Plasmodium Falciparum Dihydrofolate Reductase Research Articles

Effect of sulfadoxine-pyrimethamine chemoprophylaxis in pregnant women on selection of the new P. falciparum dhps quintuple mutant carrying the I431V mutation.

A new mutation in the Plasmodium falciparum dihydropteroate synthetase gene (pfdhps), I431V, has been identified in several countries of Central and West Africa. This mutation is mostly found in association with four other SNPs on pfdhps (S436A, A437G, A581G and A613S), forming a quintuple mutant (vagKgs) and almost always associated with the Plasmodium falciparum dihydrofolate reductase gene (pfdhfr) CirnI (C50R, N51I, S108N) triple mutant. To date, nothing is known about the impact of this new pfdhps genotype on sulfadoxine-pyrimethamine (SP) resistance. We sought to assess the prevalence of this pfdhps vagKgs quintuple mutant in two groups of pregnant women with malaria, one that took intermittent preventive treatment with sulfadoxine-pyrimethamine (IPTp-SP) and one that did not. The pfdhfr and pfdhps genes from Plasmodium falciparum isolates collected in Yaoundé (Cameroon) from pregnant women with symptomatic malaria under IPTp-SP or not, were sequenced. Of 159 patients evaluated, 70 had already taken SP during pregnancy and 89 had never taken SP. Only the vagKgs allele was significantly overrepresented in the SP+ group (21.4% versus 3.4%; P < 0.001), whereas the ISgKAA mutant, widely distributed in this area and known to be less susceptible to SP, tended to be less abundant in this group (48.6% versus 64.0%; P = 0.0503). We found a strong overrepresentation of the CirnI/vagKgs haplotype in the IPTp-SP pregnant group, suggesting a high level of resistance of this mutant to SP. This could compromise not only the effectiveness of IPTp-SP but also the seasonal malaria chemoprevention of young children, now widely implemented.

Naphthyl bearing 1,3,4-thiadiazoleacetamides targeting the parasitic folate pathway as anti-infectious agents: in silico, synthesis, and biological approach.

Malaria is still a complex and lethal parasitic infectious disease, despite the availability of effective antimalarial drugs. Resistance of malaria parasites to current treatments necessitates new antimalarials targeting P. falciparum proteins. The present study reported the design and synthesis of a series of a 2-(4-substituted piperazin-1-yl)-N-(5-((naphthalen-2-yloxy)methyl)-1,3,4-thiadiazol-2-yl)acetamide hybrids for the inhibition of Plasmodium falciparum dihydrofolate reductase (PfDHFR) using computational biology tools followed by chemical synthesis, structural characterization, and functional analysis. The synthesized compounds were evaluated for their in vitro antimalarial activity against CQ-sensitive PfNF54 and CQ-resistant PfW2 strain. Compounds T5 and T6 are the most active compounds having anti-plasmodial activity against PfNF54 with IC50 values of 0.94 and 3.46 μM respectively. Compound T8 is the most active against the PfW2 strain having an IC50 of 3.91 μM. Further, these active hybrids (T5, T6, and T8) were also evaluated for enzyme inhibition assay against PfDHFR. All the tested compounds were non-toxic against the Hek293 cell line with good selectivity indices. Hemolysis assay also showed non-toxicity of these compounds on normal uninfected human RBCs. In silico molecular docking studies were carried out in the binding pocket of both the wild-type and quadruple mutant Pf-DHFR-TS to gain further insights into probable modes of action of active compounds. ADME prediction and physiochemical properties support their drug-likeness. Additionally, they were screened for antileishmanial activity against L. donovani promastigotes to explore broader applications. Thus, this study provides molecular frameworks for developing potent antimalarials and antileishmanial agents.

Discovery of rigid biphenyl Plasmodium falciparum DHFR inhibitors using a fragment linking strategy.

