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1.  Catalysis and Sulfa Drug Resistance in Dihydropteroate Synthase 
Science (New York, N.Y.)  2012;335(6072):1110-1114.
The sulfonamide antibiotics inhibit dihydropteroate synthase (DHPS), a key enzyme in the folate pathway of bacteria and primitive eukaryotes. However, resistance mutations have severely compromised the usefulness of these drugs. Here, we report structural, computational and mutagenesis studies on the catalytic and resistance mechanisms of DHPS. By performing the enzyme-catalyzed reaction in crystalline DHPS, we have structurally characterized key intermediates along the reaction pathway. Results support an SN1 reaction mechanism via formation of a novel cationic pterin intermediate. We also show that two conserved loops generate a substructure during catalysis that creates a specific binding pocket for p-aminobenzoic acid, one of the two DHPS substrates. This substructure, together with the pterin-binding pocket, explains the roles of the conserved active site residues, and reveals how sulfonamide resistance arises.
doi:10.1126/science.1214641
PMCID: PMC3531234  PMID: 22383850
2.  Validation of Molecular Docking Programs for Virtual Screening against Dihydropteroate Synthase 
Dihydropteroate synthase (DHPS) is the target of the sulfonamide class of antibiotics and has been a validated antibacterial drug target for nearly 70 years. The sulfonamides target the p-aminobenzoic acid (pABA) binding site of DHPS and interfere with folate biosynthesis and ultimately prevent bacterial replication. However, widespread bacterial resistance to these drugs has severely limited their effectiveness. This study explores the second and more highly conserved pterin binding site of DHPS as an alternative approach to developing novel antibiotics that avoid resistance. In this study, five commonly-used docking programs, FlexX, Surflex, Glide, GOLD, and DOCK, and nine scoring functions, were evaluated for their ability to rank-order potential lead compounds for an extensive virtual screening study of the pterin binding site of B. anthracis DHPS. Their performance in ligand docking and scoring was judged by their ability to reproduce a known inhibitor conformation and to efficiently detect known active compounds seeded into three separate decoy sets. Two other metrics were used to assess performance; enrichment at 1% and 2%, and Receiver Operating Characteristic (ROC) curves. The effectiveness of post-docking relaxation prior to rescoring and consensus scoring were also evaluated. Finally, we have developed a straightforward statistical method of including the inhibition constants of the known active compounds when analyzing enrichment results to more accurately assess scoring performance, which we call the ‘sum of the sum of log rank’ or SSLR. Of the docking and scoring functions evaluated, Surflex with Surflex-Score and Glide with GlideScore were the best overall performers for use in virtual screening against the DHPS target, with neither combination showing statistically significant superiority over the other in enrichment studies or pose selection. Post-docking ligand relaxation and consensus scoring did not improve overall enrichment.
PMCID: PMC2788795  PMID: 19434845
3.  Structural Studies of Pterin-Based Inhibitors of Dihydropteroate Synthase 
Journal of medicinal chemistry  2010;53(1):166-177.
Dihydropteroate synthase (DHPS) is a key enzyme in bacterial folate synthesis and the target of the sulfonamide class of antibacterials. Resistance and toxicities associated with sulfonamides have led to a decrease in their clinical use. Compounds that bind to the pterin binding site of DHPS, as opposed to the p-amino benzoic acid (pABA) binding site targeted by the sulfonamide agents, are anticipated to bypass sulfonamide resistance. To identify such inhibitors and map the pterin binding pocket, we have performed virtual screening, synthetic, and structural studies using Bacillus anthracis DHPS. Several compounds with inhibitory activity have been identified, and crystal structures have been determined that show how the compounds engage the pterin site. The structural studies identify the key binding elements and have been used to generate a structure-activity based pharmacophore map that will facilitate the development of the next generation of DHPS inhibitors which specifically target the pterin site.
doi:10.1021/jm900861d
PMCID: PMC2804029  PMID: 19899766
4.  Crystal Structure of the 6-Hydroxymethyl-7,8-Dihydropterin Pyrophosphokinase•Dihydropteroate Synthase Bifunctional Enzyme from Francisella tularensis 
PLoS ONE  2010;5(11):e14165.
The 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) enzymes catalyze sequential metabolic reactions in the folate biosynthetic pathway of bacteria and lower eukaryotes. Both enzymes represent validated targets for the development of novel anti-microbial therapies. We report herein that the genes which encode FtHPPK and FtDHPS from the biowarfare agent Francisella tularensis are fused into a single polypeptide. The potential of simultaneously targeting both modules with pterin binding inhibitors prompted us to characterize the molecular details of the multifunctional complex. Our high resolution crystallographic analyses reveal the structural organization between FtHPPK and FtDHPS which are tethered together by a short linker. Additional structural analyses of substrate complexes reveal that the active sites of each module are virtually indistinguishable from those of the monofunctional enzymes. The fused bifunctional enzyme therefore represents an excellent vehicle for finding inhibitors that engage the pterin binding pockets of both modules that have entirely different architectures. To demonstrate that this approach has the potential of producing novel two-hit inhibitors of the folate pathway, we identify and structurally characterize a fragment-like molecule that simultaneously engages both active sites. Our study provides a molecular framework to study the enzyme mechanisms of HPPK and DHPS, and to design novel and much needed therapeutic compounds to treat infectious diseases.
doi:10.1371/journal.pone.0014165
PMCID: PMC2994781  PMID: 21152407
5.  Crystal structures of IspF from Plasmodium falciparum and Burkholderia cenocepacia: comparisons inform antimicrobial drug target assessment 
Background
2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase (IspF) catalyzes the conversion of 4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate to 2C-methyl-D-erythritol-2,4-cyclodiphosphate and cytidine monophosphate in production of isoprenoid-precursors via the methylerythritol phosphate biosynthetic pathway. IspF is found in the protozoan Plasmodium falciparum, a parasite that causes cerebral malaria, as well as in many Gram-negative bacteria such as Burkholderia cenocepacia. IspF represents a potential target for development of broad-spectrum antimicrobial drugs since it is proven or inferred as essential in these pathogens and absent from mammals. Structural studies of IspF from these two important yet distinct pathogens, and comparisons with orthologues have been carried out to generate reagents, to support and inform a structure-based approach to early stage drug discovery.
