Malaria, caused by Plasmodium protozoal parasites, remains a leading cause of morbidity and mortality. The Plasmodium parasite has a complex life cycle, with asexual and sexual forms in humans and Anopheles mosquitoes. Most antimalarials target only the symptomatic asexual blood stage. However, to ensure malaria eradication, new drugs with efficacy at multiple stages of the life cycle are necessary. We previously demonstrated that arsinothricin (AST), a newly discovered organoarsenical natural product, is a potent broad-spectrum antibiotic that inhibits the growth of various prokaryotic pathogens. Here, we report that AST is an effective multi-stage antimalarial. AST is a nonproteinogenic amino acid analog of glutamate that inhibits prokaryotic glutamine synthetase (GS). Phylogenetic analysis shows that Plasmodium GS, which is expressed throughout all stages of the parasite life cycle, is more closely related to prokaryotic GS than eukaryotic GS. AST potently inhibits Plasmodium GS, while it is less effective on human GS. Notably, AST effectively inhibits both Plasmodium erythrocytic proliferation and parasite transmission to mosquitoes. In contrast, AST is relatively nontoxic to a number of human cell lines, suggesting that AST is selective against malaria pathogens, with little negative effect on the human host. We propose that AST is a promising lead compound for developing a new class of multi-stage antimalarials.

Mosquito-transmitted Plasmodium parasites cause millions of people worldwide to suffer malaria every year. Drug-resistant Plasmodium parasites and insecticide-resistant mosquitoes make malaria hard to control. Thus, the next generation of antimalarial drugs that inhibit malaria infection and transmission are needed. We screened our Global Fungal Extract Library (GFEL) and obtained a candidate that completely inhibited Plasmodium falciparum transmission to Anopheles gambiae. The candidate fungal strain was determined as Aspergillus aculeatus. The bioactive compound was purified and identified as asperaculane B. The concentration of 50% inhibition on P. falciparum transmission (IC₅₀) is 7.89 µM. Notably, asperaculane B also inhibited the development of asexual P. falciparum with IC₅₀ of 3 µM, and it is nontoxic to human cells. Therefore, asperaculane B is a new dual-functional antimalarial lead that has the potential to treat malaria and block malaria transmission.

Background: Malaria transmission depends on infected mosquitoes and can be controlled by transmission-blocking drugs. The recently discovered FREP1-mediated malaria transmission pathway is an excellent target to screen drugs for limiting transmission. Methods: To identify candidate small molecules, we used an ELISA-based approach to analyze extracts from a fungal library for inhibition of the FREP1–parasite interaction. We isolated and determined one active compound by chromatography and crystallography, respectively. We measured the effects of the bioactive compound on malaria transmission to mosquitoes through standard membrane-feeding assays (SMFA) and on parasite proliferation in blood by culturing. Results: We discovered the ethyl acetate extract of the fungus Purpureocillium lilacinum that inhibited Plasmodium falciparum transmission to mosquitoes. Pre-exposure to the extract rendered Anopheles gambiae resistant to Plasmodium infection. Furthermore, we isolated one novel active compound from the extract and identified it as 3-amino-7,9-dihydroxy-1-methyl-6H-benzo[c]chromen-6-one, or “pulixin.” Pulixin prevented FREP1 from binding to P. falciparum-infected cell lysate. Pulixin blocked the transmission of the parasite to mosquitoes with an EC₅₀ (the concentration that gave half-maximal response) of 11 µM based on SMFA. Notably, pulixin also inhibited the proliferation of asexual-stage P. falciparum with an EC₅₀ of 47 nM. The compound did not show cytotoxic effects at a concentration of 116 µM or lower. Conclusion: By targeting the FREP1–Plasmodium interaction, we discovered that Purpureocillium lilacinum extract blocked malaria transmission. We isolated and identified the bioactive agent pulixin as a new compound capable of stopping malaria transmission to mosquitoes and inhibiting parasite proliferation in blood culture. Graphical

As part of our drug discovery program against malaria, the Penicillium janthinellum extract was discovered to inhibit P. falciparum proliferation in blood and transmission to mosquitoes. Bioactivity-guided fractionation of P. janthinellum extraction was carried out using chromatographic techniques. We determined the activities of fractions against Plasmodium falciparum asexual stage parasite proliferation in culture and sexual stage parasite transmission to mosquitoes using standard membrane feeding assays (SMFA). One active compound was isolated. Based on mass spectrometry and nuclear magnetic resonance profiles, the compound was structurally determined to be sterigmatocystin. Sterigmatocystin inhibited P. falciparum proliferation in the blood with an IC₅₀ of 34 µM and limited the sexual parasites to infect mosquitoes with an IC₅₀ of 48 µM. Meanwhile, sterigmatocystin did not show any acute toxicity to human kidney cells at a concentration of 64 µM or lower. Sterigmatocystin can be used as a drug lead for malaria control and as a probe to understand molecular mechanisms of malaria transmission.

