Falciparum Dihydrofolate Reductase Research Articles

Molecular characterization of Plasmodium falciparum antifolate resistance markers in Thailand between 2008 and 2016

BackgroundResistance to anti-malarials is a major threat to the control and elimination of malaria. Sulfadoxine–pyrimethamine (SP) anti-malarial treatment was used as a national policy for treatment of uncomplicated falciparum malaria in Thailand from 1973 to 1990. In order to determine whether withdrawal of this antifolate drug has led to restoration of SP sensitivity, the prevalence of genetic markers of SP resistance was assessed in historical Thai samples.MethodsPlasmodium falciparum DNA was collected from the Thailand–Myanmar, Thailand–Malaysia and Thailand–Cambodia borders during 2008–2016 (N = 233). Semi-nested PCR and nucleotide sequencing were used to assess mutations in Plasmodium falciparum dihydrofolate reductase (pfdhfr), P. falciparum dihydropteroate synthase (pfdhps). Gene amplification of Plasmodium falcipaurm GTP cyclohydrolase-1 (pfgch1) was assessed by quantitative real-time PCR. The association between pfdhfr/pfdhps mutations and pfgch1 copy numbers were evaluated.ResultsMutations in pfdhfr/pfdhsp and pfgch1 copy number fluctuated overtime through the study period. Altogether, 14 unique pfdhfr–pdfhps haplotypes collectively containing quadruple to octuple mutations were identified. High variation in pfdhfr–pfdhps haplotypes and a high proportion of pfgch1 multiple copy number (51% (73/146)) were observed on the Thailand–Myanmar border compared to other parts of Thailand. Overall, the prevalence of septuple mutations was observed for pfdhfr–pfdhps haplotypes. In particular, the prevalence of pfdhfr–pfdhps, septuple mutation was observed in the Thailand–Myanmar (50%, 73/146) and Thailand–Cambodia (65%, 26/40) border. In Thailand–Malaysia border, majority of the pfdhfr–pfdhps haplotypes transaction from quadruple (90%, 9/10) to quintuple (65%, 24/37) during 2008–2016. Within the pfdhfr–pfdhps haplotypes, during 2008–2013 the pfdhps A/S436F mutation was observed only in Thailand–Myanmar border (9%, 10/107), while it was not identified later. In general, significant correlation was observed between the prevalence of pfdhfr I164L (ϕ = 0.213, p-value = 0.001) or pfdhps K540E/N (ϕ = 0.399, p-value ≤ 0.001) mutations and pfgch1 gene amplification.ConclusionsDespite withdrawal of SP as anti-malarial treatment for 17 years, the border regions of Thailand continue to display high prevalence of antifolate and anti-sulfonamide resistance markers in falciparum malaria. Significant association between pfgch1 amplification and pfdhfr (I164L) or pfdhps (K540E) resistance markers were observed, suggesting a compensatory mutation.

A multicomponent reaction to design antimalarial pyridyl-indole derivatives: Synthesis, biological activities and molecular docking

Using fragment-based design strategy, new pyridyl-indole hybrids 4a-y and indole intermediates 3a-e were synthesized using multicomponent one pot reaction. The synthesized compounds were subjected to screening for antimalarial activity against chloroquine sensitive (D6) and chloroquine resistant (W2) strains of Plasmodium falciparum. Several compounds exhibited antimalarial activity with IC50 values in the range of 1.47-9.23μM, and 1.16-7.66 μM, for D6 and W2 strains, respectively. Compounds 4a, 4k and 4u showed the highest selectivity index among all the tested compounds (S.I. ranged 3.8-10). Binding interactions between the active antimalarial compounds and the active site of quadruple mutant Plasmodium falciparum dihydrofolate reductase enzyme have been investigated using molecular docking analysis.

