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

Mitcheson, DF and Bottrill, AR and Carr, K and Coxon, CR and Cano, C and Golding, BT and Griffin, RJ and Fry, AM and Doerig, C and Bayliss, R and Tobin, AB (2016) A new tool for the chemical genetic investigation of the Plasmodium falciparum Pfnek-2 NIMA-related kinase. Malaria Journal, 15 (1). ISSN 1475-2875

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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. © 2016 The Author(s).

Item Type: Article
Uncontrolled Keywords: 1108 Medical Microbiology
Subjects: Q Science > QD Chemistry
Q Science > QH Natural history > QH301 Biology
Q Science > QH Natural history > QH426 Genetics
Divisions: Pharmacy & Biomolecular Sciences
Publisher: BioMed Central LTD
Date Deposited: 29 Nov 2016 12:07
Last Modified: 06 Sep 2017 15:30
DOI or Identification number: 10.1186/s12936-016-1580-3
URI: http://researchonline.ljmu.ac.uk/id/eprint/4906

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