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

Antimalarial drug which target more than one life stage of the parasite are valuable tools in the fight against malaria. Previous generation of antifolate drugs are able to inhibit replicative stages of drug-sensitive, but not resistant parasites in humans, and mosquitoes. The lack of reliable gametocyte-producing, antifolate resistant P. falciparum hindrance the development of new antifolate compounds against mosquito stages. We used CRISPR-Cas9 technology to develop transgenic gametocyte producing P. falciparum with quadruple mutations in dhfr gene, using NF54 as a parental strain. The transgenic parasites gained pyrimethamine resistance while maintaining the gametocyte producing activity. In contrast to pyrimethamine that cannot inhibit exflagellation of the quadruple dhfr mutant parasite, the novel antifolate P218 showed a good potency for exflagellation inhibition (exflagellation IC50 10.74 {+/-} 4.22 nM). The exflagellation IC50 was 5.3 times lower than erythrocytic IC50 suggesting that the human to mosquito transmission poses as a strong barrier to prevent P218 resistant parasite among population. This study demonstrates that P218 can be considered as a highly potent tool to prevent the spread of antifolate resistant parasites. Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY Research Highlights- Transgenic gametocyte producing pyrimethamine resistant P. falciparum was generated. - P218 asexual stage IC50 in NF54-4mutPfdhfr was 56.94 {+/-} 15.69 nM. - P218 exflagellation IC50 in NF54-4mutPfdhfr was 10.74 {+/-} 4.22 nM. - P218 exflagellation IC50 in NF54-4mutPfdhfr is 5.3 times lower than erythrocytic IC50. - P218 is an invaluable tool for malaria treatment and transmission control.