Late clinical failure associated with cytochrome b codon 268 mutation during treatment of falciparum malaria with atovaquone\u2013proguanil in traveller returning from Congo

BackgroundThe drug combination atovaquone–proguanil, is recommended for treatment of uncomplicated falciparum malaria in France. Despite high efficacy, atovaquone–proguanil treatment failures have been reported. Resistance to cycloguanil, the active metabolite of proguanil, is conferred by multiple mutations in the Plasmodium falciparum dihydrofolate reductase (pfdhfr) and resistance to atovaquone by single mutation on codon 268 of the cytochrome b gene (pfcytb).Case presentationA 47-year-old female, native from Congo and resident in France, was admitted in hospital for uncomplicated falciparum malaria with parasitaemia of 0.5%, after travelling in Congo (Brazzaville and Pointe Noire). She was treated with atovaquone–proguanil (250 mg/100 mg) 4 tablets daily for 3 consecutive days. On day 5 after admission she was released home. However, many weeks after this episode, without having left France, she again experienced fever and intense weakness. On day 39 after the beginning of treatment, she consulted for fever, arthralgia, myalgia, photophobia, and blurred vision. She was hospitalized for uncomplicated falciparum malaria with a parasitaemia of 0.375% and treated effectively by piperaquine–artenimol (320 mg/40 mg) 3 tablets daily for 3 consecutive days. Resistance to atovaquone–proguanil was suspected. The Y268C mutation was detected in all of the isolates tested (D39, D42, D47). The genotyping of the pfdhfr gene showed a triple mutation (N51I, C59R, S108N) involved in cycloguanil resistance.ConclusionThis is the first observation of a late clinical failure of atovaquone–proguanil treatment of P. falciparum uncomplicated malaria associated with pfcytb 268 mutation in a traveller returning from Congo. These data confirm that the Y268C mutation is associated with delayed recrudescence 4 weeks or more after initial treatment. Although atovaquone–proguanil treatment failures remain rare, an increased surveillance is required. It is essential to declare and publish all well-documented cases of treatment failures because it is the only way to evaluate the level of resistance to atovaquone.

Molecular docking and QSAR studies for modeling the antimalarial activity of hybrids 4-anilinoquinoline-triazines derivatives with the wild-type and mutant receptor pf-DHFR

Plasmodium falciparum dihydrofolate reductase (pf-DHFR) is one of the several targets in the treatment of malaria. Double and quadruple mutations at residues 51, 59, 108, and 164 of pf-DHFR have been linked to antifolate resistance. Several efforts are underway to overcome this drug resistance and to produce potential inhibitors. In this regard, the quantitative structure-activity relationship (QSAR) and docking studies were performed for previously reported 4-anilinoquinoline and 1,3,5-triazines based molecular hybrids. The generated model showed good correlation coefficients (R2 = 0.70) and test set prediction coefficient (R2 = 0.74). These outcomes showed the good predictive competence of the established QSAR model. Based on these results we docked into active site of pf-DHFR protein with the most active (4) and the less active (5) compounds. The docking results revealed that these molecules interact specifically with SER108 and ILE164 in the pf-DHFR binding pocket as that of best active compound but also showed additional interactions with LEU40 and GLY44.

Design, synthesis and biological evaluation of 4-aminoquinoline-guanylthiourea derivatives as antimalarial agents

Guanylthiourea (GTU) has been identified as an important antifolate antimalarial pharmacophore unit, whereas, 4-amino quinolones are already known for antimalarial activity. In the present work molecules carrying 4-aminoquinoline and GTU moiety have been designed using molecular docking analysis with PfDHFR enzyme and heme unit. The docking results indicated that the necessary interactions (Asp54 and Ile14) and docking score (−9.63 to −7.36 kcal/mmol) were comparable to WR99210 (−9.89 kcal/mol). From these results nine molecules were selected for synthesis. In vitro analysis of these synthesized compounds reveal that out of the nine molecules, eight show antimalarial activity in the range of 0.61–7.55 μM for PfD6 strain and 0.43–8.04 μM for PfW2 strain. Further, molecular dynamics simulations were performed on the most active molecule to establish comparative binding interactions of these compounds and reference ligand with Plasmodium falciparum dihydrofolate reductase (PfDHFR).

