Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
High-throughput screening with the Eimeria tenella CDC2-related kinase2/cyclin complex EtCRK2/EtCYC3a.
Fernández MLS
,
Engels KK
,
Bender F
,
Gassel M
,
Marhöfer RJ
,
Mottram JC
,
Selzer PM
.
???displayArticle.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.
Fig. 1. . Bioinformatic identification of cyclin-like proteins of E. tenella. (a) Multiple sequence alignment of the potential cyclin-like E. tenella proteins (EtCYC1, EtCYC3a, EtCYC3b and EtCYC4). For EtCYC1 and EtCYC3b, only incomplete sequences could be identified. Blue-shaded residues are conserved among the four sequences, yellow-shaded residues among any three of the four sequences, grey-shaded residues among any two only. (b) Comparison of known cyclin-like sequences of P. falciparum and E. tenella. The potential cyclin-like proteins are homologous to the known cyclin-like proteins of P. falciparum. The characteristic cyclin box sequence motif is conserved among the apicomplexan cyclin-like protein sequences and is coloured blue.
Fig. 2. . Comparison of the activity and inhibition of EtCRK2–His activated either by MBP–EtCYC3a or by MBP–XlRINGO. (a) Both activator proteins were able to activate EtCRK2 in a similar manner. Values are depicted as mean±sd EtCRK2 activity values from at least four independent experiments (n≥4). (b) Percentage activity of the two protein–activator complexes in the presence of increasing concentrations of 10-Z-hymenialdisine. Eleven inhibitor concentrations from 0.02 to 200 µM were tested in the TR-FRET assay. Points represent mean values±sd from at least three independent experiments (n≥3). The IC50 values of the inhibitor for EtCRK2/XlRINGO and EtCRK2/EtCYC3a were calculated to be 0.44±0.08 and 0.93±0.40 µM, respectively.
Fig. 3. . SDS-PAGE of the co-purification of the EtCRK2–His/MPB–EtCYC3a complex. Lane 1, amylase affinity chromatography, protein complex eluate fraction; lane 2, size exclusion chromatography, eluate fraction above 100 kDa.
Fig. 4. . Filtering steps during in vitro screening and in silico hit enrichment. Three lead compounds, each being the most active representative of their respective cluster of structurally related compounds, were selected.
Fig. 5. . Top ranked docking solution for BES124764. BES124764 is docked into the predicted ATP binding pocket of EtCRK2. Arene–H interactions to Gln84 and Ile10 and the arene–arene interaction to Phe79 are shown as dashed orange lines.
Al-Sha'er,
Discovery of novel CDK1 inhibitors by combining pharmacophore modeling, QSAR analysis and in silico screening followed by in vitro bioassay.
2010, Pubmed
Al-Sha'er,
Discovery of novel CDK1 inhibitors by combining pharmacophore modeling, QSAR analysis and in silico screening followed by in vitro bioassay.
2010,
Pubmed
Aziz,
Plumbagin, a medicinal plant-derived naphthoquinone, is a novel inhibitor of the growth and invasion of hormone-refractory prostate cancer.
2008,
Pubmed
Bruno,
Different effects of staurosporine, an inhibitor of protein kinases, on the cell cycle and chromatin structure of normal and leukemic lymphocytes.
1992,
Pubmed
Chapman,
Biochemical, genetic and applied aspects of drug resistance in Eimeria parasites of the fowl.
1997,
Pubmed
Dai,
Cyclin-dependent kinase inhibitors.
2003,
Pubmed
Dalloul,
Poultry coccidiosis: recent advancements in control measures and vaccine development.
2006,
Pubmed
Doerig,
Protein kinases as drug targets in parasitic protozoa.
2002,
Pubmed
Doerig,
The cell cycle in protozoan parasites.
2000,
Pubmed
Engels,
Inhibition of Eimeria tenella CDK-related kinase 2: From target identification to lead compounds.
2010,
Pubmed
Ferby,
A novel p34(cdc2)-binding and activating protein that is necessary and sufficient to trigger G(2)/M progression in Xenopus oocytes.
1999,
Pubmed
,
Xenbase
Hofmann,
Improved techniques for the in vitro cultivation of Eimeria tenella in primary chick kidney cells.
1990,
Pubmed
Jeffrey,
Mechanism of CDK activation revealed by the structure of a cyclinA-CDK2 complex.
1995,
Pubmed
Karaiskou,
Differential regulation of Cdc2 and Cdk2 by RINGO and cyclins.
2001,
Pubmed
,
Xenbase
Kim,
Discovery of piperidinyl aminopyrimidine derivatives as IKK-2 inhibitors.
2011,
Pubmed
Kinnaird,
EtCRK2, a cyclin-dependent kinase gene expressed during the sexual and asexual phases of the Eimeria tenella life cycle.
2004,
Pubmed
Le Roch,
Activation of a Plasmodium falciparum cdc2-related kinase by heterologous p25 and cyclin H. Functional characterization of a P. falciparum cyclin homologue.
2000,
Pubmed
Malumbres,
Cell cycle, CDKs and cancer: a changing paradigm.
2009,
Pubmed
Merckx,
Identification and initial characterization of three novel cyclin-related proteins of the human malaria parasite Plasmodium falciparum.
2003,
Pubmed
Monaco,
Cyclin-dependent kinase inhibitors: cancer killers to neuronal guardians.
2003,
Pubmed
Morgan,
Cyclin-dependent kinases: engines, clocks, and microprocessors.
1997,
Pubmed
Morrison,
Evolution of the Apicomplexa: where are we now?
2009,
Pubmed
Nairn,
A novel cyclin provides a link between dopamine and RNA processing.
2001,
Pubmed
Nebreda,
CDK activation by non-cyclin proteins.
2006,
Pubmed
,
Xenbase
Olashaw,
Paradigms of growth control: relation to Cdk activation.
2002,
Pubmed
Russo,
Structural basis of cyclin-dependent kinase activation by phosphorylation.
1996,
Pubmed
Schang,
Five years of progress on cyclin-dependent kinases and other cellular proteins as potential targets for antiviral drugs.
2006,
Pubmed
Schulman,
Substrate recruitment to cyclin-dependent kinase 2 by a multipurpose docking site on cyclin A.
1998,
Pubmed
Shirley,
Live attenuated vaccines against avian coccidiosis: Success with precocious and egg-adapted lines of Eimeria.
1997,
Pubmed
Shirley,
The genome of Eimeria spp., with special reference to Eimeria tenella--a coccidium from the chicken.
2000,
Pubmed
Shirley,
The biology of avian Eimeria with an emphasis on their control by vaccination.
2005,
Pubmed
Shirley,
Challenges in the successful control of the avian coccidia.
2007,
Pubmed
Sielecki,
Cyclin-dependent kinase inhibitors: useful targets in cell cycle regulation.
2000,
Pubmed
Tamamori,
Essential roles for G1 cyclin-dependent kinase activity in development of cardiomyocyte hypertrophy.
1998,
Pubmed
Wallach,
Field application of a subunit vaccine against an enteric protozoan disease.
2008,
Pubmed
Waters,
Cyclin-dependent protein kinases as therapeutic drug targets for antimalarial drug development.
2003,
Pubmed
Williams,
The efficacy and economic benefits of Paracox, a live attenuated anticoccidial vaccine, in commercial trials with standard broiler chickens in the United Kingdom.
1999,
Pubmed