Results
Efficient recombinant protein production and crystallization protocols were developed, and high-resolution crystal structures of IspF from P. falciparum (Emphasis/Emphasis>IspF) and B. cenocepacia (BcIspF) in complex with cytidine nucleotides determined. Comparisons with orthologues, indicate a high degree of order and conservation in parts of the active site where Zn2+ is bound and where recognition of the cytidine moiety of substrate occurs. However, conformational flexibility is noted in that area of the active site responsible for binding the methylerythritol component of substrate. Unexpectedly, one structure of BcIspF revealed two molecules of cytidine monophosphate in the active site, and another identified citrate coordinating to the catalytic Zn2+. In both cases interactions with ligands appear to help order a flexible loop at one side of the active site. Difficulties were encountered when attempting to derive complex structures with other ligands.
Conclusions
High-resolution crystal structures of IspF from two important human pathogens have been obtained and compared to orthologues. The studies reveal new data on ligand binding, with citrate coordinating to the active site Zn2+ and when present in high concentrations cytidine monophosphate displays two binding modes in the active site. Ligand binding appears to order a part of the active site involved in substrate recognition. The high degree of structural conservation in and around the IspF active site suggests that any structural model might be suitable to support a program of structure-based drug discovery.
doi:10.1186/1472-6807-14-1
PMCID: PMC3927217  PMID: 24410837
Antimicrobial drug target; Isoprenoid biosynthesis; X-ray crystallography; Zn2+-dependent enzyme
6.  Structure Based Design of Novel Pyrimido[4,5-c]pyridazine Derivatives as Dihydropteroate Synthase (DHPS) Inhibitors with Increased Affinity 
ChemMedChem  2012;7(5):861-870.
Dihydropteroate synthase (DHPS) is the validated drug target for sulfonamide antimicrobial therapy. However, due to widespread drug resistance and poor tolerance, the use of the sulfonamide antibiotics is now limited. The pterin binding pocket in DHPS has a high degree of conservation and is distinct from the sulfonamide binding site, and therefore represents an attractive alternative target for the design of novel antibacterial agents. Previously, we have structurally characterized a known pyridazine inhibitor in the Bacillus anthracis DHPS pterin site and identified a number of unfavorable interactions that appear to compromise the binding. Using this structural information, a series of 4,5-dioxo-1,4,5,6-tetrahydropyrimido[4,5-c]pyridazines was designed to improve binding affinity. Most importantly, the N-methyl ring substitution was removed to improve binding within the pterin pocket and the length of the side chain carboxylic acid was optimized to fully engage the pyrophosphate binding site. These inhibitors were synthesized and evaluated by an enzyme activity assay, X-ray crystallography, isothermal calorimetry, and surface plasmon resonance to obtain a comprehensive understanding of the binding interactions from structural, kinetic and thermodynamic perspectives. This study clearly demonstrates that compounds lacking the N-methyl substitution exhibit increased inhibition of DHPS, but the beneficial effects of optimizing the side chain length were less apparent.
doi:10.1002/cmdc.201200049
PMCID: PMC3463507  PMID: 22416048
Heterocycles; Dihydropteroate synthase; Antimicrobial agents; Structure Based Drug design
7.  Dihydropteroate Synthase Mutations in Pneumocystis jiroveci Can Affect Sulfamethoxazole Resistance in a Saccharomyces cerevisiae Model 
Dihydropteroate synthase (DHPS) mutations in Pneumocystis jiroveci have been associated epidemiologically with resistance to sulfamethoxazole (SMX). Since P. jiroveci cannot be cultured, inherent drug resistance cannot be measured. This study explores the effects of these mutations in a tractable model organism, Saccharomyces cerevisiae. Based on the sequence conservation between the DHPS enzymes of P. jiroveci and S. cerevisiae, together with the structural conservation of the three known DHPS structures, DHPS substitutions commonly observed in P. jiroveci were reverse engineered into the S. cerevisiae DHPS. Those mutations, T597A and P599S, can occur singly but are most commonly found together and are associated with SMX treatment failure. Mutations encoding the corresponding changes in the S. cerevisiae dhps were made in a yeast centromere vector, p414FYC, which encodes the native yeast DHPS as part of a trifunctional protein that also includes the two enzymes upstream of DHPS in the folic acid synthesis pathway, dihydroneopterin aldolase and 2-amino-4-hydroxymethyl dihydropteridine pyrophosphokinase. A yeast strain with dhps deleted was employed as the host strain, and transformants having DHPS activity were recovered. Mutants having both T597 and P599 substitutions had a requirement for p-aminobenzoic acid (PABA), consistent with resistance being associated with altered substrate binding. These mutants could be adapted for growth in the absence of PABA, which coincided with increased sulfa drug resistance. Upregulated PABA synthesis was thus implicated as a mechanism for sulfa drug resistance for mutants having two DHPS substitutions.
doi:10.1128/AAC.48.7.2617-2623.2004
PMCID: PMC434176  PMID: 15215118
8.  Characterisation of the bifunctional dihydrofolate synthase–folylpolyglutamate synthase from Plasmodium falciparum; a potential novel target for antimalarial antifolate inhibition 
Graphical abstract
The activities of the bifunctional folate pathway enzyme dihydrofolate synthase–folylpolyglutamate synthase from Plasmodium falciparum are characterised with respect to their kinetics, substrate specificities and responses to folate analogue inhibitors.
