University of Leicester
Browse
A new tool for the chemical genetic investigation of the Plasmodium falciparum Pfnek-2 NIMA-related kinase.pdf (4.05 MB)

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

Download (4.05 MB)
journal contribution
posted on 2017-05-15, 14:10 authored by Deborah F. Mitcheson, Andrew R. Bottrill, Katherine Carr, Christopher R. Coxon, Celine Cano, Bernard T. Golding, Roger J. Griffin, Andrew M. Fry, Christian Doerig, Richard Bayliss, Andrew B. Tobin
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.

History

Citation

Malaria Journal, 2016, 15:535

Author affiliation

/Organisation/COLLEGE OF MEDICINE, BIOLOGICAL SCIENCES AND PSYCHOLOGY/MBSP Non-Medical Departments/Molecular & Cell Biology

Version

  • VoR (Version of Record)

Published in

Malaria Journal

Publisher

BioMed Central

eissn

1475-2875

Acceptance date

2016-10-28

Copyright date

2016

Available date

2017-05-15

Publisher version

https://malariajournal.biomedcentral.com/articles/10.1186/s12936-016-1580-3

Language

en

Usage metrics

    University of Leicester Publications

    Categories

    No categories selected

    Licence

    Exports

    RefWorks
    BibTeX
    Ref. manager
    Endnote
    DataCite
    NLM
    DC