Plasmodium falciparum dihydrofolate reductase (PfDHFR), a historical target for antimalarials, has been considered compromised due to resistance inducing mutations caused by pyrimethamine (PYR) overexposure. The clinical candidate P218 has demonstrated that inhibitors could efficiently target both PYR-sensitive and PYR-resistant parasites through careful drug design. Yet, P218 clinical development has been limited by its pharmacokinetic profile, incompatible with single dose regimen. Herein, we report the design of new PfDHFR inhibitors using fragment-based design, aiming at improved lipophilicity and overall drug-like properties. Fragment-based screening identified hits binding in the pABA site of the enzyme. Using structure-guided design, hits were elaborated into leads by fragment linking with 2,4-diaminopyrimidine. Resulting compounds display nM range inhibition of both drug-sensitive and resistant PfDHFR, high selectivity against the human isoform, drug-like lipophilicity and metabolic stability. Compound 4 and its ester derivative 3 kill blood stage TM4/8.2 parasite at nM concentrations while showing no toxicity against Vero cells.

Molecular Docking Studies, Synthesis and Biological Evaluation of Substituted Pyrimidine-2,4-diamines as Inhibitors of Plasmodium falciparum Dihydrofolate Reductase.

A series of 5-[(phenethylamino)methyl]pyrimidine-2,4-diamines were assessed in silico as potential inhibitors of Plasmodium falciparum dihydrofolate reductase (PfDHFR), synthesised and tested for inhibitory activity against PfDHFR in vitro. The compounds displayed promising inhibitory activity against both wild-type (Ki 1.3-243 nM) and quadruple mutant (Ki 13-208 nM) PfDHFR in the biochemical enzyme assay, but were less potent in the whole-cell P. falciparum assay (IC50 (TM4/8.2) 0.4-28 μM; IC50 (V1S) 3.7-54 μM). Further investigation into the pharmacokinetic properties of these compounds may guide the development of more potent analogues.

Inhibitory Effect of Gedunin Analogue against the Plasmodium falciparum Dihydrofolate Reductase

Objective: Plasmodium parasites are the cause of malaria. Malaria victims get infected upon being bitten by female anopheles mosquito; which transmits the parasite to the victim. The P. falciparum and P. vivax are the most active disease-causing agents of all five malaria-causing species of Plasmodium. The anti-folate drugs which were the first class of clinical antimetabolites act by disrupting metabolic pathways in which the one-carbon moiety supplied by the B9 folate vitamins is a major requirement.&#x0D; Methods: Chemical structures of the anti-folate drugs which served as the experimental control ligands were downloaded from the PubChem database and saved as PDB files while the gedunin modification was achieved using the Marvin-Sketch software.&#x0D; Results: Molecular visualization of the polar interactions with amino acid residues of the Plasmodium falciparum dihydrofolate-reductase showed that all the control ligands interacted with similar residues contrary to the interaction of the gedunin modified ligand in the same binding pocket.&#x0D; Conclusion: Results from the molecular docking study showed that gedunin and its C=O of gedunin might be better antimalarial agents; having exhibited the best binding energies with a score of -9.5 and -9.0 Kcal/mol respectively.

The prevalence of molecular markers of resistance to sulfadoxine-pyrimethamine among pregnant women at first antenatal clinic attendance and delivery in the forest-savannah area of Ghana.

