Design, synthesis and Anti-Plasmodial activity of Mortiamide-Lugdunin conjugates.

In this study, two linear and corresponding cyclic heptapeptide versions of mortiamide A-lugdunin hybrids were designed and synthesized by integrating an anti-malarial peptide epitope derived from Mortiamide A, combined with four residues known for their membrane interactions. Using this synthetic strategy, the sequence of mortiamide A was partly re-engineered with an epitope sequence of lugdunin along with an amino acid replacement using all-L and D/L configurations. Importantly, the re-engineered cyclic mortiamides with all-L (3) and D/L (4) configurations exhibited promising anti-malarial activities against the P. falciparum drug-sensitive TM4/8 strain with half-maximal inhibitory concentration (IC50) values of 6.2 ± 0.5 and 4.8 ± 0.1 µM, respectively. Additionally, they exhibited anti-malarial activities against the P. falciparum multidrug-resistant V1/S strain with IC50 values of 5.0 ± 2.6 and 3.7 ± 0.7 µM, respectively. Interestingly, a linear re-engineered mortiamide with D/L configuration (2) exhibited promising anti-malarial activities, surpassing those of the re-engineered cyclic mortiamides (3 and 4), against both the P. falciparum sensitive TM4/8 and multidrug-resistant V1/S strains with IC50 values of 3.6 ± 0.5 and 2.8 ± 0.7 µM (IC50 of Mortiamide A = 7.85 ± 0.97, 5.31 ± 0.24 µM against 3D7 and Dd2 strains) without any cytotoxicity at >100 µM. The presence of D/L forms in a linear structure significantly impacted the anti-malarial activity against both the P. falciparum sensitive TM4/8 strain and the multidrug-resistant V1/S strain.

Antimalarial target vulnerability of the putative Plasmodium falciparum methionine synthase.

Background: Plasmodium falciparum possesses a cobalamin-dependent methionine synthase (MS). MS is putatively encoded by the PF3D7_1233700 gene, which is orthologous and syntenic in Plasmodium. However, its vulnerability as an antimalarial target has not been assessed. Methods: We edited the PF3D7_1233700 and PF3D7_0417200 (dihydrofolate reductase-thymidylate synthase, DHFR-TS) genes and obtained transgenic P. falciparum parasites expressing epitope-tagged target proteins under the control of the glmS ribozyme. Conditional loss-of-function mutants were obtained by treating transgenic parasites with glucosamine. Results: DHFR-TS, but not MS mutants showed a significant proliferation defect over 96 h, suggesting that P. falciparum MS is not a vulnerable antimalarial target.

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.

Antagonistic antimalarial properties of a methoxyamino chalcone derivative and 3-hydroxypyridinones in combination with dihydroartemisinin against Plasmodium falciparum.

Background: The spread of artemisinin (ART)-resistant Plasmodium falciparum threatens the control of malaria. Mutations in the propeller domains of P. falciparum Kelch13 (k13) are strongly associated with ART resistance. Ferredoxin (Fd), a component of the ferredoxin/NADP+ reductase (Fd/FNR) redox system, is essential for isoprenoid precursor synthesis in the plasmodial apicoplast, which is important for K13-dependent hemoglobin trafficking and ART activation. Therefore, Fd is an antimalarial drug target and fd mutations may modulate ART sensitivity. We hypothesized that loss of Fd/FNR function enhances the effect of k13 mutation on ART resistance. Methods: In this study, methoxyamino chalcone (C3), an antimalarial compound that has been reported to inhibit the interaction of recombinant Fd and FNR proteins, was used as a chemical inhibitor of the Fd/FNR redox system. We investigated the inhibitory effects of dihydroartemisinin (DHA), C3, and iron chelators including deferiprone (DFP), 1-(N-acetyl-6-aminohexyl)-3-hydroxy-2-methylpyridin-4-one (CM1) and deferiprone-resveratrol hybrid (DFP-RVT) against wild-type (WT), k13 mutant, fd mutant, and k13 fd double mutant P. falciparum parasites. Furthermore, we investigated the pharmacological interaction of C3 with DHA, in which the iron chelators were used as reference ART antagonists. Results: C3 showed antimalarial potency similar to that of the iron chelators. As expected, combining DHA with C3 or iron chelators exhibited a moderately antagonistic effect. No differences were observed among the mutant parasites with respect to their sensitivity to C3, iron chelators, or the interactions of these compounds with DHA. Discussion: The data suggest that inhibitors of the Fd/FNR redox system should be avoided as ART partner drugs in ART combination therapy for treating malaria.