In silico ADMET study, docking, synthesis and antimalarial evaluation of thiazole-1,3,5-triazine derivatives as Pf-DHFR inhibitor.

Plasmodium falciparum dihydrofolate reductase (Pf-DHFR) is an essential enzyme in the folate pathway and is an important target for antimalarial drug discovery. In this study a modern approach has been undertaken to identify new hits of thiazole-1,3,5-triazine derivatives as antimalarials targeting Pf-DHFR. The library of 378 thiazole-1,3,5-triazines were designed and subjected to ADME analysis. The compounds having optimal ADME score, was then evaluated by docking against wild and mutant Pf-DHFR complex. The resultant compound after screening from above these two methods were synthesized, and assayed for in vitro antimalarial against chloroquine-sensitive (3D-7) and chloroquine resistant (Dd-2) strains of P. falciparum. Twenty compounds were identified from the dataset based on considerable AlogP98 vs. PSA_2D confidence ellipse, ADME filter and TOPKAT toxicity analysis. Majority of compounds showed interaction with Asp54, Arg59, Arg122 and Ile 164 in docking analysis. Entire set of tested derivatives exhibited considerable activity at the tested dose against sensitive strain with IC50 values varying from 10.03 to 54.58 μg/ml. Furthermore, against chloroquine resistant strain, eight compounds showed IC50 from 11.29 to 40.92 μg/ml. Compound A5 and H16 were found to be the most potent against both the strains of P. Falciparum. Results of the study suggested the possible utility of thiazole-1,3,5-triazines as new lead for identifying new class of Pf-DHFR inhibitor.

Multicomplex-based pharmacophore modeling in conjunction with multi-target docking and molecular dynamics simulations for the identification of PfDHFR inhibitors

Plasmodium falciparum dihydrofolate reductase enzyme (PfDHFR) is counted as one of the attractive and validated antimalarial drug targets. However, the point mutations in the active site of wild-type PfDHFR have developed resistance against the well-known antifolates. Therefore, there is a dire need for the development of inhibitors that can inhibit both wild-type and mutant-type DHFR enzyme. In the present contribution, we have constructed the common feature pharmacophore models from the available PfDHFR. A representative hypothesis was prioritized and then employed for the screening of natural product library to search for the molecules with complementary features responsible for the inhibition. The screened candidates were processed via drug-likeness filters and molecular docking studies. The docking was carried out on the wild-type PfDHFR (3QGT); double-mutant PfDHFR (3UM5 and 1J3J) and quadruple-mutant PfDHFR (1J3K) enzymes. A total of eight common hits were obtained from the docking calculations that could be the potential inhibitors for both wild and mutant type DHFR enzymes. Eventually, the stability of these candidates with the selected proteins was evaluated via molecular dynamics simulations. Except for SPECS14, all the prioritized candidates were found to be stable throughout the simulation run. Overall, the strategy employed in the present work resulted in the retrieval of seven candidates that may show inhibitory activity against PfDHFR and could be further exploited as a scaffold to develop novel antimalarials.Communicated by Ramaswamy H. Sarma

Molecular docking analysis of phyto-constituents from Cannabis sativa with pfDHFR.

Available antimalarial drugs have been associated with numerous side effects, which include skin rashes and myelo-suppression. Therefore, it is of interest to explore compounds from natural source having drug-like properties without side effect. This study focuses on the screening of compounds from Cannabis sativa against malaria Plasmodium falciparum dihydrofolate reductase for antimalarial properties using Glide (Schrodinger maestro 2018-1). The result showed that phytochemicals from Cannabis sativa binds with a higher affinity and lower free energy than the standard ligand with isovitexin and vitexin having a glide score of -11.485 and -10.601 respectively, sophoroside has a glide score of -9.711 which is lower than the cycloguanil (co-crystallized ligand) having a glide score of -6.908. This result gives new perception to the use of Cannabis sativa as antimicrobial agent.

