed reaction in water have similar average donor-acceptor distances. Insight into the effect of a mutation is provided by constructing the relevant free energy surfaces in terms of the generalized solute-solvent coordinates. It is shown how the mutations change the reaction coordinate and the activation barrier, and it is clarified that the corresponding changes do not reflect dynamical effects. It is also pointed out that all reactions in a condensed phase involve correlated motions (both in enzymes and in solution) and that the change of such motions upon mutations is a result of the change in the shape of the multidimensional reaction path on the solute-solvent surface, rather than the reason for the change in rate constant. Thus, as far as catalysis is concerned, the change in the activation barrier is due to the change in the electrostatic preorganization energy.

SULFADOXINE–PYRIMETHAMINE EFFICACY AND SELECTION OF PLASMODIUM FALCIPARUM DHFR MUTATIONS IN BURKINA FASO BEFORE ITS INTRODUCTION AS INTERMITTENT PREVENTIVE TREATMENT FOR PREGNANT WOMEN

Sulfadoxine-pyrimethamine efficacy was determined with a 28-day follow-up in 97 children between 6 months and 15 years of age. The polymerase chain reaction (PCR)-corrected treatment failure was 8.2% and the uncorrected was 21.6%. The presence of the dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) mutations linked to sulfadoxine-pyrimethamine resistance before and after treatment was determined by PCR-restriction fragment length polymorphism (RFLP) and by a fluorogenic PCR assay. Before treatment, the prevalence of the triple DHFR mutations was higher among the patients having had a recurrent parasitemia (either recrudescence or new infection; 28.6% versus 9.3%), although the difference was not significant (P = 0.1). The double mutation Ala-436/Gly-437 was observed in 67% of samples, whereas no Glu-540 mutation was found. After treatment, the triple DHFR mutation was found in 76.2% of patients with recurrent parasitemia, recrudescence, and new infection alike. Such high prevalence of mutant parasites indicates that sulfadoxine-pyrimethamine should not be used as monotherapy.

Stabilization of Hyperactive Dihydrofolate Reductase by Cyanocysteine-mediated Backbone Cyclization

Stabilization of an enzyme while maintaining its activity has been a major challenge in protein chemistry. Although it is difficult to simultaneously improve stability and activity of a protein by amino acid substitutions due to the activity-stability trade-off, backbone cyclization by connecting the N and C termini with a linker is promising as a general method of stabilizing a protein without affecting its activity. Recently, we created a hyperactive, methionine- and cysteine-free mutant of dihydrofolate reductase from Escherichia coli, called ANLYF, by introducing seven amino acid substitutions, which, however, destabilized the protein. Here we show that ANLYF is stabilized without a loss of its high activity by a novel backbone cyclization method for unprotected proteins. The method is based on the in vitro cyanocysteine-mediated intramolecular ligation reaction, which can be conducted with relatively high efficiency by a simple procedure and under mild conditions. We also show that the reversibility of thermal denaturation is highly improved by the cyclization. Thus, activity and stability of the protein can be separately improved by amino acid substitutions and backbone cyclization, respectively. We suggest that the cyanocysteine-mediated cyclization method is complementary to the intein-mediated cyclization method in stabilizing a protein without affecting its activity.

Emergence of mutations associated with resistance to Sulfadoxine-Pyrimethamine (SP) after single therapeutic dose: Implications on the useful therapeutic life of SP in malaria holoedemic areas

Background: Drugs with long elimination half-lives such as Sulfadoxine –Pyrimethamine (SP) maintain sub-curative levels in blood for a long time such that in high malaria transmission areas, re-infecting parasites are continuously under selection pressure for resistant genotypes. Objective: To assess SP efficacy and post therapeutic in-vivo selection for Plasmodium falciparum dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) mutations associated with SP resistance. Design: SP efficacy trial with prospective follow up for selection of parasites with DHFR and DHPS mutations at re-infection in the resistance selection period (RSP). Subjects: Children aged < 5 years attending the outpatient reproductive and child health clinic of Kibaha district hospital with uncomplicated malaria fulfilling the inclusion criteria for efficacy trials in holoendemic settings. Main outcome measures: Clinical & parasitological efficacy, pre-treatment and post-treatment prevalence of P. falciparum DHFR & DHPS mutations. Results: Very high (98.2%) clinical & parasitological cure rates. DHFR single, double or triple mutations occurred in 46.7% of pre-treatment infections; triple c108/51/59 & double c108/51 mutations being commonest. Few (15.9%) DHPS mutations occurred in pre-treatment infections at c436 and c437. DHFR & DHPS mutations were significantly higher in post- than pre- SP treatment parasites. In a Poisson regression analysis, DHFR mutations at c108, c51 & c59 and the exclusive c108/51/59 triple mutations were strongly associated with exposure to SP at re-infection. Conclusion: DHFR & DHPS mutations associated with SP resistance exist in P. falciparum infections in a background of high SP efficacy. Despite optimal dosage, in holoendemic areas, these mutations will be selected by SP at re-infection; cumulatively shortening the useful therapeutic life of SP due to resistance.