Susceptibility of Southeast Asian Plasmodium falciparum isolates to P218.

A major threat to the goal of eliminating malaria, particularly in Southeast Asia, is the spread of Plasmodium falciparum resistant to artemisinin-based combination therapies. P218 is a drug candidate designed to combat antifolate-sensitive and -resistant parasites. However, there is no evidence that P218 is effective against artemisinin-resistant P. falciparum. This report investigated the susceptibilities of 10 parasite isolates from Southeast Asia to P218 and other antimalarial drugs. All isolates with different levels of artemisinin resistance were genetically distinct from one another, although common haplotypes associated with antimalarial resistance were identified. All isolates were highly resistant to pyrimethamine, and none of them were significantly less sensitive to P218 than the pyrimethamine-resistant laboratory strain V1/S. Significant differences in sensitivity to other types of antimalarials (mefloquine, atovaquone and chloroquine) compared with V1/S were found for some isolates, although the differences were not clinically relevant. P218 is thus efficacious against multi-drug (including artemisinin-resistant P. falciparum.

N-Containing α-Mangostin Analogs via Smiles Rearrangement as the Promising Cytotoxic, Antitrypanosomal, and SARS-CoV-2 Main Protease Inhibitory Agents.

New N-containing xanthone analogs of α-mangostin were synthesized via one-pot Smiles rearrangement. Using cesium carbonate in the presence of 2-chloroacetamide and catalytic potassium iodide, α-mangostin (1) was subsequently transformed in three steps to provide ether 2, amide 3, and amine 4 in good yields at an optimum ratio of 1:3:3, respectively. The evaluation of the biological activities of α-mangostin and analogs 2-4 was described. Amine 4 showed promising cytotoxicity against the non-small-cell lung cancer H460 cell line fourfold more potent than that of cisplatin. Both compounds 3 and 4 possessed antitrypanosomal properties against Trypanosoma brucei rhodesiense at a potency threefold stronger than that of α-mangostin. Furthermore, ether 2 gave potent SARS-CoV-2 main protease inhibition by suppressing 3-chymotrypsinlike protease (3CLpro) activity approximately threefold better than that of 1. Fragment molecular orbital method (FMO-RIMP2/PCM) indicated the improved binding interaction of 2 in the 3CLpro active site regarding an additional ether moiety. Thus, the series of N-containing α-mangostin analogs prospectively enhance druglike properties based on isosteric replacement and would be further studied as potential biotically active chemical entries, particularly for anti-lung-cancer, antitrypanosomal, and anti-SARS-CoV-2 main protease applications.

Key interactions of pyrimethamine derivatives specific to wild-type and mutant P. falciparum dihydrofolate reductase based on 3D-QSAR, MD simulations and quantum chemical calculations.

Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) is an important target enzyme in malarial chemotherapy. An understanding of how novel inhibitors interact with wild-type (wtPfDHFR), quadruple-mutant (qmPfDHFR), and human (hDHFR) enzymes is required for the development of these compounds as antimalarials. This study is focused on a series of des-Cl and m-Cl phenyl analogs of pyrimethamine with various flexible 6-substituents. The interactions of these compounds with DHFR enzymes were investigated by 3 D-QSAR, MD simulations, MM-PBSA, and DFT calculations. CoMFA and CoMSIA models were developed with good predictive abilities for wtPfDHFR and qmPfDHFR. For hDHFR, CoMSIA models combined with clogP descriptor were successfully derived. Binding free energy using MM-PBSA and comparison of per residue decomposition energy analyses with the DFT method at M06-2X/6-31G ++(d,p) level of theory indicated that Asp54 and Phe58 play important roles in the binding of the most potent compound in the series (compound 27) with both wtPfDHFR and qmPfDHFR, whereas Arg59 and Arg122 were additionally found to interact with this inhibitor in qmPfDHFR. For hDHFR, the residues Glu30 and Phe34 but not Arg70, equivalent to Asp54, Phe58, and Arg122 in PfDHFR, also play role in compound 27 binding through strong hydrophobic interactions (Phe34) and hydrogen bond network with Glu30, Ile7, and Val115. From the key interactions identified in the DHFR-inhibitor complexes, a general scheme is proposed for designing new inhibitors selective for PfDHFR that is important for the development of novel antifolate antimalarials.Communicated by Ramaswamy H. Sarma.

Transcriptomic complexity of the human malaria parasite Plasmodium falciparum revealed by long-read sequencing.

The Plasmodium falciparum human malaria parasite genome is incompletely annotated and does not accurately represent the transcriptomic diversity of this species. To address this need, we performed long-read transcriptomic sequencing. 5' capped mRNA was enriched from samples of total and nuclear-fractionated RNA from intra-erythrocytic stages and converted to cDNA library. The cDNA libraries were sequenced on PacBio and Nanopore long-read platforms. 12,495 novel isoforms were annotated from the data. Alternative 5' and 3' ends represent the majority of isoform events among the novel isoforms, with retained introns being the next most common event. The majority of alternative 5' ends correspond to genomic regions with features similar to those of the reference transcript 5' ends. However, a minority of alternative 5' ends showed markedly different features, including locations within protein-coding regions. Alternative 3' ends showed similar features to the reference transcript 3' ends, notably adenine-rich termination signals. Distinguishing features of retained introns could not be observed, except for a tendency towards shorter length and greater GC content compared with spliced introns. Expression of antisense and retained intron isoforms was detected at different intra-erythrocytic stages, suggesting developmental regulation of these isoform events. To gain insights into the possible functions of the novel isoforms, their protein-coding potential was assessed. Variants of P. falciparum proteins and novel proteins encoded by alternative open reading frames suggest that P. falciparum has a greater proteomic repertoire than the current annotation. We provide a catalog of annotated transcripts and encoded alternative proteins to support further studies on gene and protein regulation of this pathogen.

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.

Genome-wide functional screening of drug-resistance genes in Plasmodium falciparum.

The global spread of drug resistance is a major obstacle to the treatment of Plasmodium falciparum malaria. The identification of drug-resistance genes is an essential step toward solving the problem of drug resistance. Here, we report functional screening as a new approach with which to identify drug-resistance genes in P. falciparum. Specifically, a high-coverage genomic library of a drug-resistant strain is directly generated in a drug-sensitive strain, and the resistance gene is then identified from this library using drug screening. In a pilot experiment using the strain Dd2, the known chloroquine-resistant gene pfcrt is identified using the developed approach, which proves our experimental concept. Furthermore, we identify multidrug-resistant transporter 7 (pfmdr7) as a novel candidate for a mefloquine-resistance gene from a field-isolated parasite; we suggest that its upregulation possibly confers the mefloquine resistance. These results show the usefulness of functional screening as means by which to identify drug-resistance genes.

Assay Development and Identification of the First Plasmodium falciparum 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase Inhibitors.

In the fight towards eradication of malaria, identifying compounds active against new drug targets constitutes a key approach. Plasmodium falciparum 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase (PfHPPK) has been advanced as a promising target, as being part of the parasite essential folate biosynthesis pathway while having no orthologue in the human genome. However, no drug discovery efforts have been reported on this enzyme. In this study, we conducted a three-step screening of our in-house antifolate library against PfHPPK using a newly designed PfHPPK-GFP protein construct. Combining virtual screening, differential scanning fluorimetry and enzymatic assay, we identified 14 compounds active against PfHPPK. Compounds' binding modes were investigated by molecular docking, suggesting competitive binding with the HMDP substrate. Cytotoxicity and in vitro ADME properties of hit compounds were also assessed, showing good metabolic stability and low toxicity. The most active compounds displayed low micromolar IC50 against drug-resistant parasites. The reported hit compounds constitute a good starting point for inhibitor development against PfHPPK, as an alternative approach to tackle the malaria parasite.