Prevalence of molecular markers of sulfadoxine\u2013pyrimethamine and artemisinin resistance in Plasmodium falciparum from Pakistan

BackgroundIn Pakistan, artesunate (AS) in combination with sulfadoxine–pyrimethamine (SP) is the recommended treatment for uncomplicated Plasmodium falciparum malaria. Monitoring molecular markers of anti-malarial drug resistance is crucial for early detection and containment of parasite resistance to treatment. Currently, no data are available on molecular markers of artemisinin resistance (K13 mutations) in P. falciparum isolates from Pakistan. In this study, the prevalence of mutations associated with SP and artemisinin resistance was estimated in different regions of Pakistan.MethodsA total of 845 blood samples that were positive for malaria parasites by microscopy or rapid diagnostic test were collected from January 2016 to February 2017 from 16 different sites in Pakistan. Of these samples, 300 were positive for P. falciparum by PCR. Polymorphisms in the P. falciparum dihydrofolate reductase (pfdhfr) and dihydropteroate synthase (pfdhps) genes were identified by pyrosequencing while polymorphisms in the propeller domain of the pfk13 gene were identified by Sanger sequencing.ResultsThe prevalence of the PfDHFR 108N and 59R mutations was 100% and 98.8%, respectively, while the prevalence of PfDHFR 50R and 51I mutations was 8.6%. No mutation was observed at PfDHFR position 164. In PfDHPS, the prevalence of mutations at positions 436, 437, and 613 was 9.9%, 45.2%, and 0.4%, respectively. No mutations were found at PfDHPS positions 540 and 581. The prevalence of double PfDHFR mutants (59R + 108N) ranged from 93.8% to 100%, while the prevalence of parasites having the PfDHFR 59R + 108N mutations in addition to the PfDHPS 437G mutation ranged from 9.5% to 83.3% across different regions of Pakistan. Nine non-synonymous and four synonymous mutations were observed in the PfK13 propeller domain, none of which correspond to mutations validated to contribute to artemisinin resistance.ConclusionThe absence of the highly resistant PfDHFR/PfDHPS quintuple mutant parasites and the lack of PfK13 mutations associated with artemisinin resistance is consistent with AS + SP being effective in Pakistan.

Standardization of virtual-screening and post-processing protocols relevant to in-silico drug discovery.

Structure-based drug discovery has emerged as a powerful tool in computational drug discovery and has gained rapid acceleration due to the development of better algorithms for high-end computation in an affordable time. Molecular docking and virtual screening methods are routinely used for the purpose but computing the ligand binding energies with inbuilt scoring functions accurately is still a limitation. Although, MMPBSA and MMGBSA are routinely employed tools for achieving accurate binding free energies, they are applied on well-equilibrated explicitly solvated systems and are computationally expensive and time-consuming. This study compares different post-processing protocols performed on an in-silico screened benchmarked P. falciparum Dihydrofolate reductase (PfDHFR) dataset with AutoDockVina. The docked and implicitly solvated complexes were subjected to (1) rescoring, (2) energy minimization and (3) Binding Estimation After Refinement (BEAR) algorithm. Subsequently, binding free energies were computed using three different tools-MMPBSA, MMGBSA and "g_mmpbsa". Surprisingly, rescoring alone displays lower accuracy than the inherent scoring function of the AutoDockVina. However, encouraging results were seen after post-processing with the other two protocols. The results suggest that MMPBSA applied on energy minimized conformations is able to achieve 42-fold reductions in computational time as opposed to the BEAR algorithm with comparable accuracy.

Exploring PfDHFR reaction surface: A combined molecular dynamics and QM/MM analysis

