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

Lookup NU author(s): Christopher Coxon, Dr Celine CanoORCiD, Emeritus Professor Bernard Golding, Professor Roger Griffin

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This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).

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.

Publication metadata

Author(s): Mitcheson DF, Bottrill AR, Carr K, Coxon CR, Cano C, Golding BT, Griffin RJ, Fry AM, Doerig C, Bayliss R, Tobin AB

Publication type: Article

Publication status: Published

Journal: Malaria Journal

Year: 2016

Volume: 15

Online publication date: 07/11/2016

Acceptance date: 28/10/2016

Date deposited: 14/12/2016

ISSN (electronic): 1475-2875

Publisher: BioMed Central Ltd

URL: http://dx.doi.org/10.1186/s12936-016-1580-3

DOI: 10.1186/s12936-016-1580-3

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