Structural Insight into Effective Inhibitors' Binding to Toxoplasma gondii Dihydrofolate Reductase Thymidylate Synthase.

Pyrimethamine (Pyr), a known dihydrofolate reductase (DHFR) inhibitor, has long been used to treat toxoplasmosis caused by Toxoplasma gondii (Tg) infection. However, Pyr is effective only at high doses with associated toxicity to patients, calling for safer alternative treatments. In this study, we investigated a series of Pyr analogues, previously developed as DHFR inhibitors of Plasmodium falciparum bifunctional DHFR-thymidylate synthase (PfDHFR-TS), for their activity against T. gondii DHFR-TS (TgDHFR-TS). Of these, a set of compounds with a substitution at the C6 position of the pyrimidine ring exhibited high binding affinities (in a low nanomolar range) against TgDHFR-TS and in vitro T. gondii inhibitory activity. Three-dimensional structures of TgDHFR-TS reported here include the ternary complexes with Pyr, P39, or P40. A comparison of these structures showed the minor steric strain between the p-chlorophenyl group of Pyr and Thr83 of TgDHFR-TS. Such a conflict was relieved in the complexes with the two analogues, P39 and P40, explaining their highest binding affinities described herein. Moreover, these structures suggested that the hydrophobic environment in the active-site pocket could be used for drug design to increase the potency and selectivity of antifolate inhibitors. These findings would accelerate the development of new antifolate drugs to treat toxoplasmosis.

Semi-Synthesis of N-Aryl Amide Analogs of Piperine from Piper nigrum and Evaluation of Their Antitrypanosomal, Antimalarial, and Anti-SARS-CoV-2 Main Protease Activities.

Piper nigrum, or black pepper, produces piperine, an alkaloid that has diverse pharmacological activities. In this study, N-aryl amide piperine analogs were prepared by semi-synthesis involving the saponification of piperine (1) to yield piperic acid (2) followed by esterification to obtain compounds 3, 4, and 5. The compounds were examined for their antitrypanosomal, antimalarial, and anti-SARS-CoV-2 main protease activities. The new 2,5-dimethoxy-substituted phenyl piperamide 5 exhibited the most robust biological activities with no cytotoxicity against mammalian cell lines, Vero and Vero E6, as compared to the other compounds in this series. Its half-maximal inhibitory concentration (IC50) for antitrypanosomal activity against Trypanosoma brucei rhodesiense was 15.46 ± 3.09 µM, and its antimalarial activity against the 3D7 strain of Plasmodium falciparum was 24.55 ± 1.91 µM, which were fourfold and fivefold more potent, respectively, than the activities of piperine. Interestingly, compound 5 inhibited the activity of 3C-like main protease (3CLPro) toward anti-SARS-CoV-2 activity at the IC50 of 106.9 ± 1.2 µM, which was threefold more potent than the activity of rutin. Docking and molecular dynamic simulation indicated that the potential binding of 5 in the 3CLpro active site had the improved binding interaction and stability. Therefore, new aryl amide analogs of piperine 5 should be investigated further as a promising anti-infective agent against human African trypanosomiasis, malaria, and COVID-19.

New Insights into Antimalarial Chemopreventive Activity of Antifolates.