Similar Trends of Pyrimethamine Resistance-Associated Mutations in Plasmodium vivax and P. falciparum

The antifolate drugs sulfadoxine and pyrimethamine are commonly used to treat Plasmodium falciparum malaria. However, they can also affect the Plasmodium vivax parasite if it coexists with P. falciparum, as both species have common drug targets. Resistance to the antifolate drugs arises due to point mutations in the target enzymes of the respective parasite. To assess the cross-species impact of antifolate drug treatment, we describe here the dihydrofolate reductase (DHFR) mutations among field isolates of P. vivax and P. falciparum. The overall DHFR mutation rate for P. vivax was lower than that for P. falciparum. However, both species of Plasmodium followed similar trends of DHFR mutations. Similar to P. falciparum, the DHFR mutation rate of P. vivax also varied from region to region. It was lower in P. vivax-dominant regions but higher in the P. falciparum-dominated areas and highest where antifolates are used as the first line of antimalarial treatment. In conclusion, the antifolate treatment of falciparum malaria is proportionately affecting the DHFR mutations of P. vivax, suggesting that the drug should be used with caution to minimize the development of cross-species resistance in the field.

Genetic characterization of the dihydrofolate reductase gene of Pneumocystis jirovecii isolates from Portugal

The aim of the present study was to evaluate the genetic variation of Pneumocystis jirovecii dihydrofolate reductase (DHFR) gene in an immunocompromised Portuguese population and to investigate the possible association between DHFR genotypes and P. jirovecii pneumonia (PcP) prophylaxis with co-trimoxazole. One hundred and thirty-eight P. jirovecii isolates were submitted to DHFR genetic characterization by PCR and sequencing. In the studied population, 72.7% of the patients presented sequences identical to the wild-type sequence of the P. jirovecii DHFR gene and 27.3% presented point substitutions. A total of nine substitution sites were identified; four synonymous substitutions at nucleotide positions 201, 272, 312 and 381 were detected in 31 patients. Five non-synonymous substitutions were observed, leading to the DHFR mutations Leu-13-->Ser, Asn-23-->Ser, Ser-31-->Phe, Met-52-->Leu and Ala-67-->Val. With the exception of the polymorphism at position 312 and the mutation at codon 52, all polymorphisms were reported in this study for the first time. Our results suggest that DHFR gene polymorphisms are frequent in the Portuguese immunocompromised population but do not seem to be associated with PcP prophylaxis failure (P = 0.748 and P = 0.730).

Molecular genetic transfection of the coccidian parasite Sarcocystis neurona

Sarcocystis neurona is an apicomplexan parasite that is the major cause of equine protozoal myeloencephalitis (EPM). The biology of this pathogen remains poorly understood in part due to unavailability of molecular genetic tools. Hence, with an objective to develop DNA transfection capabilities for S. neurona, the 5′ flanking region of the SnSAG1 gene was isolated from a genomic library and used to construct expression plasmids. In transient assays, the reporter molecules β-galactosidase (β-gal) and yellow fluorescent protein (YFP) could be detected in electroporated S. neurona, thereby confirming the feasibility of transgene expression in this organism. Stable transformation of S. neurona was achieved using a mutant dihydrofolate reductase thymidylate synthase (DHFR-TS) gene of Toxoplasma gondii that confers resistance to pyrimethamine. This selection system was used to create transgenic S. neurona that stably express β-gal and YFP. As shown in this study, these transgenic clones can be useful for analyzing growth rate of parasites in vitro and for assessing drug sensitivities. More importantly, the DNA transfection methods described herein should greatly facilitate studies examining intracellular parasitism by this important coccidian pathogen.