Passage ofPlasmodiumthrough a mosquito midgut is essential for malaria transmission. FREP1, a peritrophic matrix protein in a mosquito midgut, binds to the parasite and mediatesPlasmodiuminfection inAnopheles. The FREP1-mediatedPlasmodiuminvasion pathway is highly conserved across multiple species ofPlasmodiumandAnopheles. Through pulldown, nineP. bergheiproteins were co-precipitated with FREP1-conjugated beads. After cloning these nine genes fromP. bergheiand expressing them in insect cells, six of them were confirmed to interact with recombinant FREP1 protein. Among them,α-tubulin-1 and heat shock protein 70 (Hsp70) were highly conserved inPlasmodiumspecies with >95% identity. Thus,P. falciparum α-tubulin-1 and Hsp70 were cloned and expressed inE. colito stimulate antibody (Ab) in mice. Our results showed that anti-serum againstP. falciparum α-tubulin-1 significantly inhibitedP. falciparumtransmission toAn. gambiae, while Ab againstP. falciparumHsp70 serum did not. The polyclonal Ab against humanα-tubulin did not interfere formation of ookinetes, however, significantly reduced the number ofP. falciparumoocysts inAn. gambiaemidguts. Moreover, fluorescence microscope assays showed that anti-α-tubulin Ab bound to impermeablePlasmodiumookinete apical invasive apparatus. Therefore, we propose that the interaction betweenAnophelesFREP1 protein andPlasmodium α-tubulin-1 directs the ookinete invasive apparatus towards midgut peritrophic matrix for the efficient passage of the parasite.AnophelesFREP1 andPlasmodiumα-tubulin-1 are potential targets for blocking malaria transmission to the mosquito host.AUTHOR SUMMARY: The molecular mechanisms of malaria transmission to mosquito are not well-understood. FREP1 proteins in mosquito midget PM has been proved to mediate malaria transmission by binding to parasite ookinetes. Here we reported thatPlasmodiumparasite α-tubulin-1 is an FREP1 binding partner. We initially identified the α-tubulin-1 through the FREP1-pulldown assay; Then we clonedP. falciparumα-tubulin-1, and demonstrated that the insect cell expressed recombinantPlasmodiumα-tubulin-1 bound to FREP1in vitro; Next, mouse anti-serum againstP. falciparumα-tubulin-1 was found to inhibitP. falciparumtransmission toAn. gambiae. P. falciparumα-tubulin-1 shares >84% identical amino acid sequence with human α-tubulin, purified Ab against human α-tubulin significantly inhibited malaria transmission. Anti-human α-tubulin Ab did not interfere the gametocyte-to-ookinetes conversion. Final, we found that anti-α-tubulin Ab bound to the apical end of impermeable ookinetes. Structurally, ookinete invasive apparatus locates at the apical opening. Therefore, we propose that the interaction betweenAnophelesmidgut FREP1 protein andPlasmodiumapicalα-tubulin-1 directs the ookinete invasive apparatus towards midgut PM for the efficient parasite invasion.

Vector control is considered an effective approach to controlling diseases spread by mosquito bites. Entomopathogenic fungi are widely used in agriculture to control insect pests, and fungal metabolites can potentially be developed as effective mosquitocides. In this study, a high-throughput screening method was used to search for potential mosquitocides in the Global Fungal Extract Library (GFEL). We tested the larvicidal activity of 264 fungal ethyl acetate crude extracts against Culex pipiens quinquefasciatus. Nine fungal extracts caused moderate to high mortality rates (>50%), with two fungal extracts (58A7 and 101H12) causing a 100% mortality rate. The lethal concentrations for 50% of the population (LC₅₀) were 44.27 mg/L and 31.90 mg/L, respectively. Fraction 14 had a high mortality rate, with an LC₅₀ value of 12.13 mg/L, and was isolated from 58A7 (Fractions 1–11) and 101H12 (Fractions 12–15). Further analyses showed that Fraction 14 was made up of vermistatin and dihydrovermistatin. In a Cx. p. quinquefasciatus larvicidal bioassay, vermistatin (LC₅₀ = 28.13 mg/L) was more toxic than dihydrovermistatin (LC₅₀ = 83.87 mg/L). Our findings suggested that the active fungal extract 101H12 from Talaromyces sp. and its compound vermistatin could be developed as mosquitocides.