title: Toxoplasma gondii cyclic GMP-dependent kinase: chemotherapeutic targeting of an essential parasite protein kinase.
abstract: The trisubstituted pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl]pyridine (compound 1) has in vivo activity against the apicomplexan parasites Toxoplasma gondii and Eimeria tenella in animal models. The presumptive molecular target of this compound in E. tenella is cyclic GMP-dependent protein kinase (PKG). Native PKG purified from T. gondii has kinetic and pharmacologic properties similar to those of the E. tenella homologue, and both have been functionally expressed as recombinant proteins in T. gondii. Computer modeling of parasite PKG was used to predict catalytic site amino acid residues that interact with compound 1. The recombinant laboratory-generated mutants T. gondii PKG T761Q or T761M and the analogous E. tenella T770 alleles have reduced binding affinity for, and are not inhibited by, compound 1. By all other criteria, PKG with this class of catalytic site substitution is indistinguishable from wild-type enzyme. A genetic disruption of T. gondii PKG can only be achieved if a complementing copy of PKG is provided in trans, arguing that PKG is an essential protein. Strains of T. gondii, disrupted at the genomic PKG locus and dependent upon the T. gondii T761-substituted PKGs, are as virulent as wild type in mice. However, unlike mice infected with wild-type T. gondii that are cured by compound 1, mice infected with the laboratory-generated strains of T. gondii do not respond to treatment. We conclude that PKG represents the primary molecular target responsible for the antiparasitic efficacy of compound 1.
authors.affiliation: Department of Human and Animal Infectious Disease Research, Merck Research Laboratories, Merck and Co., Inc., Rahway New Jersey 07065, USA. robert_donald@merck.com
authors.forename: Robert G K
authors.initials: RG
authors.lastname: Donald
authors.forename: John
authors.initials: J
authors.lastname: Allocco
authors.forename: Suresh B
authors.initials: SB
authors.lastname: Singh
authors.forename: Bakela
authors.initials: B
authors.lastname: Nare
authors.forename: Scott P
authors.initials: SP
authors.lastname: Salowe
authors.forename: Judyann
authors.initials: J
authors.lastname: Wiltsie
authors.forename: Paul A
authors.initials: PA
authors.lastname: Liberator
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D030801:Animals, Genetically Modified; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D020134:Catalytic Domain / Q000235:genetics; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D016786:Eimeria tenella / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018390:Gene Targeting; D051379:Mice; D008810:Mice, Inbred C57BL; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011725:Pyridines / Q000494:pharmacology*; D011758:Pyrroles / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D014122:Toxoplasma / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D014124:Toxoplasmosis, Animal / Q000188:drug therapy
chemical_list: C452660:4-(2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl)pyridine; D000981:Antiprotozoal Agents; D004791:Enzyme Inhibitors; D011725:Pyridines; D011758:Pyrroles; D011994:Recombinant Proteins; D017869:Cyclic GMP-Dependent Protein Kinases