Abstract
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.
Highlights
The obligate intracellular apicomplexan parasites cause devastating diseases in humans and domestic animals
Using the nonradioactive time-resolved fluorescence energy transfer (TR-FRET) assay, we demonstrated the ability of EtCYC-like 3a (EtCYC3a) to activate Eimeria tenella CDC2-related kinase 2 (EtCRK2) as shown previously for XlRINGO
In order to identify potential E. tenella cyclin (EtCYC) we ran a number of BLAST database searches on the currently unfinished genomic dataset of E. tenella using a wide set of cyclins, including Plasmodium falciparum cyclin (PfCYC)1 (Le Roch et al, 2000), PfCYC2, PfCYC3 and PfCYC4 (Merckx et al, 2003) from P. falciparum, as query sequences in analogy to Engels et al (2010)
Summary
The obligate intracellular apicomplexan parasites cause devastating diseases in humans and domestic animals. Wellknown members of this phylum are Plasmodium falciparum, Toxoplasma gondii, Cryptosporidium parvum, Theileria. The most pathogenic species, E. tenella, provokes a haemorrhagic diarrhoea in young chickens, leading to a loss of weight and appetite, resorption problems, bacterial secondary infections and often to the bird’s death (Chauhan & Roy, 2007; Mehlhorn, 2008; Shirley et al, 2007). Coccidiosis causes tremendous economic losses, in excess of three billion US Dollars annually, to the world poultry industry (Dalloul & Lillehoj, 2006; Shirley et al, 2007). Prophylaxis using live-attenuated vaccines is efficient, it may have transitory adverse effects on chicken growth rate and is economically restrictive due to the high costs of these vaccines
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