The substrate to the enzyme PfDHFR (Plasmodium falciparum Dihydrofolate Reductase) is a small molecule dihydrofolate (DHF), it gets converted to tetrahydrofolate (THF) in the active site of the enzyme. The PfDHFR reaction surface involves the protonation of DHF to DHFP as an initial step before the catalytic conversion. The binding affinities of all these species (DHF, DHFP and THF) contribute to the mechanism of DHFR catalytic action. Molecular dynamics (MD) simulations and Quantum Mechanics/Molecular Mechanics (QM/MM) analysis were performed to evaluate the binding affinity and molecular recognition interactions of the substrate DHF/DHFP and the product THF, in the active site of wild-type PfDHFR (wtPfDHFR). The binding affinities of the cofactor NADPH/NADP+ were also estimated in all the three complexes. The molecular dynamics (MD) simulations of the substrate, product and cofactor in the cavities of wtPfDHFR revealed the variation of the atomic level interactions during the course of the catalytic conversion. It was found that the DHFP binds very strongly to the PfDHFR active site and pulls the cofactor NADPH closer to itself. The QM/MM analysis revealed that the binding energy of DHFP (−59.82 kcal/mol) and NADPH (−100.24 kcal/mol) in DHFP-wtPfDHFR complex, is higher in comparison to the binding energy of DHF (−38.67 kcal/mol) and NADPH (−77.53 kcal/mol) in DHF-wtPfDHFR complex and the binding energy of THF (−30.72 kcal/mol) and NADP+ (−73.72 kcal/mol) in THF-wtPfDHFR complex. The hydride ion donor-acceptor distance (DAD) analysis was also carried out. This combined MD and QM/MM analysis revealed that the protonation of DHF increases the proximity between the substrate and the cofactor, thus facilitates the reaction profile of PfDHFR.

Application of molecular docking and PSO–SVR intelligent approaches in antimalarial activity prediction of enantiomeric cycloguanil analogues

ABSTRACTA series of antifolate compounds, i.e. 1-(4-chlorophenyl)-6,6-dimethyl-1,3,5-triazine-2,4-diamine, or cycloguanil analogues, have shown effective inhibiting properties against Plasmodium falciparum dihydrofolate reductase (PfDHFR). In this work, the stereoselectivity of PfDHFR to the R and S enantiomer of cycloguanil analogues was obtained from molecular docking calculations and integrated into QSAR study to obtain a more accurate prediction model. Results indicate that PfDHFR can bind to cycloguanil analogues in the R and S enantiomers. Cycloguanil analogues with alkyl chain substituent prefer the R enantiomer over S because they do not experience steric hindrance with the Phe58 side chain, while cycloguanil analogues with phenol chain substituent prefer the S enantiomer over R because they do not experience steric hindrance with Leu46 and Met55 side chains. Particle swarm optimization and support vector regression were used to select relevant descriptors and generate the effective prediction model, with a high statistical significance level (r2training = 0.941; r2test = 0.884).

Investigation of potential anti-malarial lead candidate 2-(4-fluorobenzylthio)-5-(5-bromothiophen-2-yl)-1,3,4-oxadiazole: Insights from crystal structure, DFT, QTAIM and hybrid QM/MM binding energy analysis

A combined study involving single crystal X-ray diffraction and various theoretical approaches has been used to characterize the 2-(4-fluorobenzylthio)-5-(5-bromothiophen-2-yl)-1,3,4-oxadiazole compound. The crystal structure is primarily stabilized by intermolecular CH···π, CH⋯N and CH⋯F. A short and very rare halogen-halogen contact (Br⋯F) which adopts type I trans geometry along with the S⋯S, N⋯S contacts, which play an important role in the stabilization of the crystal packing. The importance of these contacts is established through various theoretical approaches such as QTAIM and NBO analysis. A detailed CSD analysis of Br⋯F contacts is performed to understand the geometrical preference. A detailed in silico analysis is performed to explore the binding potential of the title compound against the Plasmodium falciparum dihydrofolate reductase (PfDHFR). The results clearly suggest that the title compound may be a promising anti-malarial lead candidate by inhibiting the DHFR target and halogenated fragments of the molecule are important for the stabilization of the protein-ligand complex formation.

Molecular Modeling of Antimalarial Agents by 3D-QSAR Study and Molecular Docking of Two Hybrids 4-Aminoquinoline-1,3,5-triazine and 4-Aminoquinoline-oxalamide Derivatives with the Receptor Protein in Its Both Wild and Mutant Types.