Intermittent preventive treatment during pregnancy with sulfadoxine-pyrimethamine (IPTp-SP) is used to prevent malaria and associated unfavorable maternal and foetal outcomes in pregnancy in moderate to high malaria transmission areas. Effectiveness of IPTp-SP is, however, threatened by mutations in the Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes which confer resistance to pyrimethamine and sulfadoxine, respectively. This study determined the prevalence of molecular markers of SP resistance among pregnant women in a high malaria transmission area in the forest-savannah area of Ghana. Genomic DNA was extracted from 286 P. falciparum-positive dried blood spots obtained from pregnant women aged ≥18 years (255 at first Antenatal Care (ANC) clinic visit and 31 at delivery from 2017 to 2019) using Chelex 100. Mutations in Pfdhfr and Pfdhps genes were detected using molecular inversion probes and next generation sequencing. In the Pfdhfr gene, single nucleotide polymorphisms (SNPs) were detected in 83.1% (157/189), 92.0% (173/188) and 91.0% (171/188) at codons 51, 59, and 108 respectively in samples collected at first ANC visit, while SNPs were detected in 96.6 (28/29), 96.6% (28/29) and 96.8% (30/31) in isolates collected at delivery. The Pfdhfr triple mutant N51I, C59R and S108N (IRN) was carried by 80.5% (128/159) and 96.5% (28/29) of the typed isolates collected at ANC visit and at delivery respectively. In the Pfdhps gene, SNPs were detected in 0.6% (1/174), 76.2% (138/181), 33.2% (60/181), 1.2% (2/174), 0% (0/183), and 16.6% (27/173) at codons 431, 436, 437, 540, 581 and 613 respectively in samples collected at ANC, and 0% (0/25), 72% (18/25), 40% (10/25), 3.6% (1/25), 0% (0/29) and 7.4% (2/27) in samples collected at delivery. Quadruple mutant Pfdhfr N51I, C59R, and S108N + Pfdhps A437G (IRN-GK) was present in 25.8% (33/128) and 34.8% (8/23) of isolates at ANC and at delivery respectively. Quintuple mutant alleles Pfdhfr N51I, C59R, and S108N + Pfdhps A437G and K540E (IRN-GE) were detected in 0.8% (1/128) and 4.4% (1/23) of samples collected at ANC and at delivery respectively. No mutations were identified at Pfdhfr codons 16 or 164 or Pfdhps 581. There is a high prevalence of Pfdhfr triple mutant P. falciparum infections among pregnant women in the study area. However, prevalence of the combined Pfdhfr/Pfdhps quadruple and quintuple mutants IRN-GK and IRN-GE respectively prior to commencement of IPTp-SP were low, and no Pfdhps A581G mutant was detected, indicating that SP is still likely to be efficacious for IPTp-SP in the forest-savannah area in the middle belt of Ghana.

Microwave synthesis and antimalarial screening of novel 4-amino benzoic acid (PABA)-substituted pyrimidine derivatives as Plasmodium falciparum dihydrofolate reductase inhibitors.

The online version contains supplementary material available at 10.1007/s13205-022-03236-w.

Phthalide derivatives as dihydrofolate reductase inhibitors for malaria: molecular docking and molecular dynamics studies

Plasmodium falciparum dihydrofolate reductase enzyme (P. falciparum DHFR) is one of the vital drug targets for malaria treatment, as this protein is indispensable for nucleotide metabolic pathways. This research aimed to discover promising phthalide derivatives against both wild and mutant P. falciparum DHFR enzymes through various computational techniques. The binding affinities were investigated using molecular docking, which showed five compounds having the highest affinity scores against both enzymes compared to the reference compounds. MM-GBSA calculations displayed favourable free binding energy. Moreover, the ADMET properties of the compounds are within acceptable ranges. The stability of the ligand-protein complexes was studied by Molecular Dynamics (MD) simulations. Depending on the results obtained from this research, we propose three compounds to be hit against P. falciparum DHFR activity which could be examined experimentally. Communicated by Ramaswamy Sarma

An In Silico Study of the Interactions of Alkaloids from Cryptolepis sanguinolenta with Plasmodium falciparum Dihydrofolate Reductase and Dihydroorotate Dehydrogenase

