title: Inhibition of the SR protein-phosphorylating CLK kinases of Plasmodium falciparum impairs blood stage replication and malaria transmission.
abstract: Cyclin-dependent kinase-like kinases (CLKs) are dual specificity protein kinases that phosphorylate Serine/Arginine-rich (SR) proteins involved in pre-mRNA processing. Four CLKs, termed PfCLK-1-4, can be identified in the human malaria parasite Plasmodium falciparum, which show homology with the yeast SR protein kinase Sky1p. The four PfCLKs are present in the nucleus and cytoplasm of the asexual blood stages and of gametocytes, sexual precursor cells crucial for malaria parasite transmission from humans to mosquitoes. We identified three plasmodial SR proteins, PfSRSF12, PfSFRS4 and PfSF-1, which are predominantly present in the nucleus of blood stage trophozoites, PfSRSF12 and PfSF-1 are further detectable in the nucleus of gametocytes. We found that recombinantly expressed SR proteins comprising the Arginine/Serine (RS)-rich domains were phosphorylated by the four PfCLKs in in vitro kinase assays, while a recombinant PfSF-1 peptide lacking the RS-rich domain was not phosphorylated. Since it was hitherto not possible to knock-out the pfclk genes by conventional gene disruption, we aimed at chemical knock-outs for phenotype analysis. We identified five human CLK inhibitors, belonging to the oxo-β-carbolines and aminopyrimidines, as well as the antiseptic chlorhexidine as PfCLK-targeting compounds. The six inhibitors block P. falciparum blood stage replication in the low micromolar to nanomolar range by preventing the trophozoite-to-schizont transformation. In addition, the inhibitors impair gametocyte maturation and gametogenesis in in vitro assays. The combined data show that the four PfCLKs are involved in phosphorylation of SR proteins with essential functions for the blood and sexual stages of the malaria parasite, thus pointing to the kinases as promising targets for antimalarial and transmission blocking drugs.
authors.affiliation: Research Center for Infectious Diseases, University of Würzburg, Würzburg, Germany; Institute of Molecular Biotechnology, RWTH Aachen University, Aachen, Germany.
authors.forename: Selina
authors.initials: S
authors.lastname: Kern
authors.affiliation: Research Center for Infectious Diseases, University of Würzburg, Würzburg, Germany.
authors.forename: Shruti
authors.initials: S
authors.lastname: Agarwal
authors.affiliation: Department of Pharmacy - Center for Drug Research, Ludwig-Maximillians University, Munich, Germany.
authors.forename: Kilian
authors.initials: K
authors.lastname: Huber
authors.affiliation: Department of Pharmacy - Center for Drug Research, Ludwig-Maximillians University, Munich, Germany.
authors.forename: André P
authors.initials: AP
authors.lastname: Gehring
authors.affiliation: Department of Pharmacy - Center for Drug Research, Ludwig-Maximillians University, Munich, Germany.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Strödke
authors.affiliation: Institute of Molecular Biotechnology, RWTH Aachen University, Aachen, Germany.
authors.forename: Christine C
authors.initials: CC
authors.lastname: Wirth
authors.affiliation: Research Center for Infectious Diseases, University of Würzburg, Würzburg, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Brügl
authors.affiliation: Institute of Molecular Biotechnology, RWTH Aachen University, Aachen, Germany.
authors.forename: Liliane Onambele
authors.initials: LO
authors.lastname: Abodo
authors.affiliation: Bioinformatics, Biocenter, University of Würzburg, Würzburg, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Dandekar
authors.affiliation: INSERM U609, Global Health Institute, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland; Department of Microbiology, Monash University, Clayton, Victoria, Australia.
authors.forename: Christian
authors.initials: C
authors.lastname: Doerig
authors.affiliation: Institute of Molecular Biotechnology, RWTH Aachen University, Aachen, Germany.
authors.forename: Rainer
authors.initials: R
authors.lastname: Fischer
authors.affiliation: Department of Cell Physiology and Pharmacology, MRC Toxicology Unit, University of Leicester, Leicester, United Kingdom.
authors.forename: Andrew B
authors.initials: AB
authors.lastname: Tobin
authors.affiliation: Department of Cell Physiology and Pharmacology, MRC Toxicology Unit, University of Leicester, Leicester, United Kingdom.
authors.forename: Mahmood M
authors.initials: MM
authors.lastname: Alam
authors.affiliation: Department of Pharmacy - Center for Drug Research, Ludwig-Maximillians University, Munich, Germany.
authors.forename: Franz
authors.initials: F
authors.lastname: Bracher
authors.affiliation: Institute of Molecular Biotechnology, RWTH Aachen University, Aachen, Germany.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Pradel
mesh_terms: D000818:Animals; D000962:Antimalarials / Q000494:pharmacology*; D018507:Gene Expression Regulation, Developmental; D017125:Genes, Protozoan; D006801:Humans; D016778:Malaria, Falciparum / Q000469:parasitology / Q000517:prevention & control* / Q000635:transmission; D010766:Phosphorylation; D010963:Plasmodium falciparum / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics; D040681:Structural Homology, Protein
chemical_list: D000962:Antimalarials; D047428:Protein Kinase Inhibitors; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C066780:Clk dual-specificity kinases; C490216:SKY1 protein, S cerevisiae; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases