Plasmodium Falciparum Dihydrofolate Reductase Research Articles

Flexible 2,4-diaminopyrimidine bearing a butyrolactone as Plasmodium falciparum dihydrofolate reductase inhibitors

Recently, P218, a new flexible antifolate targeting Plasmodium falciparum dihydrofolate reductase (PfDHFR), has entered its clinical trial with good safety profile and effective Pf infection prevention. However, it carries a free carboxyl terminal, which is hydrophilic and prone to metabolic glucuronidation. Here, a new series of P218 analogues carrying butyrolactone has been synthesized with the purpose of enhancing lipophilicity and minimizing metabolic instability. The inhibition constants against the mutant PfDHFR enzymes are in sub-nanomolar level and the antimalarial activity against antifolate-resistant parasites are in the low micromolar range. The crystal structure of the most potent analogue LA1 bound enzyme complex indicates interaction with multiple residues, including Arg122 and Phe116 in the active site. In vitro log D7.4 and kinetic solubility confirmed a higher lipophilicity of this butyrolactone series as compared to P218. These outcomes suggest the possibility to further develop butyrolactone derivatives as non-carboxyl antiplasmodial antifolates.

Assessment of Saliva and Urine Performance for Antimalarial Drug Resistance Molecular Markers Study

<i>Background</i>: The malaria diagnostic tools developed to date require blood to be taken. However, certain groups in the population are reluctant to take blood samples because of their cultural habits (blood taboo), or because of the fear associated with the trauma of the injection, especially when the sample is taken repeatedly. Saliva and urine, which are not very invasive to collect, have not been widely used for malaria diagnosis. The aim of this study is to assess the performance of saliva and urine in detecting molecular markers of <i>Plasmodium falciparum </i>resistance to antimalarial drugs. <i>Methodology</i>: Blood, urine and saliva samples were collected in three different localities from 94 patients over 2 years of age with microscopically confirmed <i>Plasmodium falciparum </i>uncomplicated malaria. <i>P. falciparum </i>genomic DNA (Deoxyribonucleic acid) was then extracted and amplified using primers specific for the <i>Pfcrt (Plasmodium falciparum Chloroquine Resistance Transporter), Pfdhfr (Plasmodium falciparum dihydrofolate reductase) </i>and <i>PfK13 propeller (Plasmodium falciparum Kelch13 propeller) </i>genes. The amplification products were processed by electrophoresis and analyzed against blood, saliva and urine samples. A multivariate statistical analysis in R programming environment was performed aiming to assess the performance of blood, saliva and urine samples in detecting molecular markers of <i>P. falciparum </i>resistance. <i>Results</i>: Agarose gel electrophoresis of the amplification products of each gene detected the <i>Pfcrt </i>genes at 80.85% (76/94), <i>Pfdhfr </i>at 95.74% (90/94) and <i>PfK13 Propeller </i>at 98.93% (93/94) in blood. In saliva, gene detection levels were 50% (47/94), 69.14% (65/94) and 4.26% (4/94) respectively for the <I>K13</I> propeller, <i>Pfdhfr</i> and <i>Pfcrt</i> genes. Unlike the <i>Pfcrt </i>gene, which was not detected, 45.74% (43/94) and 38.30% (36/94) of <i>PfK13</i> Propeller and <i>pfdhfr</i> genes respectively were detected in urine. Taking blood as the reference biological sample, statistical analysis showed that unlike urine, saliva exhibited a detection performance for molecular markers of antimalarial drug resistance (<i>pfcrt,</i> <i>pfdhfr</i>, <i>pfK13</i> propeller) close to that of blood (p < 0.05). The performance of saliva and urine was also assessed on the basis of the detection of the molecular markers <i>pfdhfr</i>, <i>pfcrt </i>and <i>pfK13 </i>using ROC (<i>receiver operational characteristic</i>) analysis. The data revealed a high sensitivity of saliva compared with urine in the detection of the <i>pfdhfr</i>, <i>pfcrt </i>and <i>pfK13 propeller </i>genes. <i>Conclusion</i>: The levels of detection of molecular markers of antimalarial drug resistance studied in saliva are close to those in blood. Saliva is a high-performance biological product that could potentially be used as an alternative non-invasive sample for the study of molecular markers of Plasmodium falciparum resistance to antimalarial drugs.