Plasmodium falciparum infection dynamics and transmission potential following treatment with sulfadoxine-pyrimethamine

To investigate the overall efficacy of sulfadoxine-pyrimethamine (SP) treatment and the corresponding transmission potential for patients infected with SP-resistant Plasmodium falciparum. A mathematical model of the in-host dynamics of P. falciparum infections was used to simulate infections with parasites having different numbers of mutations in the dhfr and dhps genes and their responses to SP treatment. The treatment outcome and transmission potential of each simulated infection following SP treatment was assessed by tracking asexual parasite density and the number of days with sufficient mature gametocytes to give a > 95% probability of infecting a mosquito. The results show treatment failure only occurring in patients infected with parasites having two mutations in dihydrofolate reductase (DHFR) combined with at least two mutations in dihydropteroate synthetase (DHPS) or with parasites having a triple mutation in DHFR. Highly mutated parasites (three mutations in each gene) caused the highest clinical failure rate, while moderately mutated parasites (three mutations in DHFR plus one mutation in DHPS) produced a high rate of asymptomatic parasitological failure following SP treatment. This high rate of asymptomatic recrudescence caused the transmission potential of infections with moderately resistant parasites to exceed that of highly resistant parasites. The model output suggests that infection dynamics following SP treatment and the overall transmission potential are inherently linked. The combination of prolonged asymptomatic parasitaemia and increased transmission potential allows parasites having three mutations in DHFR, but fewer mutations in DHPS, to expand largely unnoticed.

Evolutional Design of a Hyperactive Cysteine- and Methionine-free Mutant of Escherichia coli Dihydrofolate Reductase

We developed a strategy for finding out the adapted variants of enzymes, and we applied it to an enzyme, dihydrofolate reductase (DHFR), in terms of its catalytic activity so that we successfully obtained several hyperactive cysteine- and methionine-free variants of DHFR in which all five methionyl and two cysteinyl residues were replaced by other amino acid residues. Among them, a variant (M1A/M16N/M20L/M42Y/C85A/M92F/C152S), named as ANLYF, has an approximately seven times higher k(cat) value than wild type DHFR. Enzyme kinetics and crystal structures of the variant were investigated for elucidating the mechanism of the hyperactivity. Steady-state and transient binding kinetics of the variant indicated that the kinetic scheme of the catalytic cycle of ANLYF was essentially the same as that of wild type, showing that the hyperactivity was brought about by an increase of the dissociation rate constants of tetrahydrofolate from the enzyme-NADPH-tetrahydrofolate ternary complex. The crystal structure of the variant, solved and refined to an R factor of 0.205 at 1.9-angstroms resolution, indicated that an increased structural flexibility of the variant and an increased size of the N-(p-aminobenzoyl)-L-glutamate binding cleft induced the increase of the dissociation constant. This was consistent with a large compressibility (volume fluctuation) of the variant. A comparison of folding kinetics between wild type and the variant showed that the folding of these two enzymes was similar to each other, suggesting that the activity enhancement of the enzyme can be attained without drastic changes of the folding mechanism.

Drosophila dihydrofolate reductase mutations confer antifolate resistance to mammalian cells

Antifolates, such as methotrexate, are used to inhibit dihydrofolate reductase (DHFR), an enzyme essential for the biosynthesis of thymidylate, purines, and several amino acids. DHFR sequences corresponding to mutations found in a methotrexate resistant Drosophila S3 cell line (L30Q), a methotrexate resistant fly population (K31P, Q134K), as well as predicted in silico (L22R) were expressed in Chinese Hamster Ovary (CHO) cells. The L30Q and L22R DHFRs both conferred resistance to methotrexate. L22R DHFR provided ∼200-fold resistance to methotrexate when compared to wild-type Drosophila DHFR allowing CHO L22R cells to divide in 10 μM methotrexate, a level of resistance not previously observed in any mammalian system. Constructs using this substitution in combination with other Drosophila DHFR specific residues would make excellent candidates for gene therapy and genetic markers in the treatment of certain human disorders.

