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Eukaryotic Cell, June 2002, p. 317-328, Vol. 1, No. 3
1535-9778/02/$04.00+0     DOI: 10.1128/EC.1.3.317-328.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Toxoplasma gondii Cyclic GMP-Dependent Kinase: Chemotherapeutic Targeting of an Essential Parasite Protein Kinase

Robert G. K. Donald,1* John Allocco,1 Suresh B. Singh,2 Bakela Nare,1 Scott P. Salowe,3 Judyann Wiltsie,3 and Paul A. Liberator1

Department of Human and Animal Infectious Disease Research,1 Department of Molecular Systems,2 Department of High Throughput Screening and Automation, Merck Research Laboratories, Merck and Co., Inc., Rahway New Jersey 070653

Received 14 December 2001/ Accepted 4 February 2002

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.

* Corresponding author. Mailing address: Merck Research Laboratories, P.O. Box 2000, R80Y-260, Rahway, NJ 07065-0900. Phone: (732) 594-0671. Fax: (732) 594-6708. E-mail: robert_donald{at}merck.com.

Eukaryotic Cell, June 2002, p. 317-328, Vol. 1, No. 3
1535-9778/02/$04.00+0     DOI: 10.1128/EC.1.3.317-328.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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