Purification and molecular characterization of cGMP-dependent protein kinase from Apicomplexan parasites. A novel chemotherapeutic target.
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.
Gurnett
Anne M
AM
Department of Human and Animal Infectious Disease Research, Merck Research Laboratories, Rahway, New Jersey 07065, USA. anne_gurnett@merck.com
Liberator
Paul A
PA
Dulski
Paula M
PM
Salowe
Scott P
SP
Donald
Robert G K
RG
Anderson
Jennifer W
JW
Wiltsie
Judyann
J
Diaz
Carmen A
CA
Harris
Georgiana
G
Chang
Ben
B
Darkin-Rattray
Sandra J
SJ
Nare
Bakela
B
Crumley
Tami
T
Blum
Penny Sue
PS
Misura
Andrew S
AS
Tamas
Tamas
T
Sardana
Mohinder K
MK
Yuan
Jeffrey
J
Biftu
Tesfaye
T
Schmatz
Dennis M
DM
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
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