Conformation Coupled Enzyme Catalysis: Single-Molecule and Transient Kinetics Investigation of Dihydrofolate Reductase

Ensemble kinetics and single-molecule fluorescence microscopy were used to study conformational transitions associated with enzyme catalysis by dihydrofolate reductase (DHFR). The active site loop of DHFR was labeled with a fluorescence quencher, QSY35, at amino acid position 17, and the fluorescent probe, Alexa555, at amino acid 37, by introducing cysteines at these sites with site-specific mutagenesis. The distance between the probes was such that approximately 50% fluorescence resonance energy transfer (FRET) occurred. The double-labeled enzyme retained essentially full catalytic activity, and stopped-flow studies of both the forward and reverse reactions revealed that the distance between probes increased prior to hydride transfer. A fluctuation in fluorescence intensity of single molecules of DHFR was observed in an equilibrium mixture of substrates but not in their absence. Ensemble rate constants were derived from the distributions of lifetimes observed and attributed to a reversible conformational change. Studies were carried out with both NADPH and NADPD as substrates, with no measurable isotope effect. Similar studies with a G121V mutant DHFR resulted in smaller rate constants. This mutant DHFR has reduced catalytic activity, so that the collective data for the conformational change suggest that the conformational change being observed is associated with catalysis and probably represents a conformational change prior to hydride transfer. If the change in fluorescence is attributed to a change in FRET, the distance change associated with the conformational change is approximately 1-2 A. These results are correlated with other measurements related to conformation coupled catalysis.

Plasmodium vivax dihydrofolate reductase point mutations from the Indian subcontinent

Mutations in Dihydrofolate Reductase ( dhfr) gene of Plasmodium vivax are known to be associated with resistance to antifolate drugs. To analyze the extent of these mutations in P. vivax population in India, dhfr gene was isolated and sequenced for 121 P. vivax isolates originating from different geographical regions of Indian subcontinent. These sequences were compared with the gene sequence that represent wild type sequence (accession no. X98123). P. vivax dhfr ( Pvdhfr) sequences showed limited polymorphism and about 70% isolates showed wild type dhfr sequence. A total of 36 mutations were found at 11 positions in 121 isolates. A majority of mutant isolates showed double mutations at residues 58 (S → R) and 117 (S → N), known to be associated with pyrimethamine resistance, but only 19% showed double mutations at residues 57 (F → L) and 58 (S → R). Pvdhfr alleles showing quadruple mutation (F57L, S58R, T61M andS117T) were found in two isolates. Three other mutations reported earlier at residue 13, 33 and 173 were not found in any of the Isolates. Six novel mutations at residues 38 (R → G), 93 (S → C), 109 (S → H), 131 (R → G), 159 (V → A) and 188 (I → V) were observed in seven isolates. Whether these novel mutations are linked to pyrimethamine resistance remains to be established.

Antimalarial Activities of New Pyrrolo[3,2-f]Quinazoline-1,3-Diamine Derivatives

WR227825 is an antimalarial pyrroloquinazolinediamine derivative with a high potency but a low therapeutic index. A series of carbamate, carboxamide, succinimide, and alkylamine derivatives of WR227825 were prepared to search for compounds with an improved therapeutic index. The new acetamides and imide showed potent cell growth inhibition against four clones of Plasmodium falciparum (D-6, RCS, W-2, and TM91C235), with a 50% inhibitory concentration of approximately 0.01 ng/ml, and were highly active against Plasmodium berghei, with 100% cure at doses from <0.1 mg/kg of body weight to 220 mg/kg. The carbamates and alkyl derivatives, however, showed weak activity against Plasmodium falciparum cell growth but were highly efficacious in tests against P. berghei by the Thompson test. The best compounds, bis-ethylcarbamate (compound 2a) and tetra-acetamide (3a) derivatives, further demonstrated high potency against the sporozoite Plasmodium yoelii in mice and P. falciparum and Plasmodium vivax in aotus monkeys. Against the AMRU-1 strain of P. vivax, which has four dihydrofolate reductase mutations and is highly resistant to antifolates, tetra-acetamide 3a cured the monkeys at doses of 1 and 3 mg/kg. Compound 2a cured only one out of two monkeys at 3 mg/kg. The results indicated that the new derivatives 2a and 3a not only have retained/improved the antimalarial efficacy of the parent compound WR227825 but also were less toxic to the animals used in the tests.