Unusually for a eukaryote, the malaria parasite Plasmodium falciparum expresses dihydrofolate synthase (DHFS) and folylpolyglutamate synthase (FPGS) as a single bifunctional protein. The two activities contribute to the essential pathway of folate biosynthesis and modification. The DHFS activity of recombinant PfDHFS–FPGS exhibited non-standard kinetics at high co-substrate (glutamate and ATP) concentrations, being partially inhibited by increasing concentrations of its principal substrate, dihydropteroate (DHP). Binding of DHP to the catalytic and inhibitory sites exhibited dissociation constants of 0.50 μM and 1.25 μM, respectively. DHFS activity measured under lower co-substrate concentrations, where data fitted the Michaelis–Menten equation, yielded apparent Km values of 0.88 μM for DHP, 22.8 μM for ATP and 5.97 μM for glutamate. Of the substrates tested in FPGS assays, only tetrahydrofolate (THF) was efficiently converted to polyglutamylated forms, exhibiting standard kinetics with an apparent Km of 0.96 μM; dihydrofolate, folate and the folate analogue methotrexate (MTX) were negligibly processed, emphasising the importance of the oxidation state of the pterin moiety. Moreover, MTX inhibited neither DHFS nor FPGS, even at high concentrations. Conversely, two phosphinate analogues of 7,8-dihydrofolate that mimic tetrahedral intermediates formed during DHFS- and FPGS-catalysed glutamylation were powerfully inhibitory. The Ki value of an aryl phosphinate analogue against DHFS was 0.14 μM and for an alkyl phosphinate against FPGS 0.091 μM, with each inhibitor showing a high degree of specificity. This, combined with the absence of DHFS activity in humans, suggests PfDHFS–FPGS might represent a potential new drug target in the previously validated folate pathway of P. falciparum.
doi:10.1016/j.molbiopara.2010.03.012
PMCID: PMC2877875  PMID: 20350571
Antifolate inhibitor studies; Biphasic kinetics; Folate metabolism; Methotrexate; Phosphinate analogues of folate; Substrate specificity; DHF, dihydrofolate; DHFR, dihydrofolate reductase; DHFS, dihydrofolate synthase; DHP, dihydropteroate; DHPS, dihydropteroate synthase; DTT, dithiothreitol; FPGS, folylpolyglutamate synthase; GTPCH, GTP cyclohydrolase I; HPPK, hydroxymethyldihydropterin pyrophosphokinase; MTX, methotrexate; pAB, para-aminobenzoate; PTPS, pyruvoyltetrahydropterin synthase III; PYR, pyrimethamine; SDX, sulfadoxine; THF, tetrahydrofolate
9.  Examination of Intrinsic Sulfonamide Resistance in Bacillus anthracis: A Novel Assay for Dihydropteroate Synthase 
Biochimica et biophysica acta  2008;1780(5):848-853.
Dihydropteroate synthase (DHPS) catalyzes the formation of dihydropteroate and Mg-pyrophosphate from 6-hydroxymethyl-7,8-dihydropterin diphosphate and para-aminobenzoic acid. The Bacillus anthracis DHPS is intrinsically resistant to sulfonamides. However, using a radioassay that monitors the dihydropteroate product, the enzyme was inhibited by the same sulfonamides. A continuous spectrophotometric assay for measuring the enzymatic activity of DHPS was developed and used to examine the effects of sulfonamides on the enzyme. The new assay couples the production of MgPPi to the pyrophosphate-dependent phosphofructokinase/aldolase/triose isomerase/α-glycerophosphate dehydrogenase reactions and monitoring the disappearance of NADH at 340 nm. The coupled enzyme assay demonstrates that resistance of the B. anthracis DHPS results in part from the use of the sulfonamides as alternative substrates, resulting in the formation of sulfonamide-pterin adducts, and not necessarily due to an inability to bind them.
doi:10.1016/j.bbagen.2008.02.003
PMCID: PMC2443783  PMID: 18342015
10.  A Genetic Screen for Dihydropyridine (DHP)-Resistant Worms Reveals New Residues Required for DHP-Blockage of Mammalian Calcium Channels 
PLoS Genetics  2008;4(5):e1000067.
Dihydropyridines (DHPs) are L-type calcium channel (Cav1) blockers prescribed to treat several diseases including hypertension. Cav1 channels normally exist in three states: a resting closed state, an open state that is triggered by membrane depolarization, followed by a non-conducting inactivated state that is triggered by the influx of calcium ions, and a rapid change in voltage. DHP binding is thought to alter the conformation of the channel, possibly by engaging a mechanism similar to voltage dependent inactivation, and locking a calcium ion in the pore, thereby blocking channel conductance. As a Cav1 channel crystal structure is lacking, the current model of DHP action has largely been achieved by investigating the role of candidate Cav1 residues in mediating DHP-sensitivity. To better understand DHP-block and identify additional Cav1 residues important for DHP-sensitivity, we screened 440,000 randomly mutated Caenorhabditis elegans genomes for worms resistant to DHP-induced growth defects. We identified 30 missense mutations in the worm Cav1 pore-forming (α1) subunit, including eleven in conserved residues known to be necessary for DHP-binding. The remaining polymorphisms are in eight conserved residues not previously associated with DHP-sensitivity. Intriguingly, all of the worm mutants that we analyzed phenotypically exhibited increased channel activity. We also created orthologous mutations in the rat α1C subunit and examined the DHP-block of current through the mutant channels in culture. Six of the seven mutant channels examined either decreased the DHP-sensitivity of the channel and/or exhibited significant residual current at DHP concentrations sufficient to block wild-type channels. Our results further support the idea that DHP-block is intimately associated with voltage dependent inactivation and underscores the utility of C. elegans as a screening tool to identify residues important for DHP interaction with mammalian Cav1 channels.
