Anticoccidial kinase inhibitors: identification of protein kinase targets secondary to cGMP-dependent protein kinase.
Trisubstituted pyrrole inhibitors of the essential coccidian parasite cGMP dependent protein kinase (PKG) block parasite invasion and show in vivo efficacy against Eimeria in chickens and Toxoplasma in mice. An imidazopyridine inhibitor of PKG activity with greater potency in both parasite invasion assays and in vivo activity has recently been identified. Susceptibility experiments with a Toxoplasma knock-out strain expressing a complementing compound-refractory PKG allele ('T761Q-KO'), suggest a role for additional secondary protein kinase targets. Using extracts from this engineered T. gondii strain and a radiolabeled imidazopyridine ligand, a single peak of binding activity associated with calmodulin-like domain protein kinase (CDPK1) has been identified. Like PKG, CDPK1 has been implicated in host cell invasion and exhibits sub-nanomolar sensitivity to the compound. Amino acid sequence comparisons of coccidian CDPKs and a mutational analysis reveal that the binding of the ligand to PKG and CDPK1 (but not other CDPK isoforms) is mediated by similar contacts in a catalytic site hydrophobic binding pocket, and can be blocked by analogous amino acid substitutions. Transgenic strains over-expressing a biochemically active but compound-refractory CDPK1 mutant ('G128Q') fail to show reduced susceptibility to the compound in vivo, suggesting that selective inhibition of this enzyme is not responsible for the enhanced anti-parasitic potency of the imidazopyridine analog. An alternative secondary target candidate, the alpha-isoform of casein kinase 1 (CK1alpha), shows sensitivity to the compound in the low nanomolar range. These results provide an example of the utility of the Toxoplasma model system for investigating the mechanism of action of novel anticoccidial agents.
Donald
Robert G K
RG
Department of Infectious Diseases, Merck & Co., P.O. Box 2000, R80Y-260 Rahway, NJ 07065-0900, USA.
Zhong
Tanya
T
Wiersma
Helen
H
Nare
Bakela
B
Yao
Dan
D
Lee
Anita
A
Allocco
John
J
Liberator
Paul A
PA
D000595:Amino Acid Sequence; D000818:Animals; D054729:Calcium-Calmodulin-Dependent Protein Kinase Type 1; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D047389:Casein Kinase I / Q000037:antagonists & inhibitors / Q000378:metabolism; D003049:Coccidiostats / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D016786:Eimeria tenella / Q000187:drug effects / Q000201:enzymology; D021741:Eimeriida / Q000187:drug effects* / Q000201:enzymology*; D000939:Epitopes; D005347:Fibroblasts; D006801:Humans; D007093:Imidazoles / Q000378:metabolism / Q000494:pharmacology; D007668:Kidney / Q000166:cytology; D008297:Male; D008969:Molecular Sequence Data; D047428:Protein Kinase Inhibitors / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology*; D011494:Protein Kinases / Q000378:metabolism*; D011725:Pyridines / Q000378:metabolism / Q000494:pharmacology; D011743:Pyrimidines / Q000378:metabolism / Q000494:pharmacology; D011758:Pyrroles / Q000378:metabolism / Q000494:pharmacology; D011994:Recombinant Proteins; D012867:Skin / Q000166:cytology; D014122:Toxoplasma / Q000187:drug effects / Q000201:enzymology
C452660:4-(2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl)pyridine; C513588:4-(7-((dimethylamino)methyl)-2-(4-fluorophenyl)imidazo(1,2-a)pyridin-3-yl)pyrimidin-2-amine; D003049:Coccidiostats; D000939:Epitopes; D007093:Imidazoles; D047428:Protein Kinase Inhibitors; D011725:Pyridines; D011743:Pyrimidines; D011758:Pyrroles; D011994:Recombinant Proteins; D011494:Protein Kinases; D047389:Casein Kinase I; D017869:Cyclic GMP-Dependent Protein Kinases; C517596:CAMK1 protein, human; D054729:Calcium-Calmodulin-Dependent Protein Kinase Type 1; D017871:Calcium-Calmodulin-Dependent Protein Kinases