Antifolates targeting dihydrofolate reductase (DHFR) are antimalarial compounds that have long been used for malaria treatment and chemoprevention (inhibition of infection from mosquitoes to humans). Despite their extensive applications, a thorough understanding of antifolate activity against hepatic malaria parasites, especially resistant parasites, has yet to be achieved. Using a transgenic Plasmodium berghei harboring quadruple mutant dhfr from Plasmodium falciparum (Pb::Pfdhfr-4M), we demonstrated that quadruple mutations on Pfdhfr confer complete chemoprevention resistance to pyrimethamine, the previous generation of antifolate, but not to a new class of antifolate designed to overcome the resistance, such as P218. Detailed investigation to pinpoint stage-specific chemoprevention further demonstrated that it is unnecessary for the drug to be present throughout hepatic development. The drug is most potent against the developmental stages from early hepatic trophozoite to late hepatic trophozoite, but it is not effective at inhibiting sporozoite and early hepatic stage development from sporozoite to early trophozoite. Our data show that P218 also inhibited the late hepatic-stage development, from trophozoite to mature schizonts to a lesser extent. With a single dose of 15 mg/kg of body weight, P218 prevented infection from up to 25,000 pyrimethamine-resistant sporozoites, a number equal to thousands of infectious mosquito bites. Additionally, the hepatic stage of malaria parasite is much more susceptible to antifolates than the asexual blood stage. This study provides important insights into the activity of antifolates as a chemopreventive therapeutic which could lead to a more efficient and cost-effective treatment regime.

Synthesis and evaluation of tetrahydroisoquinoline derivatives against Trypanosoma brucei rhodesiense.

Human African Trypanosomiasis (HAT) is a neglected tropical disease caused by the parasitic protozoan Trypanosoma brucei (T. b.), and affects communities in sub-Saharan Africa. Previously, analogues of a tetrahydroisoquinoline scaffold were reported as having in vitro activity (IC50 = 0.25-70.5 µM) against T. b. rhodesiense. In this study the synthesis and antitrypanosomal activity of 80 compounds based around a core tetrahydroisoquinoline scaffold are reported. A detailed structure activity relationship was revealed, and five derivatives (two of which have been previously reported) with inhibition of T. b. rhodesiense growth in the sub-micromolar range were identified. Four of these (3c, 12b, 17b and 26a) were also found to have good selectivity over mammalian cells (SI > 50). Calculated logD values and preliminary ADME studies predict that these compounds are likely to have good absorption and metabolic stability, with the ability to passively permeate the blood brain barrier. This makes them excellent leads for a blood-brain barrier permeable antitrypanosomal scaffold.

Isolation of bioactive compounds from medicinal plants used in traditional medicine: Rautandiol B, a potential lead compound against Plasmodium falciparum.

BACKGROUND: Neorautanenia mitis, Hydnora abyssinica, and Senna surattensis are medicinal plants with a variety of traditional uses. In this study, we sought to isolate the bioactive compounds responsible for some of these activities, and to uncover their other potential medicinal properties. METHODS: The DCM and ethanol extracts of the roots of N. mitis and H. abyssinica, and the leaves of S. surattensis were prepared and their phytochemical components were isolated and purified using chromatographic methods. These extracts and their pure phytochemical components were evaluated in in-vitro models for their inhibitory activities against Plasmodium falciparum, Trypanosoma brucei rhodesiense, Mycobacterium tuberculosis, α-amylase (AA), and α-glucosidase (AG). RESULTS: Rautandiol B had significant inhibitory activities against two strains of Plasmodium falciparum showing a high safety ratio (SR) and IC50 values of 0.40 ± 0.07 µM (SR - 108) and 0.74 ± 0.29 µM (SR - 133) against TM4/8.2 and K1CB1, respectively. While (-)-2-isopentenyl-3-hydroxy-8-9-methylenedioxypterocarpan showed the highest inhibitory activity against T. brucei rhodesiense with an IC50 value of 4.87 ± 0.49 µM (SR > 5.83). All crude extracts showed inhibitory activities against AA and AG, with three of the most active phytochemical components; rautandiol A, catechin, and dolineon, having only modest activities against AG with IC50 values of 0.28 mM, 0.36 mM and 0.66 mM, respectively. CONCLUSION: These studies have led to the identification of lead compounds with potential for future drug development, including Rautandiol B, as a potential lead compound against Plasmodium falciparum. The relatively higher inhibitory activities of the crude extracts against AG and AA over their isolated components could be due to the synergistic effects between their phytochemical components. These crude extracts could potentially serve as alternative inhibitors of AG and AA and as therapeutics for diabetes.

Identifying transcript 5' capped ends in Plasmodium falciparum.