Author Summary
L-type calcium channels are important drug targets because they regulate many physiological processes throughout the body. For example, L-type calcium channels regulate cardiac myocytes and vascular smooth muscle contraction. Antagonists are therefore commonly used to lower blood pressure and treat other related ailments. Despite their medical importance, the mechanism by which L-type antagonists inactivate calcium channels is not fully understood, due in large part to the lack of a channel crystal structure. Here, we present the first large-scale genetic screen for L-type calcium channel residues that are important for sensitivity to a new drug analog that we discovered called nemadipine. We performed the screen using nematodes, and then recreated similar mutations in a mammalian channel to investigate how the mutant residues alter interactions with the antagonists using electrophysiological techniques. Together, our analyses revealed eight new L-type calcium channel residues that are important for DHP-sensitivity and highlight the utility of using a simple animal model system for understanding how drugs interact with their targets.
doi:10.1371/journal.pgen.1000067
PMCID: PMC2362100  PMID: 18464914
11.  Pneumocystis jiroveci Dihydropteroate Synthase Polymorphisms Confer Resistance to Sulfadoxine and Sulfanilamide in Saccharomyces cerevisiae 
Failure of anti-Pneumocystis jiroveci prophylaxis with sulfa drugs is associated with mutations within the putative active site of the fungal dihydropteroate synthase (DHPS), an enzyme encoded by the multidomain FAS gene. This enzyme is involved in the essential biosynthesis of folic acid. The most frequent polymorphisms are two mutations leading to two amino acid changes (55Trp-Arg-57Pro to 55Ala-Arg-57Ser), observed as a single or double mutation in the same P. jiroveci isolate. In the absence of a culture method for P. jiroveci, we studied potential resistance to sulfa drugs conferred by these polymorphisms by using Saccharomyces cerevisiae as a model. Single or double mutations identical to those observed in the DHPS domain of the P. jiroveci FAS gene were introduced by in vitro site-directed mutagenesis into alleles of the S. cerevisiae FOL1 gene, which is the orthologue of the P. jiroveci FAS gene. The mutated alleles were integrated at the genomic locus in S. cerevisiae and expressed by functional complementation in a strain with a disrupted FOL1 allele. The single mutation 55Trp to 55Ala conferred resistance to sulfanilamide, whereas the single mutation 57Pro to 57Ser conferred resistance to both sulfanilamide and sulfadoxine. Both single mutations also separately conferred hypersensitivity to sulfamethoxazole and dapsone. The resistance to sulfadoxine is consistent with epidemiological data on P. jiroveci. The double mutation 55Trp-Arg-57Pro to 55Ala-Arg-57Ser conferred on S. cerevisiae a requirement for p-aminobenzoate, suggesting reduced affinity of DHPS for this substrate. This characteristic is commonly observed in mutated DHPS enzymes conferring sulfa drug resistance from other organisms. However, the double mutation conferred hypersensitivity to sulfamethoxazole, which is not in agreement with epidemiological data on P. jiroveci. Taken together, our results suggest that the DHPS polymorphisms observed in P. jiroveci confer sulfa drug resistance on this pathogen.
doi:10.1128/AAC.48.7.2610-2616.2004
PMCID: PMC434158  PMID: 15215117
12.  Development of a Pterin-based Fluorescent Probe for Screening Dihydropteroate Synthase 
Bioconjugate Chemistry  2011;22(10):2110-2117.
Dihydropteroate synthase (DHPS) is the classical target of the sulfonamide class of antimicrobial agents, whose use has been limited by widespread resistance and pharmacological side effects. We have initiated a structure-based drug design approach for the development of novel DHPS inhibitors that bind to the highly conserved and structured pterin sub-site rather than to the adjacent p-amino benzoic acid binding pocket that is targeted by the sulfonamide class of antibiotics. To facilitate these studies, a robust pterin site-specific fluorescence polarization (FP) assay has been developed and is discussed herein. These studies include the design, synthesis and characterization of two fluorescent probes, and the development and validation of a rapid DHPS FP assay. This assay has excellent DMSO tolerance and is highly reproducible as evidenced by a high Z’ factor. This assay offers significant advantages over traditional radiometric or phosphate release assays against this target, and is suitable for site-specific high throughput and fragment-based screening studies.
doi:10.1021/bc200346e
PMCID: PMC3384522  PMID: 21916405
13.  Para-Aminosalicylic Acid Acts as an Alternative Substrate of Folate Metabolism in Mycobacterium tuberculosis 
Science (New York, N.Y.)  2012;339(6115):88-91.
Folate biosynthesis is an established anti-infective target, and the antifolate para-aminosalicylic acid (PAS) was one of the first anti-infectives introduced into clinical practice based on target-based drug discovery. Fifty years later, PAS continues in use for tuberculosis. PAS is assumed to inhibit dihydropteroate synthase (DHPS) in Mycobacterium tuberculosis (M. tuberculosis) by mimicking the substrate, p-aminobenzoate (PABA). However, we found inhibition of DHPS did not inhibit growth of M. tuberculosis. Instead, PAS, unlike sulfonamides, served as a replacement substrate for DHPS. Products of PAS metabolism at this and subsequent steps in folate metabolism inhibited those enzymes, competing with their substrates. PAS is thus a prodrug that blocks growth of M. tuberculosis when its active forms are generated by enzymes in the pathway that they poison.
doi:10.1126/science.1228980
PMCID: PMC3792487  PMID: 23118010
14.  Multiple Origins and Regional Dispersal of Resistant dhps in African Plasmodium falciparum Malaria 
PLoS Medicine  2009;6(4):e1000055.
Cally Roper and colleagues analyze the distribution of sulfadoxine resistance mutations and flanking microsatellite loci to trace the emergence and dispersal of drug-resistant Plasmodium falciparum malaria in Africa.
Background
Although the molecular basis of resistance to a number of common antimalarial drugs is well known, a geographic description of the emergence and dispersal of resistance mutations across Africa has not been attempted. To that end we have characterised the evolutionary origins of antifolate resistance mutations in the dihydropteroate synthase (dhps) gene and mapped their contemporary distribution.
Methods and Findings
We used microsatellite polymorphism flanking the dhps gene to determine which resistance alleles shared common ancestry and found five major lineages each of which had a unique geographical distribution. The extent to which allelic lineages were shared among 20 African Plasmodium falciparum populations revealed five major geographical groupings. Resistance lineages were common to all sites within these regions. The most marked differentiation was between east and west African P. falciparum, in which resistance alleles were not only of different ancestry but also carried different resistance mutations.
Conclusions
Resistant dhps has emerged independently in multiple sites in Africa during the past 10–20 years. Our data show the molecular basis of resistance differs between east and west Africa, which is likely to translate into differing antifolate sensitivity. We have also demonstrated that the dispersal patterns of resistance lineages give unique insights into recent parasite migration patterns.
Editors' Summary
Background
Plasmodium falciparum, a mosquito-borne parasite that causes malaria, kills nearly one million people every year, mostly in sub-Saharan Africa. People become infected with P. falciparum when they are bitten by a mosquito that has acquired the parasite in a blood meal taken from an infected person. P. falciparum malaria, which is characterized by recurring fevers and chills, anemia (loss of red blood cells), and damage to vital organs, can be fatal within hours of symptom onset if untreated. Until recently, treatment in Africa relied on chloroquine and sulfadoxine–pyrimethamine. Unfortunately, parasites resistant to both these antimalarial drugs is now widespread. Consequently, the World Health Organization currently recommends artemisinin combination therapy for the treatment of P. falciparum malaria in Africa and other places where drug-resistant malaria is common. In this therapy, artemisinin derivatives (new fast-acting antimalarial agents) are used in combination with another antimalarial to reduce the chances of P. falciparum becoming resistant to either drug.
Why Was This Study Done?
P. falciparum becomes resistant to antimalarial drugs by acquiring “resistance mutations,” genetic changes that prevent these drugs from killing the parasite. A mutation in the gene encoding a protein called the chloroquine resistance transporter causes resistance to chloroquine, a specific group of mutations in the dihydrofolate reductase gene causes resistance to pyrimethamine, and several mutations in dhps, the gene that encodes dihydropteroate synthase, are associated with resistance to sulfadoxine. Scientists have discovered that the mutations causing chloroquine and pyrimethamine resistance originated in Asia and spread into Africa (probably multiple times) in the late 1970s and mid-1980s, respectively. These Asian-derived mutations are now common throughout Africa and, consequently, it is not possible to determine how they spread across the continent. Information of this sort would, however, help experts design effective measures to control the spread of drug-resistant P. falciparum. Because the mutations in dhps that cause sulfadoxine resistance only began to emerge in the mid-1990s, they haven't spread evenly across Africa yet. In this study, therefore, the researchers use genetic methods to characterize the geographical origins and contemporary distribution of dhps resistance mutations in Africa.
What Did the Researchers Do and Find?
The researchers analyzed dhps mutations in P. falciparum DNA from blood samples collected from patients with malaria in various African countries and searched the scientific literature for other similar studies. Together, these data show that five major variant dhps sequences (three of which contain mutations that confer various degrees of resistance to sulphadoxine in laboratory tests) are currently present in Africa, each with a unique geographical distribution. In particular, the data show that P. falciparum parasites in east and west Africa carry different resistance mutations. Next, the researchers looked for microsatellite variants in the DNA flanking the dhps gene. Microsatellites are DNA regions that contain short, repeated sequences of nucleotides. Because the number of repeats can vary and because microsatellites are inherited together with nearby genes, the ancestry of various resistance mutations can be worked out by examining the microsatellites flanking different mutant dhps genes. This analysis revealed five regional clusters in which the same resistance lineage was present at all the sites examined within the region and also showed that the resistance mutations in east and west Africa have a different ancestry.
What Do These Findings Mean?
These findings show that sulfadoxine-resistant P. falciparum has recently emerged independently at multiple sites in Africa and that the molecular basis for sulfadoxine resistance is different in east and west Africa. This latter result may have clinical implications because it suggests that the effectiveness of sulfadoxine as an antimalarial drug may vary across the continent. Finally, although many more samples need to be analyzed to build a complete picture of the spread of antimalarial resistance across Africa, these findings suggest that economic and transport infrastructures may have played a role in governing recent parasite dispersal across this continent by affecting human migration. Thus, coordinated malaria control campaigns across socioeconomically linked areas in Africa may reduce the African malaria burden more effectively than campaigns that are confined to national territories.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000055.
This study is further discussed in a PLoS Medicine Perspective by Tim Anderson
The MedlinePlus encyclopedia contains a page on malaria (in English and Spanish)
Information is available from the World Health Organization on malaria (in several languages) and on drug-resistant malaria
The US Centers for Disease Control and Prevention provide information on malaria (in English and Spanish)
Information is available from the Roll Back Malaria Partnership on its approach to the global control of malaria, and on malaria control efforts in specific parts of the world
The WorldWide Antimalarial Resistance Network is creating an international database about antimalarial drug resistance
doi:10.1371/journal.pmed.1000055
PMCID: PMC2661256  PMID: 19365539
15.  Inhibition of Pneumocystis carinii dihydropteroate synthetase by para-acetamidobenzoic acid: possible mechanism of action of isoprinosine in human immunodeficiency virus infection. 
Isoprinosine has been reported to decrease progression to AIDS, primarily by preventing Pneumocystis carinii pneumonia (PCP), in human immunodeficiency virus-infected patients, but the mechanism of action is unknown. para-Acetamidobenzoic acid (PAcBA), one component of isoprinosine, is structurally related to para-aminobenzoic acid (PABA), a precursor of de novo folate synthesis. This pathway is known to be important for P. carinii because sulfonamides, which are effective anti-P. carinii agents, inhibit incorporation of PABA into folate precursors by the enzyme dihydropteroate synthetase (DHPS). Inhibition of P. carinii DHPS by PAcBA was investigated by using two assays. In short-term cultures of P. carinii from rats, [3H]PABA incorporation into reduced folates was inhibited by both isoprinosine (mean +/- standard error 50% inhibitory concentration [IC50], 20 +/- 8.4 microM) and PAcBA free acid (IC50, 240 +/- 100 microM); a soluble PAcBA salt was more potent than PAcBA free acid alone (IC50, 29 +/- 48 microM). The activity of PAcBA free acid was confirmed in a cell-free DHPS inhibition assay (IC50, 120 +/- 120 microM). Inosine and dimethylaminopropanol, two other components of isoprinosine, were poor inhibitors of PABA incorporation (IC50, > 1,000 microM). PAcBA free acid also showed activity in inhibiting the DHPS of Toxoplasma gondii, but was a poor inhibitor of the DHPSs of Escherichia coli and Saccharomyces cerevisiae. In a rat model of PCP, the PAcBA salt administered intraperitoneally demonstrated no activity against established PCP either alone or when used in combination with trimethoprim; the lack of efficacy in this model may be due to the rapid metabolism of the drug. Prevention of PCP by PaCBA through inhibition of P. carinii DHPS may explain the activity of isoprinosine in decreasing the progression to AIDS in human immunodeficiency virus-infected patients.