The Plasmodium falciparum dihydrofolate reductase (PfDHFR) and dihydroorotate dehydrogenase (PfDHODH) are essential for Plasmodium falciparum growth and development, and have been validated as targets for the development of new antimalarial agents. Several alkaloids isolated from Cryptolepis sanguinolenta have been reported to have antiplasmodial activity, but their protein targets are unknown. Therefore, molecular docking and molecular dynamics simulations were used to investigate the interactions and stability of the alkaloids with PfDHFR and PfDHODH. Based on physicochemical characteristics, alkaloids were grouped as sterically bulky (sb) or planar (pg). Docking results revealed strong binding affinities (−6.0 to −13.4 kcal/mol) of the alkaloids against PfDHODH and various strains of PfDHFR while interacting with key residues such as Asp54 and Phe58 in PfDHFR. The pg alkaloids had high binding affinity and preference for the inhibitor binding domain over the flavin mononucleotide (FMN) binding domain in PfDHODH due to size considerations. From the molecular dynamics trajectories, protein-alkaloid complexes were stable throughout the simulation, with supporting evidence from root mean square deviations, root mean square fluctuations, radius of gyration, free binding energies, and other parameters. We report herein that biscryptolepine and cryptomisrine (sb class), as well as cryptolepinone, cryptoheptine, cryptolepine, and neocryptolepine (pg class), are capable of inhibiting PfDHFR effectively in pyrimethamine sensitive and resistant cells. Also, our results show that alkaloids of the pg class can inhibit PfDHODH as FMN decoys, as well as direct enzyme inhibitors, thereby halting crucial protein function.

Molecular Markers for Sulfadoxine/Pyrimethamine and Chloroquine Resistance in Plasmodium falciparum in Thailand.

Drug resistance is an important problem hindering malaria elimination in tropical areas. Point mutations in Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes confer resistance to antifolate drug, sulfadoxine-pyrimethamine (SP) while P. falciparum chloroquine-resistant transporter (Pfcrt) genes caused resistance to chloroquine (CQ). Decline in Pfdhfr/Pfdhps and Pfcrt mutations after withdrawal of SP and CQ has been reported. The aim of present study was to investigate the prevalence of Pfdhfr, Pfdhps, and Pfcrt mutation from 2 endemic areas of Thailand. All of 200 blood samples collected from western area (Thai-Myanmar) and southern area (Thai-Malaysian) contained multiple mutations in Pfdhfr and Pfdhps genes. The most prevalent haplotypes for Pfdhfr and Pfdhps were quadruple and double mutations, respectively. The quadruple and triple mutations of Pfdhfr and Pfdhps were common in western samples, whereas low frequency of triple and double mutations was found in southern samples, respectively. The Pfcrt 76T mutation was present in all samples examined. Malaria isolated from 2 different endemic regions of Thailand had high mutation rates in the Pfdhfr, Pfdhps, and Pfcrt genes. These findings highlighted the fixation of mutant alleles causing resistance of SP and CQ in this area. It is necessary to monitor the re-emergence of SP and CQ sensitive parasites in this area.

Prevalence of Molecular Markers of Plasmodium Falciparum Resistance to Proguanil and Pyrimethamine in Children with Haemoglobin Phenotypes SS and AA in Benin City, Nigeria

Background: Chemoprophylaxis against Plasmodium falciparum (Pf) is advocated in children with sickle cell anaemia (SCA). Among them, antifolates: proguanil and pyrimethamine had replaced initial chemoprophylactic drugs because of widespread resistance. In recent past, efficacy of these antifolates has also come under scrutiny due to increasing level of drug resistance. Specific point mutations on Plasmodium falciparum dihydrofolate reductase gene (pfdhfr) have been linked with resistance to proguanil and pyrimethamine and they can be used as markers in monitoring prevalence and level of resistance to the drugs.&#x0D; Objectives: To determine the prevalence of molecular markers of Plasmodium falciparum resistance to proguanil and pyrimethamine in children with SCA.&#x0D; Methods: A total of 146 Plasmodium falciparum isolates (71 from children with SCA and 75 from those with Haemoglobin AA: HbAA) were evaluated for point mutations and mutant haplotypes on the pfdhfr gene using nested polymerase chain reaction amplification followed by direct sequencing.&#x0D; Results: The triple (S108N+N51I+C59N) mutant haplotype was present in 100.0% and 96.0% of samples from children with SCA and HbAA respectively. S108T, A16V and 1164L mutationswere not present in both groups.&#x0D; Conclusion: High prevalence of triple mutant haplotype mediates significant resistance to pyrimethamine and implies that pyrimethamine resistance is fixed in the study locale. However, the absence of pfdhfr S108T and A16V mutations, which indicate specific resistance to proguanil but not to pyrimethamine, suggests that proguanil is still useful even in the face of pyrimethamine resistance. The threat of proguanil resistance is however real due to high prevalence of the triple mutant pfdhfr haplotype.&#x0D; Keywords: Drug-resistance; Molecular markers; Plasmodium falciparum; Proguanil; Pyrimethamine.

Microfluidic Biosensor for Rapid Nucleic Acid Quantitation Based on Hyperspectral Interferometric Amplicon-Complex Analysis.

Nucleic acid detection plays a vital role in both biomedical research and clinical medicine. The temperature circulation changes of the widely used polymerase chain reaction technique are time-consuming and technically challenging for system development. Recombinase polymerase amplification (RPA) is an isothermal method for rapid nucleic acid detection. However, current RPA amplicon detection methods are complicated and expensive and easily generate false positives, restricting the promotion of RPA techniques. In this work, a hyperspectral interferometric amplicon-complex quantitation method is presented, combined with asymmetric dipole complex strategy optical scattering analysis. GelRed dye was utilized to form amplicon-complex particles, and the Fourier domain spectrum computation contributed to complex scattering quantitation. With this method, a supporting microfluidic chip and automatic system were developed to achieve integrated, rapid, quantitative, and miniscule nucleic acid detection. The Plasmodium falciparumdhfr gene was utilized as an example for targeted nucleic acid quantitation and single nucleotide polymorphism detection. The total reaction time was decreased to merely 20 min, and the limit of detection was only 3.17 ng/μL. The minimum measurable concentration of target was 1.68 copies/μL, 31.67 times more sensitive than turbidity detection, and the single reaction chamber was only 9.33 μL. No scattering increase occurred for template-free control, and thus, false positives caused by primer dimers and nonspecific products could be avoided. The experimental results prove that the provided method and system can detect single-base mutations in the dhfr gene and is a reasonable technique for rapid, automatic, and low-cost nucleic acid detection.

Decreased Susceptibility to Dihydrofolate Reductase Inhibitors Associated With Genetic Polymorphisms in Ugandan Plasmodium falciparum Isolates.

The Plasmodium falciparum dihydrofolate reductase (PfDHFR) inhibitors pyrimethamine and cycloguanil (the active metabolite of proguanil) have important roles in malaria chemoprevention, but drug resistance challenges their efficacies. A new compound, P218, was designed to overcome resistance, but drug-susceptibility data for P falciparum field isolates are limited. We studied ex vivo PfDHFR inhibitor susceptibilities of 559 isolates from Tororo and Busia districts, Uganda, from 2016 to 2020, sequenced 383 isolates, and assessed associations between genotypes and drug-susceptibility phenotypes. Median half-maximal inhibitory concentrations (IC50s) were 42 100 nM for pyrimethamine, 1200 nM for cycloguanil, 13000 nM for proguanil, and 0.6 nM for P218. Among sequenced isolates, 3 PfDHFR mutations, 51I (100%), 59R (93.7%), and 108N (100%), were very common, as previously seen in Uganda, and another mutation, 164L (12.8%), had moderate prevalence. Increasing numbers of mutations were associated with decreasing susceptibility to pyrimethamine, cycloguanil, and P218, but not proguanil, which does not act directly against PfDHFR. Differences in P218 susceptibilities were modest, with median IC50s of 1.4 nM for parasites with mixed genotype at position 164 and 5.7 nM for pure quadruple mutant (51I/59R/108N/164L) parasites. Resistance-mediating PfDHFR mutations were common in Ugandan isolates, but P218 retained excellent activity against mutant parasites.

