title: Purification and molecular characterization of cGMP-dependent protein kinase from Apicomplexan parasites. A novel chemotherapeutic target.
abstract: The trisubstituted pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl]pyridine (Compound 1) inhibits the growth of Eimeria spp. both in vitro and in vivo. The molecular target of Compound 1 was identified as cGMP-dependent protein kinase (PKG) using a tritiated analogue to purify a approximately 120-kDa protein from lysates of Eimeria tenella. This represents the first example of a protozoal PKG. Cloning of PKG from several Apicomplexan parasites has identified a parasite signature sequence of nearly 300 amino acids that is not found in mammalian or Drosophila PKG and which contains an additional, third cGMP-binding site. Nucleotide cofactor regulation of parasite PKG is remarkably different from mammalian enzymes. The activity of both native and recombinant E. tenella PKG is stimulated 1000-fold by cGMP, with significant cooperativity. Two isoforms of the parasite enzyme are expressed from a single copy gene. NH(2)-terminal sequence of the soluble isoform of PKG is consistent with alternative translation initiation within the open reading frame of the enzyme. A larger, membrane-associated isoform corresponds to the deduced full-length protein sequence. Compound 1 is a potent inhibitor of both soluble and membrane-associated isoforms of native PKG, as well as recombinant enzyme, with an IC(50) of <1 nm.
authors.affiliation: Department of Human and Animal Infectious Disease Research, Merck Research Laboratories, Rahway, New Jersey 07065, USA. anne_gurnett@merck.com
authors.forename: Anne M
authors.initials: AM
authors.lastname: Gurnett
authors.forename: Paul A
authors.initials: PA
authors.lastname: Liberator
authors.forename: Paula M
authors.initials: PM
authors.lastname: Dulski
authors.forename: Scott P
authors.initials: SP
authors.lastname: Salowe
authors.forename: Robert G K
authors.initials: RG
authors.lastname: Donald
authors.forename: Jennifer W
authors.initials: JW
authors.lastname: Anderson
authors.forename: Judyann
authors.initials: J
authors.lastname: Wiltsie
authors.forename: Carmen A
authors.initials: CA
authors.lastname: Diaz
authors.forename: Georgiana
authors.initials: G
authors.lastname: Harris
authors.forename: Ben
authors.initials: B
authors.lastname: Chang
authors.forename: Sandra J
authors.initials: SJ
authors.lastname: Darkin-Rattray
authors.forename: Bakela
authors.initials: B
authors.lastname: Nare
authors.forename: Tami
authors.initials: T
authors.lastname: Crumley
authors.forename: Penny Sue
authors.initials: PS
authors.lastname: Blum
authors.forename: Andrew S
authors.initials: AS
authors.lastname: Misura
authors.forename: Tamas
authors.initials: T
authors.lastname: Tamas
authors.forename: Mohinder K
authors.initials: MK
authors.lastname: Sardana
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Yuan
authors.forename: Tesfaye
authors.initials: T
authors.lastname: Biftu
authors.forename: Dennis M
authors.initials: DM
authors.lastname: Schmatz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016782:Apicomplexa / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002645:Chickens / Q000469:parasitology; D003001:Cloning, Molecular; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D016786:Eimeria tenella / Q000201:enzymology*; D006801:Humans; D008024:Ligands; D008322:Mammals; D008969:Molecular Sequence Data; D010442:Peptide Chain Initiation, Translational; D015800:Protozoan Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
chemical_list: D018076:DNA, Complementary; D016054:DNA, Protozoan; D008024:Ligands; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D017869:Cyclic GMP-Dependent Protein Kinases; C569718:PRKG1 protein, human