High-throughput screening with the Eimeria tenella CDC2-related kinase2/cyclin complex EtCRK2/EtCYC3a.
The poultry disease coccidiosis, caused by infection with Eimeria spp. apicomplexan parasites, is responsible for enormous economic losses to the global poultry industry. The rapid increase of resistance to therapeutic agents, as well as the expense of vaccination with live attenuated vaccines, requires the development of new effective treatments for coccidiosis. Because of their key regulatory function in the eukaryotic cell cycle, cyclin-dependent kinases (CDKs) are prominent drug targets. The Eimeria tenella CDC2-related kinase 2 (EtCRK2) is a validated drug target that can be activated in vitro by the CDK activator XlRINGO (Xenopus laevis rapid inducer of G2/M progression in oocytes). Bioinformatics analyses revealed four putative E. tenella cyclins (EtCYCs) that are closely related to cyclins found in the human apicomplexan parasite Plasmodium falciparum. EtCYC3a was cloned, expressed in Escherichia coli and purified in a complex with EtCRK2. Using the non-radioactive time-resolved fluorescence energy transfer (TR-FRET) assay, we demonstrated the ability of EtCYC3a to activate EtCRK2 as shown previously for XlRINGO. The EtCRK2/EtCYC3a complex was used for a combined in vitro and in silico high-throughput screening approach, which resulted in three lead structures, a naphthoquinone, an 8-hydroxyquinoline and a 2-pyrimidinyl-aminopiperidine-propane-2-ol. This constitutes a promising starting point for the subsequent lead optimization phase and the development of novel anticoccidial drugs.
Institute of Microbiology and Wine Research, Johannes-Gutenberg-Universität Mainz, Becherweg 15, 55099 Mainz, Germany.|Intervet Innovation GmbH, Zur Propstei, 55270 Schwabenheim, Germany.
María L Suárez
MLS
Fernández
Intervet Innovation GmbH, Zur Propstei, 55270 Schwabenheim, Germany.
Kristin K
KK
Engels
Intervet Innovation GmbH, Zur Propstei, 55270 Schwabenheim, Germany.
Frank
F
Bender
Intervet Innovation GmbH, Zur Propstei, 55270 Schwabenheim, Germany.
Michael
M
Gassel
Intervet Innovation GmbH, Zur Propstei, 55270 Schwabenheim, Germany.
Richard J
RJ
Marhöfer
Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, 120 University Place, Glasgow G12 8TA, UK.
Jeremy C
JC
Mottram
Interfaculty Institute of Biochemistry, Eberhard Karls University Tübingen, Hoppe-Seyler-Str. 4, 72076 Tübingen, Germany.|Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, 120 University Place, Glasgow G12 8TA, UK.|Intervet Innovation GmbH, Zur Propstei, 55270 Schwabenheim, Germany.
Paul M
PM
Selzer
D000818:Animals; D000981:Antiprotozoal Agents / Q000302:isolation & purification*; D016203:CDC2 Protein Kinase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D019295:Computational Biology / Q000379:methods; D016213:Cyclins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D016786:Eimeria tenella / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000302:isolation & purification; D057166:High-Throughput Screening Assays / Q000379:methods*; D010963:Plasmodium falciparum / Q000201:enzymology*; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism
D000981:Antiprotozoal Agents; D016213:Cyclins; D004791:Enzyme Inhibitors; D015800:Protozoan Proteins; D016203:CDC2 Protein Kinase