Molecular Docking Studies of (2E)-1(1-Methyl-1H-Indol-3-yl)-3-Phenyl-Prop-2-en-one) as Antimalarial and Its Synthesis using Dimethylsulfate

Malaria is a disease with high prevalence in tropical and subtropical areas. The case of parasite resistance to proguanil, an antimalarial drug, has prompted the discovery of new drug candidates through molecular docking studies. In this study, we used the derivative of chalcone (2E)-1-(1-methyl-1H-indol-3-yl)-3-phenyl-prop-2-en-1-one as a test ligand and some comparison ligands to see its interaction with Plasmodium falciparum dihydrofolate reductase - thymidylate synthase (PfDHFR-TS) enzyme. Furthermore, the compound (2E)-1-(1-methyl-1H-indol-3-yl)-3-phenyl prop-2-en-1-one was synthesized by reacting (2E)-1-(1H-indol-3-yl)-3-phenyl prop-2-en-1-one and dimethyl sulfate through an N-alkylation reaction. The (2E)-1-(1-methyl-1H-indol-3-yl)-3-phenyl-prop-2-en-1-one has the higher ΔG bind (-7.53 kcal/mole) than native ligan WR99210(-8.83 kcal/mole), but has the lower than proguanil (-6.75 kcal/mole) and (E)-3-(3,4-dimethoxy phenyl)-1-(2-hydroxy-4-methoxy-5-(prenyl)phenyl)-prop-2-en-1-one (-7.29 kcal/mole). However, there was only pi interaction of amino acid residue of the PfDHFR-TS ligand complexes (PHE58, ILE112, LEU119, ALA16, ILE14), in contrast to the WR99210 and proguanil complexes. This is similar to the interaction of the (E)-3-(3,4-dimethoxyphenyl)-1-(2-hydroxy-4-methoxy-5-(prenyl)phenyl)-prop-2-en-1-one-PfDHFR-TS complex which is active as an antimalarial in vitro, so that the test ligand still has a chance to be used as an antimalarial candidate. The compound (2E)-1-(1-methyl-1H-indol-3-yl)-3-phenyl prop-2-en-1-one was successfully synthesized with 41.55% yield.

Single Nucleotide Polymorphisms of Pfdhfr and Pfdhps Genes: Implications for Malaria Prophylactic Strategies in Maiduguri, Northeast Nigeria

Background. The success of Intermittent Preventive Treatment in Pregnancy (IPTp), Intermittent Preventive Treatment in Infancy (IPTi), and Seasonal Malaria Chemoprevention (SMC) depends on sulfadoxine-pyrimethamine (SP) efficacy. Objective. The study determined Single Nucleotide Polymorphisms (SNPs) of Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) in Maiduguri, Northeast Nigeria. Materials and Methods. Giemsa-stained blood smears, capillary blood, and dried blood spot samples were collected from 63 subjects with uncomplicated malaria in Maiduguri between May and October 2018. Plasmodium species was determined and parasite density (PD) was estimated using the smears. Genomic DNA (gDNA) of P. falciparum was extracted from the dried blood spot samples using QIAamp DNA Mini Kit. The gDNA was subjected to nested PCR followed by restriction fragment length polymorphism (RFLP) to determine SNPs at Pfdhfr codons N51I, C59R, and S108N and Pfdhps codons S436A/F, A437G, and K540E. Results. The subjects’ mean age ± standard deviation was 23.6 ± 8.7 (2.0–67.0) years with a geometric mean PD of 8,948 (2,100–13,400) asexual parasites/µl blood. SNPs prevalence at any of the six Pfdhfr and Pfdhps codons was 85.7% (54/63); the prevalence was higher ( p &lt; 0.05 ) in Pfdhfr (82.5%; 52/63) than Pfdhps (58.7%; 37/63). Pfdhfr allele 108N (82.5%; 52/63) was the highest ( p &lt; 0.05 ) mutant when compared with alleles 51I (60.3%; 38/63) and 59R (66.7%; 42/63). Triple Pfdhfr mutation was observed in 60.3% (38/63) of the isolates and was higher ( p &lt; 0.05 ) among female subjects and SP recipients. Prevalence of Pfdhps allele 436A (28.6%; 18/63) was similar ( p &gt; 0.05 ) to allele 437G (34.9%; 22/63), with double mutation recorded in 4.8% (3/63). K540E mutation was not observed. Conclusion. Pfdhfr and Pfdhps mutations observed in Maiduguri are suggestive of SP resistance level, and this could constitute a setback to malaria prophylactic strategies in the region if unchecked. Thus, there is a need to investigate the clinical efficacy of SP.

