title: Development of a transgenic Plasmodium berghei line (Pb pfpkg) expressing the P. falciparum cGMP-dependent protein kinase, a novel antimalarial drug target.
abstract: With the inevitable selection of resistance to antimalarial drugs in treated populations, there is a need for new medicines to enter the clinic and new targets to progress through the drug discovery pipeline. In this study we set out to develop a transgenic rodent model for testing inhibitors of the Plasmodium falciparum cyclic GMP-dependent kinase in vivo. A model was needed that would allow us to investigate whether differences in amino acid sequence of this enzyme between species influences in vivo efficacy. Here we report the successful development of a transgenic P. berghei line in which the cyclic GMP-dependent protein kinase (PKG) was replaced by the P. falciparum orthologue. We demonstrate that the P. falciparum orthologue was able to functionally complement the endogenous P. berghei pkg gene throughout blood stage development and early sexual development. However, subsequent development in the mosquito was severely compromised. We show that this is due to a defect in the female lineage of the transgenic by using genetic crosses with both male and female deficient P. berghei lines. This defect could be due to expression of a female-specific target in the mosquito stages of P. berghei that cannot be phosphorylated by the P. falciparum kinase. Using a previously reported anti-coccidial inhibitor of the cyclic GMP-dependent protein kinase, we show no difference in in vivo efficacy between the transgenic and control P. berghei lines. This in vivo model will be useful for screening future generations of cyclic GMP-dependent protein kinase inhibitors and allowing us to overcome any species-specific differences in the enzyme primary sequence that would influence in vivo efficacy in the rodent model. The approach will also be applicable to in vivo testing of other antimalarial compounds where the target is known.
authors.affiliation: Centre for Genetics and Genomics, School of Life Sciences, Queens Medical Centre, University of Nottingham, Nottingham, United Kingdom.
authors.forename: Rita
authors.initials: R
authors.lastname: Tewari
authors.affiliation: Centre for Genetics and Genomics, School of Life Sciences, Queens Medical Centre, University of Nottingham, Nottingham, United Kingdom.
authors.forename: Eva-Maria
authors.initials: EM
authors.lastname: Patzewitz
authors.affiliation: Centre for Genetics and Genomics, School of Life Sciences, Queens Medical Centre, University of Nottingham, Nottingham, United Kingdom.
authors.forename: Benoit
authors.initials: B
authors.lastname: Poulin
authors.affiliation: Department of Pathogen Molecular Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London, United Kingdom.
authors.forename: Lindsay
authors.initials: L
authors.lastname: Stewart
authors.affiliation: Department of Pathogen Molecular Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, London, United Kingdom.
authors.forename: David A
authors.initials: DA
authors.lastname: Baker
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D000962:Antimalarials / Q000627:therapeutic use; D009033:Culicidae / Q000235:genetics / Q000469:parasitology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D016778:Malaria, Falciparum / Q000235:genetics* / Q000469:parasitology / Q000628:therapy; D058990:Molecular Targeted Therapy; D010962:Plasmodium berghei / Q000201:enzymology / Q000235:genetics* / Q000472:pathogenicity; D010963:Plasmodium falciparum / Q000201:enzymology / Q000235:genetics* / Q000472:pathogenicity
chemical_list: D000962:Antimalarials; D017869:Cyclic GMP-Dependent Protein Kinases