Synthesis, In Silico Study and Antimalarial Activity of Triazolo[1,5‐a]pyrimidine Derivatives

AbstractThe present study mainly focused on synthesizing novel triazolo[1,5‐a]pyrimidine‐6‐carboxamide derivatives using multicomponent reaction. Synthesized derivatives were duly characterized using spectroscopic methods of analysis FT‐IR and mass spectroscopy, and 1H NMR and 13C NMR confirmed the structure of compounds. Further, the potential of the synthesized compounds against Plasmodium falciparum dihydrofolate reductase (Pf‐DHFR) inhibitors was evaluated by in vitro antimalarial activity, and the results of the study showed moderate to good antimalarial profile of synthesized entities. In silico study of all the synthesized compounds was carried out using Schrödinger LLC‐2020‐3 Software to explain the binding mode and interactions between molecules and pf DHFR enzyme. To study the drug likeliness of molecules, 3D QSAR and Pharmacokinetic studies have been carried out, and the results obtained showed a good pharmacokinetics profile of two compounds, namely AMP‐24 and AMP‐28, having good IC50 values concerning standard drugs. Further, the in vitro enzyme inhibition assay results suggested that the synthesized compound interacts nicely with the enzyme and might be used as a potent antimalarial agent.

Plasmodium falciparum dhps and dhfr markers of resistance to sulfadoxine–pyrimethamine five years (2016–2020) after the implementation of seasonal malaria chemoprevention in Cameroon

Background Antimalarial drug resistance is a major challenge in the fight against malaria. Cameroon implemented seasonal malaria chemoprevention (SMC) with sulfadoxine–pyrimethamine and amodiaquine (SPAQ) to over 1.5 million children aged 3–59 months from 2016, raising concerns whether drug pressure may lead to a selection of known parasite resistance mutations. This study aimed at assessing the profiles of plasmodium falciparum dihydrofolate reductase (DHFR) and plasmodium falciparum dihydropteroate synthase (DHPS) gene mutations that encode enzyme targeting SP before and 5 years after the introduction of SMC in the northern part of Cameroon. Methods Dried blood spots were prepared from symptomatic P. falciparum-positive children prior to SPAQ administration in 2016 and after the SMC round of 2020. DNA was extracted using the Chelex-100 method, and dhfr and dhps mutations were determined after a nested polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) technique and agarose gel electrophoresis. Results 405 children with acute uncomplicated malaria were recruited. Of 405 samples, 201/405 (49.63%) were collected in 2016 and 204/405 (50.37%) were collected in 2020. High levels of mutant alleles S108N, C59R, N51I of dhfr were obtained both in 2016 and 2020 (174 (100%), 166 (95.4%), 131 (75.3%)); (140 (99.4%), 131 (92.2%), 114 (80.3%)) while the frequency of dhps mutant alleles in the A437G and K540E loci stood at 93 (51.9%) and 6 (3.4%) in 2016 and 73 (52.5%) and 4 (2.8%) in 2020, respectively. The quintuple resistant haplotype IRNGE was found in two (1.1%) and one (0.7%) in 2016 and 2020, respectively. No significant difference was observed in the frequency of the studied mutations between the two time points, although we noted a rise in the resistance conferring haplotype IRNG in 2020. Conclusions Continuous monitoring is recommended to preempt the widespread occurrence of high-grade resistance bearing parasites in the northern regions of Cameroon.

Plasmodium ovale spp dhfr mutations associated with reduced susceptibility to pyrimethamine in sub-Saharan Africa: a retrospective genetic epidemiology and functional study