Polymorphisms in Plasmodium falciparum dihydropteroate synthetase and dihydrofolate reductase genes in Nigerian children with uncomplicated malaria using high-resolution melting technique

In 2005, the Nigerian Federal Ministry of Health revised the treatment policy for uncomplicated malaria with the introduction of artemisinin-based combination therapies (ACTs). This policy change discouraged the use of Sulphadoxine-pyrimethamine (SP) as the second-line treatment of uncomplicated falciparum malaria. However, SP is used as an intermittent preventive treatment of malaria in pregnancy (IPTp) and seasonal malaria chemoprevention (SMC) in children aged 3–59 months. There have been increasing reports of SP resistance especially in the non-pregnant population in Nigeria, thus, the need to continually monitor the efficacy of SP as IPTp and SMC by estimating polymorphisms in dihydropteroate synthetase (dhps) and dihydrofolate reductase (dhfr) genes associated with SP resistance. The high resolution-melting (HRM) assay was used to investigate polymorphisms in codons 51, 59, 108 and 164 of the dhfr gene and codons 437, 540, 581 and 613 of the dhps gene. DNA was extracted from 271 dried bloodspot filter paper samples obtained from children (< 5 years old) with uncomplicated malaria. The dhfr triple mutant I51R59N108, dhps double mutant G437G581 and quadruple dhfr I51R59N108 + dhps G437 mutant haplotypes were observed in 80.8%, 13.7% and 52.8% parasites, respectively. Although the quintuple dhfr I51R59N108 + dhps G437E540 and sextuple dhfr I51R59N108 + dhps G437E540G581 mutant haplotypes linked with in-vivo and in-vitro SP resistance were not detected, constant surveillance of these haplotypes should be done in the country to detect any change in prevalence.

Discovery of new non-pyrimidine scaffolds as Plasmodium falciparum DHFR inhibitors by fragment-based screening

In various malaria-endemic regions, the appearance of resistance has precluded the use of pyrimidine-based antifolate drugs. Here, a three-step fragment screening was used to identify new non-pyrimidine Plasmodium falciparum dihydrofolate reductase (PfDHFR) inhibitors. Starting from a 1163-fragment commercial library, a two-step differential scanning fluorimetry screen identified 75 primary fragment hits. Subsequent enzyme inhibition assay identified 11 fragments displaying IC50 in the 28-695 μM range and selectivity for PfDHFR. In addition to the known pyrimidine, three new anti-PfDHFR chemotypes were identified. Fragments from each chemotype were successfully co-crystallized with PfDHFR, revealing a binding in the active site, in the vicinity of catalytic residues, which was confirmed by molecular docking on all fragment hits. Finally, comparison with similar non-hit fragments provides preliminary input on available growth vectors for future drug development.