PMCID: PMC187944  PMID: 7687120
16.  Amino Acid Repetitions in the Dihydropteroate Synthase of Streptococcus pneumoniae Lead to Sulfonamide Resistance with Limited Effects on Substrate Km 
Sulfonamide resistance in Streptococcus pneumoniae is due to changes in the chromosomal folP (sulA) gene coding for dihydropteroate synthase (DHPS). The first reported laboratory-selected sulfonamide-resistant S. pneumoniae isolate had a 6-bp repetition, the sul-d mutation, leading to a repetition of the amino acids Ile66 and Glu67 in the gene product DHPS. More recently, clinical isolates showing this and other repetitions have been reported. WA-5, a clinical isolate from Washington State, contains a 6-bp repetition in the folP gene, identical to the sul-d mutation. The repetition was deleted by site-directed mutagenesis. Enzyme kinetic measurements showed that the deletion was associated with a 35-fold difference in Ki for sulfathiazole but changed the Km for p-aminobenzoic acid only 2.5-fold and did not significantly change the Km for 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine pyrophosphate. The enzyme characteristics of the deletion variant were identical to those of DHPS from a sulfonamide-susceptible strain. DHPS from clinical isolates with repetitions of Ser61 had very similar enzyme characteristics to the DHPS from WA-5. The results confirm that the repetitions are sufficient for development of a resistant enzyme and suggest that the fitness cost to the organism of developing resistance may be very low.
doi:10.1128/AAC.45.3.805-809.2001
PMCID: PMC90378  PMID: 11181365
17.  Anti-folate drug resistance in Africa: meta-analysis of reported dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) mutant genotype frequencies in African Plasmodium falciparum parasite populations 
Malaria Journal  2010;9:247.
Background
Mutations in the dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes of Plasmodium falciparum are associated with resistance to anti-folate drugs, most notably sulphadoxine-pyrimethamine (SP). Molecular studies document the prevalence of these mutations in parasite populations across the African continent. However, there is no systematic review examining the collective epidemiological significance of these studies. This meta-analysis attempts to: 1) summarize genotype frequency data that are critical for molecular surveillance of anti-folate resistance and 2) identify the specific challenges facing the development of future molecular databases.
Methods
This review consists of 220 studies published prior to 2009 that report the frequency of select dhfr and dhps mutations in 31 African countries. Maps were created to summarize the location and prevalence of the highly resistant dhfr triple mutant (N51I, C59R, S108N) genotype and dhps double mutant (A437G and K540E) genotype in Africa. A hierarchical mixed effects logistic regression was used to examine the influence of various factors on reported mutant genotype frequency. These factors include: year and location of study, age and clinical status of sampled population, and reporting conventions for mixed genotype data.
Results
A database consisting of dhfr and dhps mutant genotype frequencies from all African studies that met selection criteria was created for this analysis. The map illustrates particularly high prevalence of both the dhfr triple and dhps double mutant genotypes along the Kenya-Tanzania border and Malawi. The regression model shows a statistically significant increase in the prevalence of both the dhfr triple and dhps double mutant genotypes in Africa.
Conclusion
Increasing prevalence of the dhfr triple mutant and dhps double mutant genotypes in Africa are consistent with the loss of efficacy of SP for treatment of clinical malaria in most parts of this continent. Continued assessment of the effectiveness of SP for the treatment of clinical malaria and intermittent preventive treatment in pregnancy is needed. The creation of a centralized resistance data network, such as the one proposed by the WorldWide Antimalarial Resistance Network (WWARN), will become a valuable resource for planning timely actions to combat drug resistant malaria.
doi:10.1186/1475-2875-9-247
PMCID: PMC2940896  PMID: 20799995
18.  Sequential Metabolism of 7-Dehydrocholesterol to Steroidal 5,7-Dienes in Adrenal Glands and Its Biological Implication in the Skin 
PLoS ONE  2009;4(2):e4309.
Since P450scc transforms 7-dehydrocholesterol (7DHC) to 7-dehydropregnenolone (7DHP) in vitro, we investigated sequential 7DHC metabolism by adrenal glands ex vivo. There was a rapid, time- and dose-dependent metabolism of 7DHC by adrenals from rats, pigs, rabbits and dogs with production of more polar 5,7-dienes as detected by RP-HPLC. Based on retention time (RT), UV spectra and mass spectrometry, we identified the major products common to all tested species as 7DHP, 22-hydroxy-7DHC and 20,22-dihydroxy-7DHC. The involvement of P450scc in adrenal metabolic transformation was confirmed by the inhibition of this process by DL-aminoglutethimide. The metabolism of 7DHC with subsequent production of 7DHP was stimulated by forscolin indicating involvement of cAMP dependent pathways. Additional minor products of 7DHC metabolism that were more polar than 7DHP were identified as 17-hydroxy-7DHP (in pig adrenals but not those of rats) and as pregna-4,7-diene-3,20-dione (7-dehydroprogesterone). Both products represented the major identifiable products of 7DHP metabolism in adrenal glands. Studies with purified enzymes show that StAR protein likely transports 7DHC to the inner mitochondrial membrane, that 7DHC can compete effectively with cholesterol for the substrate binding site on P450scc and that the catalytic efficiency of 3βHSD for 7DHP (Vm/Km) is 40% of that for pregnenolone. Skin mitochondria are capable of transforming 7DHC to 7DHP and the 7DHP is metabolized further by skin extracts. Finally, 7DHP, its photoderivative 20-oxopregnacalciferol, and pregnenolone exhibited biological activity in skin cells including inhibition of proliferation of epidermal keratinocytes and melanocytes, and melanoma cells. These findings define a novel steroidogenic pathway: 7DHC→22(OH)7DHC→20,22(OH)27DHC→7DHP, with potential further metabolism of 7DHP mediated by 3βHSD or CYP17, depending on mammalian species. The 5–7 dienal intermediates of the pathway can be a source of biologically active vitamin D3 derivatives after delivery to or production in the skin, an organ intermittently exposed to solar radiation.