Mutations in the Plasmodium falciparum dhfr gene confer resistance to pyrimethamine, which is widely used for malaria chemoprevention in Africa. We aimed to evaluate the frequency and evolution of dhfr mutations in Plasmodium ovale spp in Africa and their functional consequences, which are incompletely characterised. We analysed dhfr mutations and their frequencies in P ovale spp isolates collected between Feb 1, 2004, and Aug31, 2023, from the French National Malaria Reference Centre collection and from field studies in Benin, Gabon, and Kenya. Genetic patterns of positive selection were investigated. Full-length recombinant wild-type and mutant DHFR enzymes from both P ovale curtisi and P ovale wallikeri were expressed in bacteria to test whether the most common mutations reduced pyrimethamine susceptibility. We included 518 P ovale spp samples (314 P ovale curtisi and 204 P ovale wallikeri). In P ovale curtisi, Ala15Ser-Ser58Arg was the most common dhfr mutation (39%; 124 of 314 samples). In P ovale wallikeri, dhfr mutations were less frequent, with Phe57Leu-Ser58Arg reaching 17% (34 of 204 samples). These two mutants were the most prevalent in central and east Africa and were fixed in Kenyan isolates. We detected six and four other non-synonymous mutations, representing 8% (24 isolates) and 2% (five isolates) of the P ovale curtisi and Povale wallikeri isolates, respectively. Whole-genome sequencing and microsatellite analyses revealed reduced genetic diversity around themutant pocdhfr and powdhfr genes. The mutant DHFR proteins showed structural changes at the pyrimethamine binding site in-silico, confirmed by a 4-times increase in pyrimethamine half-maximal inhibitory concentration in an Escherichia coli growth assay for the Phe57Leu-Ser58Arg mutant and 50-times increase for the Ala15Ser-Ser58Arg mutant, compared with the wild-type counterparts. The widespread use of sulfadoxine-pyrimethamine for malaria chemoprevention might have exerted fortuitous selection pressure for dhfr mutations in P ovale spp. This calls for closer monitoring of dhfr and dhps mutations in P ovale spp. French Ministry of Health, Agence Nationale de la Recherche, and Global Emerging Infections Surveillance branch of the Armed Forces Health Surveillance Division.

Sub-microscopic Plasmodium falciparum infections and multiple drug resistant single nucleotide polymorphic alleles in pregnant women from southwestern Nigeria

ObjectivesThe study evaluated sub-microscopic malaria infections in pregnancy using two malaria Rapid Diagnostic Tests (mRDTs), microscopy and RT-PCR and characterized Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and Plasmodium falciparum dihydropteroate synthase (Pfdhps) drug resistant markers in positive samples.MethodsThis was a cross sectional survey of 121 pregnant women. Participants were finger pricked, blood drops were collected for rapid diagnosis with P. falciparum histidine-rich protein 11 rapid diagnostic test kit and the ultra-sensitive Alere Pf malaria RDT, Blood smears for microscopy and dried blood spots on Whatman filter paper for molecular analysis were made. Real time PCR targeting the var acidic terminal sequence (varATS) gene of P. falciparum was carried out on a CFX 96 real time system thermocycler (BioRad) in discriminating malaria infections. For each run, laboratory strain of P. falciparum 3D7 and nuclease free water were used as positive and negative controls respectively. Additionally, High resolution melt analyses was employed for genotyping of the different drug resistance markers.ResultsOut of one hundred and twenty-one pregnant women sampled, the SD Bioline™ Malaria Ag P.f HRP2-based malaria rapid diagnostic test (mRDT) detected eight (0.06%) cases, the ultra-sensitive Alere™ malaria Ag P.f rapid diagnostic test mRDT had similar outcome in the same samples as detected by the HRP2-based mRDT. Microscopy and RT-PCR confirmed four out of the eight infections detected by both rapid diagnostic tests as true positive and RT-PCR further detected three false negative samples by the two mRDTs providing a sub-microscopic malaria prevalence of 3.3%. Single nucleotide polymorphism in Pfdhps gene associated with sulphadoxine resistance revealed the presence of S613 mutant genotypes in three of the seven positive isolates and isolates with mixed wild/mutant genotype at codon A613S. Furthermore, four mixed genotypes at the A581G codon were also recorded while the other Pfdhps codons (A436G, A437G and K540E) showed the presence of wild type alleles. In the Pfdhfr gene, there were mutations in 28.6%, 28.6%, and 85.7% at the I51, R59 and N108 codons respectively. Mixed wild and mutant type genotypes were also observed in 28.6% each of the N51I, and C59R codons. For the Pfcrt, two haplotypes CVMNK and CVIET were observed. The SVMNT was altogether absent. Triple mutant CVIET 1(14.3%) and triple mutant + wild genotype CVIET + CVMNK 1(14.3%) were observed. The Pfmdr1 haplotypes were single mutants YYND 1(14.3%); NFND 1(14.3%) and double mutants YFND 4(57.1%); YYDD 1(14.3%).