Molecular Docking and QSAR Study of Chalcone and Pyrimidine Derivatives as Potent Anti-Malarial Agents against &lt;i&gt;Plasmodium falciparum&lt;/i&gt;

A data set of chalcone and pyrimidine derivatives with anti-malarial activity against Plasmodium falciparum was employed in investigating the quantitative structure-activity relationship (QSAR). Molecular docking study was performed for plasmodium falciparum dihydrofolate reductase (PfDHFR-TS). Genetic function approximation (GFA) technique was used to identify the descriptors that have influence on anti-malarial activity. The most influencing molecular descriptors identified include thermodynamics, structural and physical descriptors. Generated model was found to be good based on correlation coefficient, LOF, rm2 and rcv2 values. Nrotb, solubility, polarizibility may have negative influence on antimalarial activity or play an important role in growth inhibition of Plasmodium falciparum. The QSAR models so constructed provide fruitful insights for the future development of anti-malarial agents.

Molecular Docking and QSAR Study of Chalcone and Pyrimidine Derivatives as Potent Anti-Malarial Agents against &lt;i&gt;Plasmodium falciparum&lt;/i&gt;

A data set of chalcone and pyrimidine derivatives with anti-malarial activity against Plasmodium falciparum was employed in investigating the quantitative structure-activity relationship (QSAR). Molecular docking study was performed for plasmodium falciparum dihydrofolate reductase (PfDHFR-TS). Genetic function approximation (GFA) technique was used to identify the descriptors that have influence on anti-malarial activity. The most influencing molecular descriptors identified include thermodynamics, structural and physical descriptors. Generated model was found to be good based on correlation coefficient, LOF, rm2 and rcv2 values. Nrotb, solubility, polarizibility may have negative influence on antimalarial activity or play an important role in growth inhibition of Plasmodium falciparum. The QSAR models so constructed provide fruitful insights for the future development of anti-malarial agents.

Changes in the frequencies of Plasmodium falciparum dhps and dhfr drug-resistant mutations in children from Western Kenya from 2005 to 2018: the rise of Pfdhps S436H

BackgroundSulfadoxine-pyrimethamine (SP) is the only anti-malarial drug formulation approved for intermittent preventive treatment in pregnancy (IPTp). However, mutations in the Plasmodium falciparum dhfr (Pfdhfr) and dhps (Pfdhps) genes confer resistance to pyrimethamine and sulfadoxine, respectively. Here, the frequencies of SP resistance-associated mutations from 2005 to 2018 were compared in samples from Kenyan children with malaria residing in a holoendemic transmission region.MethodsPartial sequences of the Pfdhfr and Pfdhps genes were amplified and sequenced from samples collected in 2005 (n = 81), 2010 (n = 95), 2017 (n = 43), and 2018 (n = 55). The frequency of known mutations conferring resistance to pyrimethamine and sulfadoxine were estimated and compared. Since artemisinin-based combination therapy (ACT) is the current first-line treatment for malaria, the presence of mutations in the propeller domain of P. falciparum kelch13 gene (Pfk13) linked to ACT-delayed parasite clearance was studied in the 2017/18 samples.ResultsAmong other changes, the point mutation of Pfdhps S436H increased in frequency from undetectable in 2005 to 28% in 2017/18. Triple Pfdhfr mutant allele (CIRNI) increased in frequency from 84% in 2005 to 95% in 2017/18, while the frequency of Pfdhfr double mutant alleles declined (allele CICNI from 29% in 2005 to 6% in 2017/18, and CNRNI from 9% in 2005 to undetectable in 2010 and 2017/18). Thus, a multilocus Pfdhfr/Pfdhps genotype with six mutations (HGEAA/CIRNI), including Pfdhps S436H, increased in frequency from 2010 to 2017/18. Although none of the mutations associated with ACT-delayed parasite clearance was observed, the Pfk13 mutation A578S, the most widespread Pfk13 SNP found in Africa, was detected in low frequency (2.04%).ConclusionsThere were changes in SP resistance mutant allele frequencies, including an increase in the Pfdhps S436H. Although these patterns seem consistent with directional selection due to drug pressure, there is a lack of information to determine the actual cause of such changes. These results suggest incorporating molecular surveillance of Pfdhfr/Pfdhps mutations in the context of SP efficacy studies for intermittent preventive treatment in pregnancy (IPTp).