Epidemiology of drug‐resistant malaria in Republic of Congo: using molecular evidence for monitoring antimalarial drug resistance combined with assessment of antimalarial drug use

In Congo, urgent efforts are needed to help with the revision of the national antimalarial drug policy. Despite its high resistance level, chloroquine (CQ) is still extensively used as the first-line treatment for uncomplicated Plasmodium falciparum malaria. The study was conducted in children under 5 years with uncomplicated malaria in Pointe-Noire and Brazzaville, the two largest cities that contain approximately 60% of the population of Congo. We investigated by polymerized chain reaction and sequencing methods the frequency distribution of molecular markers for antimalarial drug resistance, including mutations in P. falciparum chloroquine resistance transporter (pfcrt) gene associated with CQ resistance and mutations in dihydrofolate reductase (dhfr) and dihydropteroate synthetase (dhps) genes conferring resistance to sulphadoxine/pyrimethamine (SP) among pre-treatment P. falciparum isolates, as well as assessing antimalarial drug use in the community. pfcrt (K76T) mutation was present in most isolates (96.4%, n = 138) and high frequency (69.2%, n = 133) of triple-mutant dhfr-S108N, N51I, C59R was observed. The quintuple mutant (dhfr-S108N, N51I, C59R and dhps-A437G or S436A, K540E) considered as molecular marker for SP treatment failure was not found because dhps-K540E mutation was absent in isolates tested; this is a clear evidence for the excellent efficacy of SP that we previously described in the same population. The complete absence of the dhps-K540E mutation is a deterrent component for using this molecular marker as an early warning tool for SP resistance testing in that population. Poor compliance issues related to the antimalarial drug use including inappropriate manufacturing practices reported in this study require intensive attention and should be taken into account when implementing drug policy change. If Congo changes its treatment policy from CQ to SP monotherapy, this will not last long. The strategy of combining SP with other affordable and effective antimalarial drugs such as the artemisinin derivatives to improve efficacy and to delay the development of parasite resistance is essential.

A simple dual selection for functionally active mutants of Plasmodium falciparum dihydrofolate reductase with improved solubility

Sufficient solubility of the active protein in aqueous solution is a prerequisite for crystallization and other structural studies of proteins. In this study, we have developed a simple and effective in vivo screening system to select for functionally active proteins with increased solubility by using Plasmodium falciparum dihydrofolate reductase (pfDHFR), a well-known malarial drug target, as a model. Prior to the dual selection process, pfDHFR was fused to green fluorescent protein (GFP), which served as a reporter for solubility. The fusion gene was used as a template for construction of mutated DNA libraries of pfDHFR. Two amino acids with large hydrophobic side chains (Y35 and F37) located on the surface of pfDHFR were selected for site-specific mutagenesis. Additionally, the entire pfDHFR gene was randomly mutated using error-prone PCR. During the first step of the dual selection, mutants with functionally active pfDHFR were selected from two libraries by using bacterial complementation assay. Fluorescence signals of active mutants were subsequently measured and five mutants with increased GFP signal, namely Y35Q + F37R, Y35L + F37T, Y35G + F37L and Y35L + F37R from the site-specific mutant library and K27E from the random mutant library, were recovered. The mutants were expressed, purified and characterized as monofunctional pfDHFR following excision of GFP. Our studies indicated that all mutant pfDHFRs exhibited kinetic properties similar to that of the wild-type protein. For comparison of protein solubility, the maximum concentrations of mutant enzymes prior to aggregation were determined. All mutants selected in this study exhibited 3- to 6-fold increases in protein solubility compared with the wild-type protein, which readily aggregated at 2 mg/ml. The dual selection system we have developed should be useful for engineering functionally active protein mutants with sufficient solubility for functional/structural studies and other applications.

Tolerance by Selective In Vivo Expansion of Foreign Major Histocompatibility Complex-Transduced Autologous Bone Marrow1

Application of gene therapy to induce antigen-specific immune tolerance could be important for transplantation or treatment of autoimmune diseases. Hematopoietic stem cell-based gene therapy has been hampered by relatively weak gene expression in vivo and loss of transduced cells over time. Selective expansion of transduced hematopoietic stem cells has been accomplished by incorporating the dihydrofolate reductase (DHFR) gene into the gene transfer vector. To assess whether this strategy could be applied to transplantation, we constructed a retroviral vector plasmid (KA274) containing the cDNA encoding human leukocyte antigen (HLA)-A2.1 and a tyr22 mutant DHFR and generated vesicular stomatitis virus-G-pseudotyped recombinant retrovirus by transfection into 293GPG cells. Bone marrow cells from C57BL/6 mice were infected with KA274 at a multiplicity of infection of 100, and transplanted into lethally irradiated syngeneic mice. After transplantation with transduced bone marrow, the proportion of peripheral blood cells expressing HLA-A2 ranged from 3.2% to 38% and increased 2- to 4.9-fold after selection for DHFR-expressing cells using trimetrexate and nitrobenzylmercaptpurine riboside 5' monophosphate. HLA-A2 expression remained above pretreatment levels throughout the study. Cytotoxic spleen cells from reconstituted mice lysed third-party HLA-B7-expressing targets but were unable to lyse HLA-A2-expressing targets. All KA274 reconstituted C57BL/6 mice accepted skin grafts from HLA-A2.1 transgenic mice for more than 245 days but rejected third-party Balb/c skin grafts in 12 days. Long-term transgene expression and immunologic tolerance to retrovirus-encoded HLA-A2, equivalent to that obtained by donor bone marrow transplantation, was accomplished, and selective expansion of transduced bone marrow cells was induced using DHFR as a selectable marker.

