SummaryA sensitive molecular assay was developed to quantify male and female Plasmodium falciparum gametocytes. Its application in 2 clinical trials demonstrates that the early effects of primaquine may be due to gametocyte fitness rather than sex ratio.
Summary
The search for antimalarial chemotypes with modes of action unrelated to existing drugs has intensified with the recent failure of first-line therapies across Southeast Asia. Here, we show that the trisubstituted imidazole MMV030084 potently inhibits hepatocyte invasion by
Plasmodium
sporozoites, merozoite egress from asexual blood stage schizonts, and male gamete exflagellation. Metabolomic, phosphoproteomic, and chemoproteomic studies, validated with conditional knockdown parasites, molecular docking, and recombinant kinase assays, identified cGMP-dependent protein kinase (PKG) as the primary target of MMV030084. PKG is known to play essential roles in
Plasmodium
invasion of and egress from host cells, matching MMV030084's activity profile. Resistance selections and gene editing identified tyrosine kinase-like protein 3 as a low-level resistance mediator for PKG inhibitors, while PKG itself never mutated under pressure. These studies highlight PKG as a resistance-refractory antimalarial target throughout the
Plasmodium
life cycle and promote MMV030084 as a promising
Plasmodium
PKG-targeting chemotype.
Summary
Emerging resistance to first-line antimalarial combination therapies threatens malaria treatment and the global elimination campaign. Improved therapeutic strategies are required to protect existing drugs and enhance treatment efficacy. We report that the piperazine-containing compound ACT-451840 exhibits single-digit nanomolar inhibition of the Plasmodium falciparum asexual blood stages and transmissible gametocyte forms. Genome sequence analyses of in vitro-derived ACT-451840-resistant parasites revealed single nucleotide polymorphisms in pfmdr1, which encodes a digestive vacuole membrane-bound ATP-binding cassette transporter known to alter P. falciparum susceptibility to multiple first-line antimalarials. CRISPR-Cas9 based gene editing confirmed that PfMDR1 point mutations mediated ACT-451840 resistance. Resistant parasites demonstrated increased susceptibility to the clinical drugs lumefantrine, mefloquine, quinine and amodiaquine. Stage V gametocytes harboring Cas9-introduced pfmdr1 mutations also acquired ACT-451840 resistance. These findings reveal that PfMDR1 mutations can impart resistance to compounds active against asexual blood stages and mature gametocytes. Exploiting PfMDR1 resistance mechanisms provides new opportunities for developing disease-relieving and transmission-blocking antimalarials.
New
reliable and cost-effective antimalarial drug screening assays
are urgently needed to identify drugs acting on different stages of
the parasite Plasmodium falciparum,
and particularly those responsible for human-to-mosquito transmission,
that is, the P. falciparum gametocytes.
Low Z′ factors, narrow dynamic ranges, and/or
extended assay times are commonly reported in current gametocyte assays
measuring gametocyte-expressed fluorescent or luciferase reporters,
endogenous ATP levels, activity of gametocyte enzymes, or redox-dependent
dye fluorescence. We hereby report on a dual-luciferase gametocyte
assay with immature and mature P. falciparum gametocyte stages expressing red and green-emitting luciferases
from Pyrophorus plagiophthalamus under
the control of the parasite sexual stage-specific pfs16 gene promoter. The assay was validated with reference antimalarial
drugs and allowed to quantitatively and simultaneously measure stage-specific
drug effects on parasites at different developmental stages. The optimized
assay, requiring only 48 h incubation with drugs and using a cost-effective
luminogenic substrate, significantly reduces assay cost and time in
comparison to state-of-the-art analogous assays. The assay had a Z′ factor of 0.71 ± 0.03, and it is suitable
for implementation in 96- and 384-well microplate formats. Moreover,
the use of a nonlysing d-luciferin substrate significantly
improved the reliability of the assay and allowed one to perform,
for the first time, P. falciparum bioluminescence
imaging at single-cell level.
Plasmodium falciparum gametocytes, specifically the mature stages, are the only malaria parasite stage in humans transmissible to the mosquito vector. Anti-malarial drugs capable of killing these forms are considered essential for the eradication of malaria and tools allowing the screening of large compound libraries with high predictive power are needed to identify new candidates. As gametocytes are not a replicative stage it is difficult to apply the same drug screening methods used for asexual stages. Here we propose an assay, based on high content imaging, combining “classic” gametocyte viability readout based on gametocyte counts with a functional viability readout, based on gametocyte activation and the discrimination of the typical gamete spherical morphology. This simple and rapid assay has been miniaturized to a 384-well format using acridine orange staining of wild type P. falciparum 3D7A sexual forms, and was validated by screening reference antimalarial drugs and the MMV Malaria Box. The assay demonstrated excellent robustness and ability to identify quality hits with high likelihood of confirmation of transmission reducing activity in subsequent mosquito membrane feeding assays.
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