title: Human dihydrofolate reductase influences the sensitivity of the malaria parasite Plasmodium falciparum to ketotifen - A cautionary tale in screening transgenic parasites.
abstract: Ketotifen has recently been reported to inhibit the growth of both asexual and sexual malaria parasites. A parasite transporter, PfgABCG2, has been implicated in its mechanism of action. Human dihydrofolate reductase (hDHFR) is the most commonly used selectable marker to create transgenic Plasmodium falciparum cell lines. Growth assays using transgenic P. falciparum parasites with different selectable markers revealed that the presence of hDHFR rather than the absence of PfgABCG2 is responsible for a shift in the parasite's sensitivity to ketotifen. Employing a range of in vitro assays and liquid chromatography-mass spectrometry we show that ketotifen influences hDHFR activity, but it is not metabolised by the enzyme. Our data also highlights potential pitfalls when functionally characterising transgenic parasites.
authors.affiliation: Research School of Biology, The Australian National University, Canberra, ACT, 2601, Australia.
authors.forename: Phuong N
authors.initials: PN
authors.lastname: Tran
authors.affiliation: Research School of Biology, The Australian National University, Canberra, ACT, 2601, Australia.
authors.forename: Cameron J
authors.initials: CJ
authors.lastname: Tate
authors.affiliation: Research School of Biology, The Australian National University, Canberra, ACT, 2601, Australia.
authors.forename: Melanie C
authors.initials: MC
authors.lastname: Ridgway
authors.affiliation: Research School of Biology, The Australian National University, Canberra, ACT, 2601, Australia; Medical School, The Australian National University, Canberra, ACT, 2601, Australia.
authors.forename: Kevin J
authors.initials: KJ
authors.lastname: Saliba
authors.affiliation: Research School of Biology, The Australian National University, Canberra, ACT, 2601, Australia.
authors.forename: Kiaran
authors.initials: K
authors.lastname: Kirk
authors.affiliation: Research School of Biology, The Australian National University, Canberra, ACT, 2601, Australia. Electronic address: alex.maier@anu.edu.au.
authors.forename: Alexander G
authors.initials: AG
authors.lastname: Maier
mesh_terms: D000962:Antimalarials / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical; D015870:Gene Expression; D006801:Humans; D007665:Ketotifen / Q000494:pharmacology*; D021261:Parasitic Sensitivity Tests; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism*
chemical_list: D000962:Antimalarials; D011994:Recombinant Proteins; D013762:Tetrahydrofolate Dehydrogenase; D007665:Ketotifen
keywords: Folate; Ketotifen fumarate; Malaria; Plasmodium falciparum; Selectable marker; Transfection; hDHFR