Identification of novel peptide inhibitors of Plasmodium falciparum dihydrofolate reductase (PfDHFR): molecular docking and MD simulation studies

The presence of drug-resistant variants of Plasmodium parasites within the population has presented a substantial obstacle to the eradication of Malaria. As a result, numerous research groups have directed their efforts towards creating new medication candidates that specifically target parasites. In this study, our main objective was to identify tri-peptide inhibitors for Plasmodium falciparum Dihydrofolate Reductase (PfDHFR) with the aim of finding a new peptide that exhibits superior binding properties compared to the current inhibitor, WR99210. In order to achieve this objective, a virtual library consisting of 8000 tripeptides was generated and subjected to computational screening against wild-type PfDHFR. The purpose of this screening was to discover the most effective binders at the active site. The four most optimal tripeptides identified (Trp-Trp-Glu, Trp-Phe-Tyr, Phe-Trp-Trp, Tyr-Trp-Trp) exhibited significant non-covalent interactions inside the active site of PfDHFR and had binding energies ranging from −9.5 to −9.0 kcal/mol and WR99210 had a binding energy of −6.2 kcal/mol. A 250 ns Molecular Dynamics (MD) simulation was performed to investigate the kinetic and thermodynamic characteristics of the protein-ligand complexes. The Root Mean Square Deviation (RMSD) values for the optimal tripeptides fell within the allowed range, indicating the stability of the ligands inside the protein complex. The Ki value for the most effective tripeptide was 0.3482 µM, whereas WR99210 had a Ki value of 1.02 µM. This article presents the initial discovery of peptide inhibitors targeting PfDHFR. In this text, we provide a comprehensive explanation of the interactions that occur between peptides and the enzyme. Communicated by Ramaswamy H. Sarma

Combined 3D-QSAR, molecular docking, ADMET, and drug-likeness scoring of novel diaminodihydrotriazines as potential antimalarial agents

Plasmodium falciparum dihydrofolate reductase (PfDHFR) is a promising therapeutic target for antimalarial interventions. Diaminodihydrotriazine derivatives have demonstrated their efficacy as antimalarial agents by targeting PfDHFR inhibition. In this study, we used a multifaceted approach including 3D-QSAR, in silico pharmacokinetic/ADMET evaluation, molecular docking techniques, and molecular dynamics simulation to explore a series of diaminodihydrotriazine compounds, to identify new antimalarial drug candidates featuring the diaminodihydrotriazine scaffold. Using comparative molecular similarity indices analysis (CoMSIA), we built a robust 3D-QSAR model exhibiting favorable statistical parameters, with Q2 and R2 values of 0.553 and 0.981, respectively. External validation using a test set yielded an R2pre value of 0.787, confirming the model's predictive ability. In addition, we performed comprehensive ADMET and drug similarity profile analyses for potent designed compounds, yielding promising results. Molecular docking studies further complemented our investigation facilitating the elucidation of optimal interaction modes between these inhibitors and their target receptor. Additionally, molecular dynamics simulation was conducted for the promising compound 8a. This study not only provides valuable insights into the design of new antimalarial agents but also highlights the importance of computational methods for accelerating drug discovery processes, particularly in the context of infectious disease control.

In-silico studies of hydroxyxanthone derivatives as potential pfDHFR and pfDHODH inhibitor by molecular docking, molecular dynamics simulation, MM-PBSA calculation and pharmacokinetics prediction

The investigation of hydroxyxanthone derivatives has been conducted, including molecular docking, molecular dynamics simulation MM-PBSA binding energy calculation, and pharmacokinetics prediction of the potential plasmodium falciparum dihydrofolate reductase (pfDHFR) and plasmodium falciparum dihydroorotate dehydrogenase (pfDHODH) inhibitor. The Docking result showed that compound 1,3,6,7-tetrahydroxy-5,8-bis(3-methyl-2-buten-1-yl)-9H-xanthen-9-one (X16) was found to be the best ligand with good inhibitory action against pfDHFR. Meanwhile, the pfDHODH protein was compounded 1,3,6,7-tetrahydroxy-5,8-dinitro-9H-xanthen-9-one (X14). Additionally, the hydroxyxanthone X16 complex showed more excellent stability in the molecular dynamics simulation of the pfDHFR protein than the ligand WR99210 and chloroquine. The MM-PBSA calculation showed that compound X16 had lower binding energy than ligand WR99210. However, 1,3-dihydroxy-8-(3-methyl-2-buten-1-yl)-9H-xanthen-9-one (X4), 1,3,6,7-tetrahydroxy-8-nitro-9H-xanthen-9-one (X10), 1,3,6,7-tetrahydroxy-9-oxo-9H-xanthene-8-sulfonic acid (X11), and 1,3,6,7-tetrahydroxy-5,8-dinitro-9H-xanthen-9-one (X14) complexes were shown to be more stable than chloroquine and to have the same stability when compared to the native ligand A26, according to a molecular dynamics simulation conducted in pfDHODH protein. The MM-PBSA calculation showed that compound X14 had lower binding energy than ligand A26. The hydroxyxanthones X4, X10–11, X14, and X16 fulfill Lipinski's rule parameters in terms of physicochemical and ADMET qualities and parameters related to absorption, distribution, metabolism, excretion, and toxicity tests. To sum up, hydroxyxanthones X4, X10–11, X14, and X16 have the potential to be antimalarial medications, but more in vivo and in vitro testing is needed to confirm this.