Identification of Amino Acids Required for the Functional Up-regulation of Human Dihydrofolate Reductase Protein in Response to Antifolate Treatment

Human dihydrofolate reductase (DHFR) protein levels rapidly increase upon exposure to methotrexate, a potent inhibitor of this enzyme. A model to explain this increase proposes that DHFR inhibits its own translation by binding to its cognate mRNA and that methotrexate disrupts the DHFR protein-mRNA complex allowing its translation to resume. In the present study, Chinese hamster ovary cells lacking DHFR were transfected with wild type and mutants of human DHFR to identify amino acids that are essential for increases in DHFR in response to methotrexate. Glu-30, Leu-22, and Ser-118 were involved in the up-regulation of DHFR protein levels by methotrexate and certain other antifolates. Cells transfected with E30A, L22R, and S118A mutants that did not respond to methotrexate up-regulation had higher basal levels of DHFR, consistent with the model, i.e. lack of feedback regulation of these enzymes. Although cells containing the S118A mutant enzyme had higher levels of DHFR and had catalytic activity similar to that of wild type DHFR, they had the same sensitivity to the cytotoxicity of methotrexate, as were cells with wild type DHFR. This finding provides evidence that the adaptive up-regulation of DHFR by methotrexate contributes to the decreased sensitivity to this drug. Based on these observations, a new model is proposed whereby DHFR exists in two conformations, one bound to DHFR mRNA and the other bound to NADPH. The mutants that are not up-regulated by methotrexate are unable to bind their cognate mRNA.

Mechanisms of Resistance of Malaria Parasites to Antifolates

Antifolate antimalarial drugs interfere with folate metabolism, a pathway essential to malaria parasite survival. This class of drugs includes effective causal prophylactic and therapeutic agents, some of which act synergistically when used in combination. Unfortunately, the antifolates have proven susceptible to resistance in the malaria parasite. Resistance is caused by point mutations in dihydrofolate reductase and dihydropteroate synthase, the two key enzymes in the folate biosynthetic pathway that are targeted by the antifolates. Resistance to these drugs arises relatively rapidly in response to drug pressure and is now common worldwide. Nevertheless, antifolate drugs remain first-line agents in several sub-Saharan African countries where chloroquine resistance is widespread, at least partially because they remain the only affordable, effective alternative. New antifolate combinations that are more effective against resistant parasites are being developed and in one case, recently introduced into use. Combining these antifolates with drugs that act on different targets in the parasite should greatly enhance their effectiveness as well as deter the development of resistance. Molecular epidemiological techniques for monitoring parasite drug resistance may contribute to development of strategies for prolonging the useful therapeutic life of this important class of drugs.

Why has the dihydrofolate reductase 164 mutation not consistently been found in Africa yet?

Resistance to the antifolate sulfadoxine-pyrimethamine (SP), the current mass-treatment antimalarial drug, is associated with selection of point mutations in dihydrofolate reductase and dihydropteroate synthase. Among these mutations, the leucine 164 dihydrofolate reductase mutation (Leu-164) is associated with higher levels of SP resistance; this mutation is also associated with a decrease in the efficacy of chlorproguanil/dapsone, a newly developed antifolate antimalarial drug. Leu-164 has been detected in Southeast Asia and South America, regions where SP is no longer effective. Surprisingly, this mutation has not yet been detected in Africa, using the standard protocol based on PCR-RFLP, despite high SP resistance. In this paper, we discuss briefly the reasons why Leu-164 has not yet been selected in Africa and we propose a means that may slow down the selection of this mutation.