Affiliation:
1. Department of Human and Animal Infectious Disease Research
2. Department of Molecular Systems
3. Department of High Throughput Screening and Automation, Merck Research Laboratories, Merck and Co., Inc., Rahway New Jersey 07065
Abstract
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.
Publisher
American Society for Microbiology
Subject
Molecular Biology,General Medicine,Microbiology
Cited by
125 articles.
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