Pyrimidine derivatives: Their significance in the battle against malaria, cancer and viral infections

Pyrimidine derivatives play a significant role in combating viral infections, malaria, and cancer due to their diverse pharmacological properties. Pyrimidine derivatives, particularly nucleoside analogs, have been widely used as antiviral agents. They interfere with viral replication by inhibiting DNA or RNA synthesis.Examples include drugs like acyclovir (used against herpes viruses), lamivudine (for HIV and hepatitis B), and remdesivir (for COVID-19). These compounds help to manage and treat various viral infections, reducing their severity and spread. Pyrimidine derivatives are key components of antimalarial drugs like pyrimethamine and proguanil. They target the parasite Plasmodium falciparum's dihydrofolate reductase enzyme, essential for its survival. These drugs have been instrumental in combating malaria, a major global health concern, by inhibiting the growth of the malaria parasite within the human body. Pyrimidine analogs are used in chemotherapy to target rapidly dividing cancer cells. Drugs like 5-fluorouracil (5-FU) and cytarabine inhibit DNA synthesis in cancer cells, leading to cell death. These derivatives have been crucial in the treatment of various cancers, helping to slow down tumor growth and improve patient outcomes. Overall, this article aims to consolidate existing knowledge on the topic, synthesize relevant information, and contribute to a better understanding of the potential applications of pyrimidine derivatives in the fields of oncology, virology, and tropical medicine.

“Design, Synthesis, and Molecular Docking Analysis of Novel Benzo[4,5]imidazo[1,2-a]pyrimidine Hybrids Targeting Plasmodium falciparum DHFR: A Comprehensive In Silico and Biological Study”

Synthesis of Benzo[4,5]imidazo[1,2-a]pyrimidine derivatives using an environmentally friendly Biginelli-type reaction in ionic liquid DIPEAc was performed. Remarkably, this ionic liquid exhibits efficient recyclability over five cycles. Synthesized compounds underwent rigorous in vitro antimalarial efficacy screening, complemented by computational and in vitro studies, assessing their potential as Plasmodium falciparum dihydrofolate reductase (Pf-DHFR) inhibitors. A robust 3D-QSAR model was established and validated, elucidating structure-activity relationships. Estimated ADMET descriptors highlighted favorable pharmacokinetics, suggesting their role in new antimalarial drug development. This study exemplifies the synergy of sustainable synthesis, enzyme inhibition, and pharmacokinetic profiling, promising a transformative outlook for antimalarial innovation.

Peripheral and Placental Prevalence of Sulfadoxine-Pyrimethamine Resistance Markers in Plasmodium falciparum among Pregnant Women in Southern Province, Rwanda.

Intermittent preventive therapy during pregnancy (IPTp) with sulfadoxine-pyrimethamine (SP) is recommended in areas of moderate to high malaria transmission intensity. As a result of the increasing prevalence of SP resistance markers, IPTp-SP was withdrawn from Rwanda in 2008. Nonetheless, more recent findings suggest that SP may improve birthweight even in the face of parasite resistance, through alternative mechanisms that are independent of antimalarial effects. The prevalence of single nucleotide polymorphisms in Plasmodium falciparum dihydropteroate synthase (pfdhps) and dihydrofolate reductase (pfdhfr) genes associated with SP resistance among 148 pregnant women from 2016 to 2018 within Rwanda's Southern Province (Huye and Kamonyi districts) was measured using a ligase detection reaction-fluorescent microsphere assay. The frequency of pfdhps K540E, A581G, and the quintuple (pfdhfr N51I + C59R + S108N/pfdhps A437G + K540E) and sextuple (pfdhfr N51I + C59R + S108N/pfdhps A437G + K540E + A581G) mutant genotypes was 90%, 38%, 75%, and 28%, respectively. No significant genotype difference was seen between the two districts, which are approximately 50 km apart. Observed agreements for matched peripheral to placental blood were reported and found to be 207 of 208 (99%) for pfdhfr and 239 of 260 (92%) for pfdhps. The peripheral blood sample did not miss any pfdhfr drug-resistant mutants or pfdhps except at the S436 loci. At this level of the sextuple mutant, the antimalarial efficacy of SP for preventing low birthweight is reduced, although overall SP still exerts a nonmalarial benefit during pregnancy. This study further reveals the need to intensify preventive measures to sustain malaria control in Rwanda to keep the overall incidence of malaria during pregnancy low.

