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

Mitcheson, D. F. et al. (2016) A new tool for the chemical genetic investigation of the Plasmodium falciparum Pfnek-2 NIMA-related kinase. Malaria Journal, 15(1), 535. (doi: 10.1186/s12936-016-1580-3) (PMID:27821169) (PMCID:PMC510031)

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

• Item Type: Articles
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Tobin, Andrew
• Authors: 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., and Tobin, A. B.
• College/School: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Journal Name: Malaria Journal
• Publisher: BioMed Central
• ISSN: 1475-2875
• ISSN (Online): 1475-2875
• Copyright Holders: Copyright © 2016 The Authors
• First Published: First published in Malaria Journal 15(1):535
• Publisher Policy: Reproduced under a Creative Commons License