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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.

SUBMITTER: Tran PN

PROVIDER: S-EPMC5050295 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Human dihydrofolate reductase influences the sensitivity of the malaria parasite Plasmodium falciparum to ketotifen - A cautionary tale in screening transgenic parasites.

Tran Phuong N PN Tate Cameron J CJ Ridgway Melanie C MC Saliba Kevin J KJ Kirk Kiaran K Maier Alexander G AG

International journal for parasitology. Drugs and drug resistance 20160922 3

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 ...[more]

PMID: 27705841

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Project description:Plasmodium falciparum thymidylate synthase-dihydrofolate reductase (TS-DHFR) is an essential enzyme in nucleotide biosynthesis and a validated molecular drug target in malaria. Because P. falciparum TS and DHFR are highly homologous to their human counterparts, existing active-site antifolate drugs can have dose-limiting toxicities. In humans, TS and DHFR are two separate proteins. In P. falciparum, however, TS-DHFR is bifunctional, with both TS and DHFR active sites on a single polypeptide chain of the enzyme. Consequently, P. falciparum TS-DHFR contains unique distant or nonactive regions that might modulate catalysis: (1) an N-terminal tail and (2) a linker region tethering DHFR to TS, and encoding a crossover helix that forms critical electrostatic interactions with the DHFR active site. The role of these nonactive sites in the bifunctional P. falciparum TS-DHFR is unknown. We report the first in-depth, pre-steady-state kinetic characterization of the full-length, wild-type (WT) P. falciparum TS-DHFR enzyme and probe the role of distant, nonactive regions through mutational analysis. We show that the overall rate-limiting step in the WT P. falciparum TS-DHFR enzyme is TS catalysis. We further show that if TS is in an activated (liganded) conformation, the DHFR rate is 2-fold activated, from 60 s-1 to 130 s-1 in the WT enzyme. The TS rate is also reciprocally activated by approximately 1.5-fold if DHFR is in an activated, ligand-bound conformation. Mutations to the linker region affect neither catalytic rate nor domain-domain communication. Deletion of the N-terminal tail, although in a location remote from the active site, decreases the DHFR single rate and the bifunctional TS-DHFR rate by a factor of 2. The 2-fold activation of the DHFR rate by TS ligands remains intact, although even the activated N-terminal mutant has just half the DHFR activity of the WT enzyme. However, the reciprocal communication between TS active site and DHFR ligands is impaired in N-terminal mutants. Surprisingly, deletion of the analogous N-terminal tail in Leishmania major TS-DHFR causes a 3-fold enhancement of the DHFR rate from approximately 14 s-1 to approximately 40 s-1. In summary, our results demonstrate a complex interplay of domain-domain communication and nonactive-site modulation of catalysis in P. falciparum TS-DHFR. Furthermore, each parasitic TS-DHFR is activated by unique mechanisms, modulated by their nonactive site regions. Finally, our studies suggest the N-terminal tail of P. falciparum TS-DHFR is a highly selective, novel target for potential antifolate development in malaria.

Dataset Information

Human dihydrofolate reductase influences the sensitivity of the malaria parasite Plasmodium falciparum to ketotifen - A cautionary tale in screening transgenic parasites.

Publications

Human dihydrofolate reductase influences the sensitivity of the malaria parasite Plasmodium falciparum to ketotifen - A cautionary tale in screening transgenic parasites.

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