doi:10.1371/journal.pone.0004309
PMCID: PMC2629546  PMID: 19190754
19.  Sulfonamide Resistance in Streptococcus pyogenes Is Associated with Differences in the Amino Acid Sequence of Its Chromosomal Dihydropteroate Synthase 
Sulfonamide resistance in recent isolates of Streptococcus pyogenes was found to be associated with alterations of the chromosomally encoded dihydropteroate synthase (DHPS). There were 111 different nucleotides (13.8%) in the genes found in susceptible and resistant isolates, respectively, resulting in 30 amino acid changes (11.3%). These substantial changes suggested the possibility of a foreign origin of the resistance gene, in parallel to what has already been found for sulfonamide resistance in Neisseria meningitidis. The gene encoding DHPS was linked to at least three other genes encoding enzymes of the folate pathway. These genes were in the order GTP cyclohydrolase, dihydropteroate synthase, dihydroneopterin aldolase, and hydroxymethyldihydropterin pyrophosphokinase. The nucleotide differences in genes from resistant and susceptible strains extended from the beginning of the GTP cyclohydrolase gene to the end of the gene encoding DHPS, an additional indication for gene transfer in the development of resistance. Kinetic measurements established different affinities for sulfathiazole for DHPS enzymes isolated from resistant and susceptible strains.
PMCID: PMC105745  PMID: 9593127
20.  Molecular Determination of Point Mutation Haplotypes in the Dihydrofolate Reductase and Dihydropteroate Synthase of Plasmodium falciparum in Three Districts of Northern Tanzania 
The antimalarial combination of sulfadoxine and pyrimethamine (SP) was introduced as first-line treatment for uncomplicated malaria in Tanzania during 2001 following 18 years of second-line use. The genetic determinants of in vitro resistance to the two drugs individually are shown to be point mutations at seven sites in the dihydrofolate reductase gene (dhfr) conferring resistance to pyrimethamine and five sites in the dihydropteroate synthase (dhps) gene conferring resistance to sulfadoxine. Different combinations of mutations within each gene confer differing degrees of insensitivity, but information about the frequency with which allelic haplotypes occur has been lacking because of the complicating effects of multiple infection. Here we used a novel high-throughput sequence-specific oligonucleotide probe-based approach to examine the present resistance status of three Plasmodium falciparum populations in northern Tanzania. By using surveys of asymptomatic infections and screening for the presence of all known point mutations in dhfr and dhps genes, we showed that just five dhfr and three dhps allelic haplotypes are present. High frequencies of both triple-mutant dhfr and double-mutant dhps mutant alleles were found in addition to significant interregional heterogeneity in allele frequency. In vivo studies have shown that the cooccurrence of three dhfr mutations and two dhps mutations in an infection prior to treatment is statistically predictive of treatment failure. We have combined data for both loci to determine the frequency of two-locus genotypes. The triple-dhfr/double-dhps genotype is present in all three regions with frequencies ranging between 30 and 63%, indicating that treatment failure rates are likely to be high.
doi:10.1128/AAC.47.4.1347-1354.2003
PMCID: PMC152520  PMID: 12654669
21.  Structures of Mycobacterium tuberculosis folylpolyglutamate synthase complexed with ADP and AMPPCP 
Crystal structures of M. tuberculosis folylpolyglutamate synthetase complexed with two nucleotides have been determined at 2.0 and 2.3 Å resolution, revealing an active-site loop movement and associated changes that influence substrate binding.
Folate derivatives are essential vitamins for cell growth and replication, primarily because of their central role in reactions of one-carbon metabolism. Folates require polyglutamation to be efficiently retained within the cell and folate-dependent enzymes have a higher affinity for the polyglutamylated forms of this cofactor. Polyglutamylation is dependent on the enzyme folylpolyglutamate synthetase (FPGS), which catalyzes the sequential addition of several glutamates to folate. FPGS is essential for the growth and survival of important bacterial species, including Mycobacterium tuberculosis, and is a potential drug target. Here, the crystal structures of M. tuberculosis FPGS in complex with ADP and AMPPCP are reported at 2.0 and 2.3 Å resolution, respectively. The structures reveal a deeply buried nucleotide-binding site, as in the Escherichia coli and Lactobacillus casei FPGS structures, and a long extended groove for the binding of folate substrates. Differences from the E. coli and L. casei FPGS structures are seen in the binding of a key divalent cation, the carbamylation state of an essential lysine side chain and the adoption of an ‘open’ position by the active-site β5–α6 loop. These changes point to coordinated events that are associated with dihydropteroate/folate binding and the catalysis of the new amide bond with an incoming glutamate residue.