Evolution and spread of Plasmodium falciparum mutations associated with resistance to sulfadoxine–pyrimethamine in central Africa: a cross-sectional study

Efficacy of sulfadoxine-pyrimethamine, the malaria chemoprophylaxis used in pregnant women, and in children when combined with amodiaquine, is threatened by the accumulation of mutations in the Plasmodium falciparum dihydropteroate synthase (pfdhps) and dihydrofolate reductase (pfdhfr) genes. Data on the prevalence of resistant alleles in central Africa and the new pfdhps I431V mutation, particularly associated with other mutations to form the pfdhps vagKgs allele, are scarce. We explored the frequency and geographical distribution of pfdhps and pfdhfr mutations in central Africa in 2014-18, and assessed the evolutionary origin of the vagKgs allele. Samples were collected at 18 health-care centres in seven countries (Angola, Cameroon, Central African Republic, Democratic Republic of the Congo, Gabon, Nigeria, and Republic of the Congo) from patients who showed possible symptoms of malaria between March 1, 2014, and Oct 31, 2018. Samples that were positive for P falciparum were transported to a laboratory in Toulouse, France, and genotyped. The frequency of pfdhfr and pfdhps mutations was studied in 1749 samples. Microsatellites in pfdhps flanking regions and whole-genome analysis compared with parasite genomes from the data-sharing network MalariaGEN were performed on samples carrying the vagKgs allele. Mapping of the prevalence of single nucleotide polymorphisms and corresponding alleles of pfdhfr and pfdhps showed a substantial spread of alleles associated with sulfadoxine-pyrimethamine resistance in central Africa during the 2014-18 period, especially an increase going west to east in pfdhps alleles carrying the K540E and A581G mutations. A high prevalence of the pfdhps I431V mutation was observed in Cameroon (exceeding 50% in the northern region) and Nigeria. Genomic analysis showed a recent African emergence and a clonal expansion of the most frequent pfdhps vagKgs allele. Reduced sulfadoxine-pyrimethamine efficacy due to increased resistance is a worrying situation, especially because the malaria transmission level is high in central Africa. Although the resistance phenotype remains to be confirmed, the emergence and spread of the vagKgs allele in west and central Africa could challenge the use of sulfadoxine-pyrimethamine. Toulouse Institute for Infectious and Inflammatory Diseases.

Hybrid PABA-glutamic acid conjugated 1,3,5-triazine derivatives: Design, synthesis, and antimalarial activity screening targeting Plasmodium falciparum dihydro folate reductase enzyme.

Despite the successful reduction in the malaria health burden in recent years, it continues to remain a significant global health problem mainly because of the emerging resistance to first-line treatments. Also because of the disruption in malaria prevention services during the COVID-19 pandemic, there was an increase in malaria cases in 2021 compared to 2020. Hence, the present study outlined the in silico study, synthesis, and antimalarial evaluation of 1,3,5-triazine hybrids conjugated with PABA-glutamic acid. Docking study revealed higher binding energy compared to the originally bound ligand WR99210, predominant hydrogen bond interaction, and involvement of key amino acid residues, like Arg122, Ser120, and Arg59. Fourteen compounds were synthesized using traditional and microwave synthesis. The in vitro antimalarial evaluation against chloroquine-sensitive 3D7 and resistant Dd2 strain of Plasmodium falciparum showed a high to moderate activity range. Compounds C1 and B4 showed high efficacy against both strains and a further study revealed that compound C1 is non-cytotoxic against the HEK293 cell line with no acute oral toxicity. In vivo, study was performed for the most potent antimalarial compound C1 to optimize the research work and found to be effectively suppressing parasitemia of Plasmodium berghei strain in the Swiss albino mice model.

