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



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.



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.
Department of Molecular and Cell Biology, University of Leicester, Lancaster Road, Leicester, LE1 9HN, UK.
Deborah F
DF
Mitcheson
Protein and Nucleic Acid Chemistry Laboratory, University of Leicester, Hodgkin Building, Leicester, LE1 9HN, UK.
Andrew R
AR
Bottrill
Department of Molecular and Cell Biology, University of Leicester, Lancaster Road, Leicester, LE1 9HN, UK.
Katherine
K
Carr
School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University, 2 Rodney Street, Liverpool, L3 5UX, UK.
Christopher R
CR
Coxon
School of Chemistry, Bedson Building, Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, NE1 7RU, UK.
Celine
C
Cano
School of Chemistry, Bedson Building, Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, NE1 7RU, UK.
Bernard T
BT
Golding
School of Chemistry, Bedson Building, Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, NE1 7RU, UK.
Roger J
RJ
Griffin
Department of Molecular and Cell Biology, University of Leicester, Lancaster Road, Leicester, LE1 9HN, UK.
Andrew M
AM
Fry
Department of Microbiology, Building 76, Monash University, Wellington Road, Clayton, VIC, 3800, Australia.
Christian
C
Doerig
Astbury 6.108a, School of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, UK.
Richard
R
Bayliss
Centre for Translational Pharmacology, Institute of Molecular Cell and Systems Biology, University of Glasgow, Glasgow, G12 8QQ, UK. tba@leicester.ac.uk.
Andrew B
AB
Tobin
D004791:Enzyme Inhibitors / Q000378:metabolism*; D013058:Mass Spectrometry; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism*; D000072379:NIMA-Related Kinases / Q000235:genetics / Q000378:metabolism*; D010963:Plasmodium falciparum / Q000201:enzymology*; D011687:Purines / Q000378:metabolism*; D013449:Sulfonamides / Q000378:metabolism*; D000096926:Benzenesulfonamides
D004791:Enzyme Inhibitors; D050505:Mutant Proteins; D000072379:NIMA-Related Kinases; D011687:Purines; D013449:Sulfonamides; C000620884:4-(6-ethynyl-9H-purin-2-ylamino)benzenesulfonamide; D000096926:Benzenesulfonamides
Chemical genetics; Covalent modification; Mass spectrometry; NIMA-related protein kinase (Nek); Plasmodium falciparum