A new tool for the chemical genetic investigation of the Plasmodium falciparum Pfnek-2 NIMA-related kinase

Deborah F. Mitcheson, Andrew R. Bottrill, Katherine Carr, Christopher R. Coxon, Céline Cano, Bernard T. Golding, Roger J. Griffin, Andrew M. Fry, Christian Doerig, Richard Bayliss, Andrew B. Tobin

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Abstract

Background: Examining essential biochemical pathways in Plasmodium falciparum presents serious challenges, as standard molecular techniques such as siRNA cannot be employed in this organism, and generating gene knock-outs of essential proteins requires specialized conditional approaches. In the study of protein kinases, pharmacological inhibition presents a feasible alternative option. However, as in mammalian systems, inhibitors often lack the desired selectivity. Described here is a chemical genetic approach to selectively inhibit Pfnek-2 in P. falciparum, a member of the NIMA-related kinase family that is essential for completion of the sexual development of the parasite.

Results: Introduction of a valine to cysteine mutation at position 24 in the glycine rich loop of Pfnek-2 does not affect kinase activity but confers sensitivity to the protein kinase inhibitor 4-(6-ethynyl-9H-purin-2-ylamino) benzene sulfonamide (NCL-00016066). Using a combination of in vitro kinase assays and mass spectrometry, (including phosphoproteomics) the study shows that this compound acts as an irreversible inhibitor to the mutant Pfnek2 likely through a covalent link with the introduced cysteine residue. In particular, this was shown by analysis of total protein mass using mass spectrometry which showed a shift in molecular weight of the mutant kinase in the presence of the inhibitor to be precisely equivalent to the molecular weight of NCL-00016066. A similar molecular weight shift was not observed in the wild type kinase. Importantly, this inhibitor has little activity towards the wild type Pfnek-2 and, therefore, has all the properties of an effective chemical genetic tool that could be employed to determine the cellular targets for Pfnek-2. 

Conclusions: Allelic replacement of wild-type Pfnek-2 with the mutated kinase will allow for targeted inhibition of Pfnek-2 with NCL-00016066 and hence pave the way for comparative studies aimed at understanding the biological role and transmission-blocking potential of Pfnek-2.

Original languageEnglish
Article number535
JournalMalaria Journal
Volume15
Issue number1
DOIs
Publication statusPublished - 7 Nov 2016

Keywords

  • Chemical genetics
  • Covalent modification
  • Mass spectrometry
  • NIMA-related protein kinase (Nek)
  • Plasmodium falciparum

ASJC Scopus subject areas

  • Parasitology
  • Infectious Diseases

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  • Cite this

    Mitcheson, D. F., Bottrill, A. R., Carr, K., Coxon, C. R., Cano, C., Golding, B. T., Griffin, R. J., Fry, A. M., Doerig, C., Bayliss, R., & Tobin, A. B. (2016). A new tool for the chemical genetic investigation of the Plasmodium falciparum Pfnek-2 NIMA-related kinase. Malaria Journal, 15(1), [535]. https://doi.org/10.1186/s12936-016-1580-3