Oplodiol and nitidine as potential inhibitors of Plasmodium falciparum dihydrofolate reductase: insights from a computational study

Many natural products have been shown to possess antiplasmodial activities, but their protein targets are unknown. This work employed molecular docking and molecular dynamics simulations to explore the inhibitory activity of some antiplasmodial natural products against wild-type and mutant strains of Plasmodium falciparum dihydrofolate reductase (PfDHFR). From the molecular docking study, 6 ligands preferentially bind at the active site of the DHFR domain with binding energies ranging from −6.4 to −9.5 kcal/mol. Interactions of compounds with MET55 and PHE58 were mostly observed in the molecular docking study. From the molecular dynamics study, the binding of 2 of the ligands—nitidine and oplodiol—was observed to be stable against all tested strains of PfDHFR. The average binding free energy of oplodiol in complex with the various PfDHFR strains was −93.701 kJ/mol whereas that of nitidine was −106.206 kJ/mol. The impressive in silico activities of the 2 compounds suggest they could be considered for development as potential antifolate agents. Communicated by Ramaswamy H. Sarma

Mutant DHFR Prevalence and Pyrimethamine Resistance among Asymptomatic Adolescents in Kwara State

Plasmodium falciparum malaria remains a leading public health problem in sub-Saharan Africa and its control is seriously challenged by drug resistance. Resistance to sulfadoxine-pyrimethamine is mediated by point mutations in genes encoding the target enzymes dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps). Blood from a total of 176 subjects aged 11-19 years from Ilorin East and Irepodun Local Government areas (LGAs) was examined for mutant alleles of the Plasmodium falciparum dihydrofolate reductase gene by polymerase chain reaction (PCR) at codon 51 and 108 respectively. Rapid Diagnostic Test kits were used for the malaria test. Of the 176 participants, 73 (41.5%) subjects tested positive for malaria parasite while 95 (54.0%) tested negative. For both N51I and S108N SNPscreening, mutant alleles were dominant over the wild type. The Iponrin community (Ilorin East LGA) recorded the highest percentage of mutant alleles 14 (33.33%) of the N51I SNP, while the Igbonla community (Irepodun LGA) had the least number of mutant alleles 8 (19.04%). On the other hand, the Alakuko community recorded the highest number of alleles 14 (31.80%) for S108N mutant while the Igbonla community (Irepodun LGA) had the least number of mutant alleles 8 (18.18%). There was widespread pyrimethamine resistance among the studied population and the malaria parasite remains persistent among the studied population. Therefore, there is a need for monitoring antimalarial drug resistance in Nigeria for prompt management of the antimalarial drug resistance menace.

In silico screening of phytochemicals from Dissotis rotundifolia against Plasmodium falciparum Dihydrofolate Reductase

BackgroundMalaria remains a major health concern in developing countries with high morbidity and mortality, especially in pregnant women and infants. A major obstacle to the treatment of malaria is a low effectiveness and an increase resistance of the parasite to antimalarial drugs. As a result, there is an ongoing demand for new and potent antimalarial drugs. Medicinal plants remain a potential source for the development of new antimalarial drugs. Amongst them is Dissotis rotundifolia is an ethnomedical important plant used in West Africa to treat malaria. PurposeThis study aimed at identifying new potential antifolates by virtually screening phytochemicals characterized from the whole plant methanolic extract of D. rotundifolia against Plasmodium falciparum Dihydrofolate Reductase (PfDHFR). MethodsLC-ESI-Q-TOF-MS analysis was employed to identify the phytochemicals present in the whole plant methanolic extract of D. rotundifolia. These phytochemicals were docked against the catalytic site of PfDHFR. The docking protocol was evaluated using the Area Under the Curve (AUC) of a Receiver Operating Characteristic (ROC) curve. The binding mechanisms and the drug-likeness of the phytochemicals were characterized. A 100 ns Molecular Dynamics (MD) simulation and Molecular Mechanics-Poisson Boltzmann Surface Area (MM-PBSA) calculations were utilized to analyze the stability, the energy decomposition per residue and the binding free energy of the potential leads. ResultsTwenty nine phytochemicals were characterized and docked against PfDHFR. Dimethylmatairesinol, flavodic acid, sakuranetin, and sesartemin were identified as potential leads with binding affinities of -8.4, -8.9, -8.6, and -8.9 kcal/mol respectively, greater than a stringent threshold of -8.0 kcal/mol. The potential leads also interacted hydrophobically with critical residue Phe58. A novel critical residue, Leu46 was identified to be crucial in the catalytic activity of PfDHFR. The potential leads were also predicted to be anti-protozoal with a probability of active (Pa) value ranging from 0.319 to 0.537. ConclusionThis study elucidates the potential inhibition of PfDHFR by dimethylmatairesinol, flavodic acid, sakuranetin and sesartemin present in D. rotundifolia. These compounds are druglike, do not violate Lipinski's rule of five, have a high binding affinity to PfDHFR, and interact with crucial residues involved in the catalytic activity PfDHFR. Dimethylmatairesinol, flavodic acid, sakuranetin and sesartemin could therefore be further investigated and developed as new antifolate drugs for malaria.

