Characterization of Plasmodium falciparum cGMP-dependent protein kinase (PfPKG): antiparasitic activity of a PKG inhibitor.
Cyclic GMP-dependent protein kinase (PKG) has been biochemically and genetically validated in Toxoplasma gondii as a primary target responsible for the antiparasitic activity of the trisubstituted pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H pyrrol-3-yl] pyridine (Compound 1) [Biftu T, Feng D, Ponpipom M, et al. Synthesis and SAR of 2,3-diarylpyrrole inhibitors of parasite cGMP-dependent protein kinase as novel anticoccidial agents. Bioorg Med Chem Lett 2005;15:3296-301; Gurnett AM, Liberator PA, Dulski PM, et al. Purification and molecular characterization of cGMP-dependent protein kinase from Apicomplexan parasites. A novel chemotherapeutic target. J Biol Chem 2002;277:15913-22; Donald RGK, Allocco J, Singh SB, et al. Toxoplasma gondii cyclic GMP-dependent kinase: Chemotherapeutic targeting of an essential parasite protein kinase. Eukaryotic Cell 2002;1:317-28; Nare B, Allocco J, Liberator PA, Donald RGK. Evaluation of a cyclic GMP-dependent protein kinase inhibitor in treatment of murine Toxoplasmosis: Gamma interferon is required for efficacy. Antimicrob Agents Chemother 2002;46:300-7]. Compound 1 inhibits the growth of several related protozoan parasites of the subphylum Apicomplexa. Native PKG activity has been partially purified by cGMP-affinity and MonoQ ion exchange chromatography from Plasmodium falciparum (PfPKG). Biochemical fractions enriched for a 98kDa protein detected using anti-PKG antisera, contain cGMP-induced protein kinase activity that is sensitive to inhibition by Compound 1. To enable a more thorough characterization of PfPKG we expressed a synthetic cDNA incorporating T. gondii codon preference (Pf(Tg)PKG) in T. gondii parasites. The protein kinase activity of purified recombinant Pf(Tg)PKG is stimulated by cGMP, with significant cooperativity as demonstrated by a Hill coefficient of 2. Both substrate preference and inhibition of Pf(Tg)PKG kinase activity by Compound 1 are similar to that seen with native PfPKG, as well as PKG enzymes from Eimeria spp. and T. gondii. We conclude that PfPKG has biochemical and pharmacological properties that are similar to previously characterized apicomplexan PKG enzymes. Compound 1 is active against blood cell stages of P. falciparum cultured in vitro. In a Plasmodium berghei mouse model of infection, Compound 1 delays the onset of parasitemia but does not cure the parasite infection.
Diaz
Carmen A
CA
Department of Human and Animal Infectious Research, Merck Research Laboratories, Merck & Co. Inc., P.O. Box 2000, Rahway, NJ 07065-0900, USA. carmen_diaz@merck.com
Allocco
John
J
Powles
Mary Ann
MA
Yeung
Lai
L
Donald
Robert G K
RG
Anderson
Jennifer W
JW
Liberator
Paul A
PA
D000818:Animals; D000977:Antiparasitic Agents / Q000737:chemistry / Q000494:pharmacology* / Q000627:therapeutic use; D001483:Base Sequence / Q000235:genetics; D002478:Cells, Cultured; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D004195:Disease Models, Animal; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology* / Q000627:therapeutic use; D004912:Erythrocytes / Q000469:parasitology; D016650:Fluorescein-5-isothiocyanate; D015870:Gene Expression; D008018:Life Cycle Stages / Q000502:physiology; D016778:Malaria, Falciparum / Q000188:drug therapy; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000187:drug effects / Q000201:enzymology* / Q000254:growth & development; D016415:Sequence Alignment; D014162:Transfection / Q000379:methods
D000977:Antiparasitic Agents; D004791:Enzyme Inhibitors; D017869:Cyclic GMP-Dependent Protein Kinases; D016650:Fluorescein-5-isothiocyanate