Abstract
ABSTRACTWe used a sensitization approach that involves replacement of the gatekeeper residue in a protein kinase with one with a different side chain. The activity of the enzyme with a bulky gatekeeper residue, such as methionine, cannot be inhibited using bumped kinase inhibitors (BKIs). Here, we have used this approach to study Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1). The methionine gatekeeper substitution, T145M, although it led to a 47% reduction in transphosphorylation, was successfully introduced into the CDPK1 locus using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9. As methionine is a bulky residue, BKI 1294 had a 10-fold-greater effect in vitro on the wild-type enzyme than on the methionine mutant. However, in contrast to in vitro data with recombinant enzymes, BKI 1294 had a slightly greater inhibition of the growth of CDPK1 T145M parasites than the wild type. Moreover, the CDPK1 T145M parasites were more sensitive to the action of compound 2 (C2), a specific inhibitor of protein kinase G (PKG). These results suggest that a reduction in the activity of CDPK1 due to methionine substitution at the gatekeeper position is compensated through the direct action of PKG or of another kinase under the regulation of PKG. The transcript levels of CDPK5 and CDPK6 were significantly upregulated in the CDPK1 T145M parasites. The increase in CDPK6 or some other kinase may compensate for decrease in CDPK1 activity during invasion. This study suggests that targeting two kinases may be more effective in chemotherapy to treat malaria so as not to select for mutations in one of the enzymes.
Highlights
We used a sensitization approach that involves replacement of the gatekeeper residue in a protein kinase with one with a different side chain
We have investigated the effect of gatekeeper substitution on the activity of Plasmodium falciparum calciumdependent protein kinase 1 (PfCDPK1)
Sixty amino acid residues in the kinase domain of Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1) to PfCDPK5 were aligned with Toxoplasma gondii CDPK1 (TgCDPK1) as a template
Summary
We used a sensitization approach that involves replacement of the gatekeeper residue in a protein kinase with one with a different side chain. The activity of the enzyme with a bulky gatekeeper residue, such as methionine, cannot be inhibited using bumped kinase inhibitors (BKIs). The CDPK1 T145M parasites were more sensitive to the action of compound 2 (C2), a specific inhibitor of protein kinase G (PKG) These results suggest that a reduction in the activity of CDPK1 due to methionine substitution at the gatekeeper position is compensated through the direct action of PKG or of another kinase under the regulation of PKG. Mutant parasites with methionine gatekeeper residue compensate the reduced activity of CDPK1 through the action of PKG possibly by upregulation of CDPK6 or some other kinase. We have investigated the effect of gatekeeper substitution on the activity of Plasmodium falciparum calciumdependent protein kinase 1 (PfCDPK1). PfCDPK4 was successfully disrupted in the blood-stage parasite, suggesting a redundant role or none for asexual proliferation of the parasite [6, 11]
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