Modeling studies using 3D-QSAR and molecular docking methods were performed on a set of 34 hybrids of 4-aminoquinoline derivatives previously studied as effective antimalarial agents of wild type and quadruple mutant Plasmodium falciparum dihydrofolate reductase (DHFR). So, the famous mathematical method multiple linear regression (MLR) was explored to build the QSAR model. The DFT-B3LYP method with the basis set 6-31G was used to calculate the quantum chemical descriptors, chosen to represent the electronic descriptors of molecular structures. On the contrary, the MM2 method was used to calculate lipophilic, geometrical, physicochemical, and steric descriptors. The QSAR model tested with artificial neural network (ANN) method shows high performance towards its predictability. The predicted model was confirmed by three validation methods: leave-one-out (LOO) cross validation, Y-randomization, and validation external. The molecular docking study of three compounds 9, 11, and 26 on both wild and quadruple mutant types of pf-DHFR-TS as the protein target helps to understand more and then predict the binding modes with the binding sites.

Prevention of Malaria in Pregnancy

Malaria remains one of the most preventable causes of adverse birth outcomes. Intermittent preventive treatment in pregnancy (IPTp) with sulfadoxine-pyrimethamine is used to prevent malaria, but resistance to this drug combination has decreased its efficacy and new alternatives are needed. In Africa, a meta-analysis showed three-course or monthly IPTp with sulfadoxine-pyrimethamine to be safe and more effective than the original two-course sulfadoxine-pyrimethamine strategy, prompting WHO to update its policy in 2012. Although resistance to sulfadoxine-pyrimethamine reduces the parasitological efficacy of IPTp, this drug combination remains associated with reduced incidence of low birthweight in areas where prevalence of parasites with quintuple Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthetase (Pfdhps) mutations is greater than 90%. Nevertheless, its effectiveness is compromised in women infected with sextuple mutant parasites. Six trials of IPTp showed that neither amodiaquine, mefloquine, nor chloroquine-azithromycin are suitable replacements for sulfadoxine-pyrimethamine because of poor tolerability. Furthermore, four trials showed that intermittent screening and treatment with the current generation of malaria rapid diagnostic tests was not a suitable alternative strategy to IPTp with sulfadoxine-pyrimethamine, even in areas with high prevalence of quintuple mutations. Two trials showed that IPTp with dihydroartemisinin-piperaquine was well tolerated, effective, and acceptable for IPTp, with monthly regimens being the most effective. Coverage of IPTp and insecticide-treated nets continues to lag behind targets. The key barriers to uptake are well documented, and many are open to intervention. Outside of Africa, a single trial suggests a potential role for integrated approaches that combine sulfadoxine-pyrimethamine with azithromycin for IPTp in areas of Papua New Guinea where malaria transmission is high. Modelling analysis suggests the importance of the prevention of malaria early in pregnancy and the need to protect pregnant women declines more slowly than the rate at which transmission declines. Improved funding has led to an increase in the number of prevention trials in the past decade, showing the value of more sustained protection with monthly IPTp regimens. There is a need for confirmatory trials of the safety, efficacy, and feasibility of IPTp with dihydroartemisinin-piperaquine, for studies of intermittent screening and treatment with more sensitive rapid diagnostic tests, for studies of integrated strategies for malaria and other co-infections, and for studies of prevention strategies for malaria in pregnant women who are HIV-positive and living outside of Africa. Additional research is required on how to improve uptake of WHO's updated policy on IPTp with sulfadoxine-pyrimethamine and insecticide-treated nets.