Molecular Markers of Sulfadoxine-Pyrimethamine Resistance in Samples from Children with Uncomplicated Plasmodium falciparum at Three Sites in Angola in 2019.

Sulfadoxine-pyrimethamine (SP) is used for prevention of malaria in pregnant women in Angola. We sequenced the Plasmodium falciparum dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes, implicated in SP resistance, in samples collected during a 2019 study of artemisinin-based combination therapy efficacy in Benguela, Lunda Sul, and Zaire provinces. A total of 90 day 0 and day of failure samples were individually sequenced, while 508 day 0 samples from participants without recurrent parasitemia were pooled after DNA extraction into 61 pools. The N51I, C59R, and S108N pfdhfr mutations and A437G pfdhps mutations were present at high proportions in all provinces (weighted allele frequencies, 62% to 100%). The K540E pfdhps mutation was present at lower proportions (10% to 14%). The A581G pfdhps mutation was only observed in Zaire, at a 4.6% estimated prevalence. The I431V and A613S mutations were also only observed in Zaire, at a prevalence of 2.8% to 2.9%. The most common (27% to 66%) reconstructed haplotype in all three provinces was the canonical quadruple pfdhfr pfdhps mutant. The canonical quintuple mutant was absent in Lunda Sul and Benguela and present in 7.9% of samples in Zaire. A single canonical sextuple (2.6%) mutant was observed in Zaire Province. Proportions of the pfdhps K540E and A581G mutations were well below the World Health Organization thresholds for meaningful SP resistance (prevalence of 95% for K540E and 10% for A581G). Samples from therapeutic efficacy studies represent a convenient source of samples for monitoring SP resistance markers.

A snapshot of the prevalence of dihydropteroate synthase-431V mutation and other sulfadoxine-pyrimethamine resistance markers in Plasmodium falciparum isolates in Nigeria

BackgroundMalaria is a major public health issue with substantial risks among vulnerable populations. Currently, the World Health Organization (WHO) recommends SP-IPTp in the second and third trimesters. However, the efficacy of SP-IPTp is threatened by the emergence of sulfadoxine-pyrimethamine resistant malaria parasites due to single nucleotide polymorphisms in the Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthetase genes. This study aimed to assess the current prevalence of Pfdhfr/Pfdhps mutations in P. falciparum isolates collected from individuals residing in Ile-Ife, Nigeria, and also present maps of the prevalence of Pfdhps 431V and 581G within Nigeria and surrounding countries.MethodsBetween October 2020 and April 2021, samples were collected as dried blood spots among 188 participants who showed malaria positivity with a histidine-rich-protein-based rapid diagnostic test (RDT). Nested PCR assays were used to confirm falciparum in the samples with RDT positivity, and to amplify fragments of the Pfdhfr/Pfdhps genes followed by targeted amplicon sequencing. Published data since 2007 on the prevalence of the Pfdhps genotypes in Nigeria and the neighbouring countries were used to produce maps to show the distribution of the mutant genotypes.ResultsOnly 74 and 61 samples were successfully amplified for the Pfdhfr and Pfdhps genes, respectively. At codons resulting in N51I, C59R, and S108N, Pfdhfr carried mutant alleles of 97.3% (72/74), 97.3% (72/74) and 98.6% (73/74), respectively. The Pfdhps gene carried mutations at codons resulting in amino acid changes at 431–436-437–540-581–613; I431V [45.9%, (28/61)], A581G [31.1% (19/61)] and A613S [49.2% (30/61)]. Constructed haplotypes were mainly the triple Pfdhfr mutant 51I-59R-108N (95.9%), and the most common haplotypes observed for the Pfdhps gene were the ISGKAA (32.8%), ISGKGS (8.2%), VAGKAA (14.8%), VAGKAS (9.8%) and VAGKGS (14.8%). In the context of the previously published data, a high prevalence of 431V/581G mutations was found in the study population. It seems quite evident that the Pfdhps 431V, 581G and 613S often co-occur as Pfdhps-VAGKGS haplotype.ConclusionThis study showed that the prevalence of VAGKGS haplotype seems to be increasing in prevalence. If this is similar in effect to the emergence of 581G in East Africa, the efficacy of SP-IPTp in the presence of these novel Pfdhps mutants should be re-assessed.

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.