title: Identifying antimalarial compounds targeting dihydrofolate reductase-thymidylate synthase (DHFR-TS) by chemogenomic profiling.
abstract: The mode of action of many antimalarial drugs is unknown. Chemogenomic profiling is a powerful method to address this issue. This experimental approach entails disruption of gene function and phenotypic screening for changes in sensitivity to bioactive compounds. Here, we describe the application of reverse genetics for chemogenomic profiling in Plasmodium. Plasmodium falciparum parasites harbouring a transgenic insertion of the glmS ribozyme downstream of the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene were used for chemogenomic profiling of antimalarial compounds to identify those which target DHFR-TS. DHFR-TS expression can be attenuated by exposing parasites to glucosamine. Parasites with attenuated DHFR-TS expression were significantly more sensitive to antifolate drugs known to target DHFR-TS. In contrast, no change in sensitivity to other antimalarial drugs with different modes of action was observed. Chemogenomic profiling was performed using the Medicines for Malaria Venture (Switzerland) Malaria Box compound library, and two compounds were identified as novel DHFR-TS inhibitors. We also tested the glmS ribozyme in Plasmodium berghei, a rodent malaria parasite. The expression of reporter genes with downstream glmS ribozyme could be attenuated in transgenic parasites comparable with that obtained in P. falciparum. The chemogenomic profiling method was applied in a P. berghei line expressing a pyrimethamine-resistant Toxoplasma gondii DHFR-TS reporter gene under glmS ribozyme control. Parasites with attenuated expression of this gene were significantly sensitised to antifolates targeting DHFR-TS, but not other drugs with different modes of action. In conclusion, these data show that the glmS ribozyme reverse genetic tool can be applied for identifying primary targets of antimalarial compounds in human and rodent malaria parasites.
authors.affiliation: National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Thailand Science Park, Thanon Phahonyothin, Tambon Khlong Neung, Amphoe Khlong Luang, Pathum Thani 12120, Thailand.
authors.forename: Aiyada
authors.initials: A
authors.lastname: Aroonsri
authors.affiliation: National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Thailand Science Park, Thanon Phahonyothin, Tambon Khlong Neung, Amphoe Khlong Luang, Pathum Thani 12120, Thailand; Department of Pharmacology and Therapeutics, University of Ibadan, Ibadan, Nigeria.
authors.forename: Olugbenga
authors.initials: O
authors.lastname: Akinola
authors.affiliation: National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Thailand Science Park, Thanon Phahonyothin, Tambon Khlong Neung, Amphoe Khlong Luang, Pathum Thani 12120, Thailand.
authors.forename: Navaporn
authors.initials: N
authors.lastname: Posayapisit
authors.affiliation: National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Thailand Science Park, Thanon Phahonyothin, Tambon Khlong Neung, Amphoe Khlong Luang, Pathum Thani 12120, Thailand.
authors.forename: Warangkhana
authors.initials: W
authors.lastname: Songsungthong
authors.affiliation: National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Thailand Science Park, Thanon Phahonyothin, Tambon Khlong Neung, Amphoe Khlong Luang, Pathum Thani 12120, Thailand.
authors.forename: Chairat
authors.initials: C
authors.lastname: Uthaipibull
authors.affiliation: National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Thailand Science Park, Thanon Phahonyothin, Tambon Khlong Neung, Amphoe Khlong Luang, Pathum Thani 12120, Thailand.
authors.forename: Sumalee
authors.initials: S
authors.lastname: Kamchonwongpaisan
authors.affiliation: Department of Pharmacology and Therapeutics, University of Ibadan, Ibadan, Nigeria.
authors.forename: Grace O
authors.initials: GO
authors.lastname: Gbotosho
authors.affiliation: National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Thailand Science Park, Thanon Phahonyothin, Tambon Khlong Neung, Amphoe Khlong Luang, Pathum Thani 12120, Thailand.
authors.forename: Yongyuth
authors.initials: Y
authors.lastname: Yuthavong
authors.affiliation: National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), 113 Thailand Science Park, Thanon Phahonyothin, Tambon Khlong Neung, Amphoe Khlong Luang, Pathum Thani 12120, Thailand. Electronic address: philip@biotec.or.th.
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Shaw
mesh_terms: D000818:Animals; D000962:Antimalarials / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004912:Erythrocytes / Q000469:parasitology; D005260:Female; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D015870:Gene Expression; D006801:Humans; D020128:Inhibitory Concentration 50; D051379:Mice; D008807:Mice, Inbred BALB C; D030781:Organisms, Genetically Modified; D010957:Plasmids; D010962:Plasmodium berghei / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D016337:RNA, Catalytic / Q000187:drug effects; D013047:Specific Pathogen-Free Organisms; D013762:Tetrahydrofolate Dehydrogenase / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D014162:Transfection
chemical_list: D000962:Antimalarials; D005493:Folic Acid Antagonists; D016337:RNA, Catalytic; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase
keywords: Antifolates; Chemogenomic profiling; Dihydrofolate reductase-thymidylate synthase; Drug; Malaria; Plasmodium; glmS ribozyme