Characterization of Plasmodium falciparum cGMP-dependent protein kinase (PfPKG): Antiparasitic activity of a PKG inhibitor

Abstract 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)-1 H 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 ( Pf PKG). Biochemical fractions enriched for a 98 kDa 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 Pf PKG 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 Pf PKG, as well as PKG enzymes from Eimeria spp. and T. gondii . We conclude that Pf PKG 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.