BACKGROUND: The genome of the human malaria parasite Plasmodium falciparum is poorly annotated, in particular, the 5' capped ends of its mRNA transcripts. New approaches are needed to fully catalog P. falciparum transcripts for understanding gene function and regulation in this organism. METHODS: We developed a transcriptomic method based on next-generation sequencing of complementary DNA (cDNA) enriched for full-length fragments using eIF4E, a 5' cap-binding protein, and an unenriched control. DNA sequencing adapter was added after enrichment of full-length cDNA using two different ligation protocols. From the mapped sequence reads, enrichment scores were calculated for all transcribed nucleotides and used to calculate P-values of 5' capped nucleotide enrichment. Sensitivity and accuracy were increased by combining P-values from replicate experiments. Data were obtained for P. falciparum ring, trophozoite and schizont stages of intra-erythrocytic development. RESULTS: 5' capped nucleotide signals were mapped to 17,961 non-overlapping P. falciparum genomic intervals. Analysis of the dominant 5' capped nucleotide in these genomic intervals revealed the presence of two groups with distinctive epigenetic features and sequence patterns. A total of 4,512 transcripts were annotated as 5' capped based on the correspondence of 5' end with 5' capped nucleotide annotated from full-length cDNA data. DISCUSSION: The presence of two groups of 5' capped nucleotides suggests that alternative mechanisms may exist for producing 5' capped transcript ends in P. falciparum. The 5' capped transcripts that are antisense, outside of, or partially overlapping coding regions may be important regulators of gene function in P. falciparum.

5-Phenoxy Primaquine Analogs and the Tetraoxane Hybrid as Antimalarial Agents.

The rapid emergence of drug resistance to the current antimalarial agents has led to the urgent need for the discovery of new and effective compounds. In this work, a series of 5-phenoxy primaquine analogs with 8-aminoquinoline core (7a-7h) was synthesized and investigated for their antimalarial activity against Plasmodium falciparum. Most analogs showed improved blood antimalarial activity compared to the original primaquine. To further explore a drug hybrid strategy, a conjugate compound between tetraoxane and the representative 5-phenoxy-primaquine analog 7a was synthesized. In our work, the hybrid compound 12 exhibited almost a 30-fold increase in the blood antimalarial activity (IC50 = 0.38 ± 0.11 µM) compared to that of primaquine, with relatively low toxicity against mammalian cells (SI = 45.61). Furthermore, we found that these 5-phenoxy primaquine analogs and the hybrid exhibit significant heme polymerization inhibition, an activity similar to that of chloroquine, which could contribute to their improved antimalarial activity. The 5-phenoxy primaquine analogs and the tetraoxane hybrid could serve as promising candidates for the further development of antimalarial agents.

Transgenic pyrimethamine-resistant plasmodium falciparum reveals transmission-blocking potency of P218, a novel antifolate candidate drug.

Antimalarial drugs capable of targeting multiple parasite stages, particularly the transmissible stages, can be valuable tools for advancing the malaria elimination agenda. Current antifolate drugs such as pyrimethamine can inhibit replicative parasite stages in both humans and mosquitoes, but antifolate resistance remains a challenge. The lack of reliable gametocyte-producing, antifolate-resistant Plasmodium falciparum laboratory strain hinders the study of new antifolate compounds that can overcome antifolate resistance including development stages in the mosquito. We used clustered regularly interspaced short palindromic repeats-Cas9 genome editing to develop a transgenic gametocyte-producing strain of P. falciparum with quadruple mutations (N51I, C59R, S108N, I164L) in the dihydrofolate reductase (dhfr) gene, using NF54 as a parental strain. The transgenic parasites exhibited pyrimethamine resistance while maintaining their gametocyte-producing activity. We then demonstrated that pyrimethamine could no longer inhibit male gametocyte exflagellation in the transgenic parasite. In contrast, P218, the novel antifolate, designed to overcome antifolate resistance, potently inhibited exflagellation. The exflagellation IC50 of P218 was five times lower than the asexual stage half maximal inhibitory concentration (IC50), suggesting a strong barrier for transmission of P218-resistant parasites. The transgenic gametocyte-producing, pyrimethamine-resistant parasite is a robust system for evaluating novel antifolate compounds against non-asexual stage development.

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.