doi:10.1107/S0907444908012262
PMCID: PMC2631112  PMID: 18566510
Mycobacterium tuberculosis; folate metabolism; nucleotide binding; conformational change; cobalt binding
22.  Towards an Understanding of the Mechanism of Pyrimethamine-Sulfadoxine Resistance in Plasmodium falciparum: Genotyping of Dihydrofolate Reductase and Dihydropteroate Synthase of Kenyan Parasites 
The antifolate combination of pyrimethamine (PM) and sulfadoxine (SD) is the last affordable drug combination available for wide-scale treatment of falciparum malaria in Africa. Wherever this combination has been used, drug-resistant parasites have been selected rapidly. A study of PM-SD effectiveness carried out between 1997 and 1999 at Kilifi on the Kenyan coast has shown the emergence of RI and RII resistance to PM-SD (residual parasitemia 7 days after treatment) in 39 out of 240 (16.25%) patients. To understand the mechanism that underlies resistance to PM-SD, we have analyzed the dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) genotypes of 81 patients. Fifty-one samples were obtained, before treatment, from patients who remained parasite free for at least 7 days after treatment. For a further 20 patients, samples were obtained before treatment and again when they returned to the clinic with parasites 7 days after PM-SD treatment. Ten additional isolates were obtained from patients who were parasitemic 7 days after treatment but who were not sampled before treatment. More than 65% of the isolates (30 of 46) in the initial group had wild-type or double mutant DHFR alleles, and all but 7 of the 47 (85%) had wild-type DHPS alleles. In the paired (before and after treatment) samples, the predominant combinations of DHFR and DHPS alleles before treatment were of triple mutant DHFR and double mutant DHPS (41% [7 of 17]) and of double mutant DHFR and double mutant DHPS (29% [5 of 17]). All except one of the posttreatment isolates had triple mutations in DHFR, and most of these were “pure” triple mutants. In these isolates, the combination of a triple mutant DHFR and wild-type DHPS was detected in 6 of 29 cases (20.7%), the combination of a triple mutant DHFR and a single mutant (A437G) DHPS was detected in 4 of 29 cases (13.8%), and the combination of a triple mutant DHFR and a double mutant (A437G, L540E) DHPS was detected in 16 of 29 cases (55.2%). These results demonstrate that the triply mutated allele of DHFR with or without mutant DHPS alleles is associated with RI and RII resistance to PM-SD. The prevalence of the triple mutant DHFR-double mutant DHPS combination may be an operationally useful marker for predicting the effectiveness of PM-SD as a new malaria treatment.
PMCID: PMC89803  PMID: 10722502
23.  Exploring the folate pathway in Plasmodium falciparum 
Acta tropica  2005;94(3):191-206.
As in centuries past, the main weapon against human malaria infections continues to be intervention with drugs, despite the widespread and increasing frequency of parasite populations that are resistant to one or more of the available compounds. This is a particular problem with the lethal species of parasite, Plasmodium falciparum, which claims some two million lives per year as well as causing enormous social and economic problems. Amongst the antimalarial drugs currently in clinical use, the antifolates have the best defined molecular targets, namely the enzymes dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS), which function in the folate metabolic pathway. The products of this pathway, reduced folate cofactors, are essential for DNA synthesis and the metabolism of certain amino acids. Moreover, their formation and interconversions involve a number of other enzymes that have not as yet been exploited as drug targets. Antifolates are of major importance as they currently represent the only inexpensive regime for combating chloroquine-resistant malaria, and are now first-line drugs in a number of African countries. Aspects of our understanding of this pathway and antifolate drug resistance are reviewed here, with a particular emphasis on approaches to analysing the details of, and balance between, folate biosynthesis by the parasite and salvage of pre-formed folate from exogenous sources.
doi:10.1016/j.actatropica.2005.04.002
PMCID: PMC2720607  PMID: 15845349
Antifolate drugs; Drug resistance; Folate metabolism; Folate salvage; Gene disruption; Metabolic labelling
24.  Synthesis of Bi-substrate State Mimics of Dihydropteroate Synthase as Potential Inhibitors and Molecular Probes 
Bioorganic & medicinal chemistry  2010;19(3):1298-1305.
The increasing emergence of resistant bacteria drives us to design and develop new antimicrobial agents. Pursuant to that goal, a new targeting approach of the dihydropteroate synthase enzyme, which serves as the site of action for the sulfonamide class of antimicrobial agents, is being explored. Using structural information, a new class of transition state mimics has been designed and synthesized that have the capacity to bind to the pterin, phosphate and para-amino binding sites. The design, synthesis and evaluation of these compounds as inhibitors of Bacillus anthracis dihydropteroate synthase is described herein. Outcomes from this work have identified the first trivalent inhibitors of dihydropteroate synthase whose activity displayed slow binding inhibition. The most active compounds in this series contained an oxidized pterin ring. The binding of these inhibitors was modeled into the dihydropteroate synthase active site and demonstrated a good correlation with the observed bioassay data, as well as provided important insight for the future design of higher affinity transition state mimics.
doi:10.1016/j.bmc.2010.12.003
PMCID: PMC3147184  PMID: 21216602
dihydropteroate synthase; antimicrobial agent; sulfonamide; transition state analog
25.  Plasmodium falciparum Isolates in India Exhibit a Progressive Increase in Mutations Associated with Sulfadoxine-Pyrimethamine Resistance 
The combination of sulfadoxine-pyrimethamine (SP) is used as a second line of therapy for the treatment of uncomplicated chloroquine-resistant Plasmodium falciparum malaria. Resistance to SP arises due to certain point mutations in the genes for the dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) enzymes of the parasite. We have analyzed these mutations in 312 field isolates of P. falciparum collected from different parts of India to assess the effects of drug pressure. The rate of mutation in the gene for DHFR was found to be higher than that in the gene for DHPS, although the latter had mutations in more alleles. There was a temporal rise in the number of isolates with double dhfr mutations and single dhps mutations, resulting in an increased total number of mutations in the loci for DHFR and DHPS combined over a 5-year period. During these 5 years, the number of isolates with drug-sensitive genotypes decreased and the number of isolates with drug-resistant genotypes (double DHFR mutations and a single DHPS mutation) increased significantly. The number of isolates with the triple mutations in each of the genes for the two enzymes (for a total of six mutations), however, remained very low, coinciding with the very low rate of SP treatment failure in the country. There was a regional bias in the mutation rate, as isolates from the northeastern region (the state of Assam) showed higher rates of mutation and more complex genotypes than isolates from the other regions. It was concluded that even though SP is prescribed as a second line of treatment in India, the mutations associated with SP resistance continue to be progressively increasing.
doi:10.1128/AAC.48.3.879-889.2004
PMCID: PMC353157  PMID: 14982779

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