High-throughput screening with the Eimeria tenella CDC2-related kinase2/cyclin complex EtCRK2/EtCYC3a

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.

Review of triazine antiprotozoal drugs used in veterinary medicine

“…As triazines have significant anticoccidial effects on Eimeria spp., a pathogen of coccidiosis in birds, they are commonly used in fowls, such as chickens and turkeys. However, they have faced significant problems related to drug resistance in recent years (Conway, Mathis, Johnson, Schwartz, & Baldwin, ; Fernandez et al., ; Kreiner, Worliczek, Tichy, & Joachim, ; Wang et al., ). A number of Eimeria strains have developed resistance to most drugs, thereby rendering the anticoccidials ineffective and leading to clinical failure (Abbas, Iqbal, Blake, Khan, & Saleemi, ; Allen & Fetterer, ; Harfoush, Hegazy, Soliman, & Amer, ; Zhang, Wang, Ruan, & An, ).…”

Trypanothione Reductase: A Target Protein for a Combined In Vitro and In Silico Screening Approach

“…A typical target-based approach starts either with high throughput screening of large libraries of small molecules [ 17 ] or with in silico experiments like a virtual screening to create a focused data set containing in silico hits which are subsequently tested by in vitro assays [ 18 ], [ 19 ], reducing screening costs significantly. Although several crystal structures of TR are available, their applicability for common structure-based virtual screening campaigns is inappropriate compared to other druggable protein targets like proteases [ 20 ], [ 21 ], [ 22 ], [ 23 ] or kinases [ 24 ], [ 25 ], [ 26 ]. TR has a very wide and featureless active site with approximate dimensions of 15 x 15 x 20 Å ( Fig 2 ) [ 27 ], [ 28 ].…”

Protein Kinases as Suitable Targets for Combating Eimeria spp

“…While a putative Etcyc1 gene fragment was identified, the full gene sequence could not be recovered. The EtCRK2/EtCYC3a complex was active and comparable to that of the EtCRK2/XlRINGO complex [53]. While the full-length sequence was available for EtCYC3a, only a partial sequence for EtCYC3b could be retrieved.…”

“…IC 50 -values and K i -values denote activity data on the isolated protein target in the EtCRK2/EtCYC3a complex[53]. IC 50 -values and K i -values denote activity data on the isolated protein target in the EtCRK2/EtCYC3a complex[53].…”