High prevalence of dihydrofolate reductase gene mutations in Plasmodium falciparum parasites among pregnant women in Nigeria after reported use of sulfadoxine-pyrimethamine

This study assesses the prevalence of asymptomatic Plasmodium falciparum parasitemia positivity and P. falciparum dihydrofolate reductase (pfdhfr) mutations in parasite isolates among pregnant women in Southwest Nigeria. Plasmodium falciparum parasitemia was confirmed by microscopy and nested PCR in 200 pregnant women attending antenatal care. The prevalence of pfdhfr polymorphisms was determined by direct sequencing of the gene fragments containing the C50R, N51I, C59R, S108N, and I164L mutations. Information on the use of antimalarial drugs and methods applied to prevent malaria were obtained by a questionnaire. The prevalence of asymptomatic P. falciparum infection was 30% (60/200). The frequency of the pfdhfr triple-mutant alleles (N51I, C59R, and S108N) was 63% (38/60); none of the isolates carried the I164L mutation. Among the investigated pregnant women, 40% used un-prescribed antimalarials such as dihydroartemisinin (18%), chloroquine (14%) or pyrimethamine (9%), while only 20.5% used sulfadoxine-pyrimethamine for prevention and 39.5% did not use any drug. The prevalence of P. falciparum parasitemia (37%) was higher among pregnant women who had not taken any antimalarial drugs. A significant difference in the prevalence of the pfdhfr triple-mutant alleles was observed among women who took SP (90%) compared to those who did not take any drug (82%) and women who took dihydroartemisinin (67%) p = 0.007). Poor adherence to the World Health Organisation (WHO) strategies for malaria prevention among pregnant women was observed in addition to high prevalence of pfdhfr mutations. These findings underline the need to improve control of malaria among pregnant women in the study area.

Molecular identification of sulfadoxine-pyrimethamine resistance in malaria infected women who received intermittent preventive treatment in the Democratic Republic of Congo

BackgroundPoint mutations in Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes which confer resistance to sulfadoxine-pyrimethamine (SP) occur at increasing rates. The present study aimed to identify Pfdhfr and Pfdhps mutations in P. falciparum isolates recovered from women who received two doses of SP during pregnancy in Bandundu, the Democratic Republic of Congo (DRC).MethodsA total of 48 women with confirmed P. falciparum infection were enrolled in the study. Finger-prick blood samples that were collected on filter paper at the time of delivery were used for DNA isolation. Pfdhfr and Pfdhps genes were amplified by a nested PCR protocol. DNA sequencing was performed on both strands, and the point mutations were analysed.ResultsAll of the 48 (100.0%) P. falciparum isolates carried at least one polymorphism in both genes. The wild-type haplotypes of Pfdhfr (CNCSI [C50, N51, C59, S108, I164]) and Pfdhps (SAKAA [S436, A437, K540, A581, A613]) were not observed in the study. In Pfdhfr, N51I (85.4%), C59R (60.4%), and S108N (100.0%) polymorphisms were detected. Triple mutation (CIRNI) (mutant amino acids are underlined) was the most prevalent (47.9%) Pfdhfr haplotype. In the study, all P. falciparum isolates (100.0%) harboured the A437G allele in Pfdhps gene. Also, K540E and A581G polymorphisms were observed in one (2.1%) isolate. Single mutant haplotype (SGKAA) was detected in 97.9% of the isolates. Mutant Pfdhfr and Pfdhps allele combinations revealed quintuple (CICNI-SGEGA; 2.1%), quadruple (CIRNI-SGKAA; 47.9%), triple (CICNI-SGKAA; 35.4%, CNRNI-SGKAA; 12.5%), and double (CNCNI-SGKAA; 2.1%) haplotypes.ConclusionsIn the study, the rate of SGEGA haplotype was low (2.1%). Although K540E and A581G alleles are more common in Eastern Africa, a distinct lineage of SGEGA is also present in the DRC, which is located in Central Africa. This haplotype is associated with decreased efficacy of SP in pregnant women and infants, therefore, it should be carefully considered in the DRC and SP resistance should be routinely monitored.

