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J. Biol. Chem., Vol. 277, Issue 18, 15913-15922, May 3, 2002

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Purification and Molecular Characterization of cGMP-dependent Protein Kinase from Apicomplexan Parasites
A NOVEL CHEMOTHERAPEUTIC TARGET^*

Anne M. Gurnett^§¶, Paul A. Liberator^§, Paula M. Dulski, Scott P. Salowe, Robert G. K. Donald, Jennifer W. Anderson, Judyann Wiltsie, Carmen A. Diaz, Georgiana Harris^**, Ben Chang^**, Sandra J. Darkin-Rattray, Bakela Nare, Tami Crumley, Penny Sue Blum, Andrew S. Misura, Tamas Tamas, Mohinder K. Sardana, Jeffrey Yuan^§§, Tesfaye Biftu^¶¶, and Dennis M. Schmatz

From the Departments of  Human and Animal Infectious Disease Research,  High Throughput Screening and Automation, ^** Biochemistry, ^§§ Bioinformatics, and ^¶¶ Medicinal Chemistry, Merck Research Laboratories, Rahway, New Jersey 07065 and the  Department of Biological Chemistry, Merck Research Laboratories, West Point, Pennsylvania 19486

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 ~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. NH₂-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 IC₅₀ of <1 nM.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AF411961, AF413570, AF413571, AF465543, and AF465544.

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