Novel dual inhibitors against FP-2 and PfDHFR as potential antimalarial agents: Design, synthesis and biological evaluation

Resistance to malaria parasites has quickly developed to almost all used antimalarial drugs. Cysteine protease falcipain-2 (FP-2) and Plasmodium falciparum dihydrofolate reductase (PfDHFR) have crucial roles, which are absolutely necessary, in the parasite life cycle. In this study, based on the uniform pharmacophores of reported PfDHFR inhibitors and the first-generation dual inhibitors against FP-2 and PfDHFR, we identified a novel series of dual inhibitors through fragments assembly. Lead optimization led to the identification of 14, which showed potent inhibition against FP-2 and PfDHFR enzyme (IC50=6.8±1.8μmol/L and IC50=8.8±0.3μmol/L) and P. falciparum 3D7 strain (IC50=2.9μmol/L). Additionally, 14 exhibited more potent inhibition to the proliferation of chloroquine-resistant P. falciparum Dd2 strain (IC50=1.1μmol/L) than pyrimethamine (IC50>10μmol/L), and 14 displayed micromolar inhibitory activities against two clinical isolated strains Fab9 (IC50=2.6μmol/L) and GB4 (IC50=1.0μmol/L). Collectively, these data demonstrated that 14 might be a good lead compound for the treatment of malaria.

Discovery of new antimalarial agents: Second-generation dual inhibitors against FP-2 and PfDHFR via fragments assembely

Malaria parasites are a leading cause of worldwide mortality from infectious disease. Cysteine protease falcipain-2 (FP-2) and Plasmodium falciparum dihydrofolate reductase (PfDHFR) play vital roles, which are absolutely essential, in the parasite life cycle. In this study, based on the structures of uniform fragments of reported PfDHFR inhibitors and the first-generation dual inhibitors against FP-2 and PfDHFR, we identified a novel series of dual inhibitors through fragments assembly. Lead optimization led to the discovery of 24, which showed high potency against FP-2 (IC50 = 10.0 µM), PfDHFR (IC50 = 84.1 nM), P. falciparum 3D7 (IC50 = 53.1 nM), clinical isolated strains Fab9 (IC50 = 14.2 nM) and GB4 (IC50 = 23.4 nM). The in vivo inhibition assays against P. berghei in 10 days indicated 24 had a more beneficial effect on the growth inhibition of P. berghei than artemisinin and an identical effect with pyrimethamine. Additionally, 24 moderately inhibited the proliferation of chloroquine-resistant P. falciparum Dd2 strain. Collectively, these data revealed that 24 could be an excellent lead compound as FP-2 and PfDHFR dual inhibitor for the treatment of malaria.

In Vitro Antimalarial Activity and Cytotoxity of 20 Ethanolic Crude Extracts from Thai Herbs Against Plasmodium Falciparum TM267

Objective: To determine the antimalarial activity of ethanol crude extracts from 20 Thai herbs against Plasmodiumfalciparum (P. falciparum) chloroquine-resistant strain TM267. Molecular docking of the active compounds from the selected Thaiherbs were analyzed with Plasmodium falciparum dihydrofolate reductase (PfDHFR).Material and Method: An in vitro study of antimalarial activity against P. falciparum TM267 was done using a parasitelactate dehydrogenese assay, and the cytotoxic effects of extracts were tested against Vero cells using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The 50% inhibitory concentration (IC50) and 50%cytotoxicity concentration were calculated from the dose-response curves. Molecular docking and post-dockingwere analyzed with the x-ray crystal structure of PfDHFR-thymidylate synthase complexed with pyrimethamine,nicotinamide adenine dinucleotide phosphate and deoxyuridylate.Results: Of these, the Plumbago indica L. root extract showed high antimalarial activity, with an IC50 value of 3.7μg/ml and less cytotoxicity when tested against Vero cells, followed by the Citrus hystrix DC. fruit extract, Vitex trifoliaLinn. root extract, Ocimum sanctum L. leave extract, of Allium sativum L. bulb extract and Salacia chinensis L. stem extract,respectively. All 7 active compounds reported from these herbal extracts had high docking scores against PfDHFR.The Citrusoside C from Citrus hystrix DC. had the highest docking score.Conclusion: It could be purposed that there were active compounds in Plumbago indica L., Vitex trifolia Linn. and Citrus hystrix DC. which are potential inhibitors against malaria that could bind to the active site of PfDHFR. However, the active Citrusosides from Citrus hystrix DC. should be further investigated for their effectiveness against malaria.