Inhibition of Eimeria tenella CDK‐Related Kinase 2: From Target Identification to Lead Compounds

Abstract

Apicomplexan parasites encompass several human-pathogenic as well as animal-pathogenic protozoans like Plasmodium falciparum, Toxoplasma gondii, and Eimeria tenella. E. tenella is the causative agent of coccidiosis a disease of chickens, which causes tremendous economic losses to the world poultry industry. Considerable increase of drug resistance makes it necessary to develop and pursue new therapeutic strategies. Cyclin-dependent kinases (CDKs) are key molecules in the regulation of the cell cycle and are therefore prominent target proteins in parasitic diseases. Bioinformatic analysis revealed four potential CDK-like proteins of which one -E. tenella CDKrelated kinase 2 (EtCRK2) -is already cloned, expressed and characterized. [1] Using the CDK specific inhibitor Flavopiridol in EtCRK2 enzyme assays and schizont maturation assays we could chemically validate CDK-like proteins as potential drug targets. An X-ray crystal structure of human CDK2 (HsCDK2) served as template to built protein models of EtCRK2 by comparative homology modeling. Structural differences in the ATP-binding site between EtCRK2 and HsCDK2 as well as chicken CDK3 have been addressed for the optimization of selective ATPcompetitive inhibitors. Virtual screening and "wet-bench" high throughput screening campaigns on large compound libraries resulted in an initial set of hit compounds. These compounds were further analyzed and characterized leading to a set of four promising lead compounds inhibiting EtCRK2.

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

“…There are a high number of anti-cancer drugs that interfere with cell cycle regulation, leading to the death of the rapidly dividing cells (Tamamori et al , 1998; Doerig et al , 2002; Monaco & Vallano, 2003; Malumbres & Barbacid, 2009). Therefore, we, like others, predict that the disruption of the parasite’s cell cycle will lead to its death (Engels et al , 2010; Waters & Geyer, 2003). …”

“…We were able to clone one of the identified E. tenella cyclins (EtCYCs) (EtCYC3a) and demonstrated that its protein product was able to activate EtCRK2, in a similar manner to that shown with the non-cyclin activator Xenopus laevis rapid inducer of G2/M progression in oocytes (XlRINGO) (Engels et al , 2010; Ferby et al , 1999). In a combined in vitro and in silico high-throughput screening approach, using real (3514 compounds) and virtual (approx.…”

“…E. tenella CDC2-related kinase 2 (EtCRK2) is by analogy assumed to play a similar key role in Eimeria (Kinnaird et al , 2004; Engels et al , 2010). It has been chemically validated using the synthetic flavone flavopiridole, a specific CDK inhibitor (Dai & Grant, 2003).…”

“…Concentrations below 80 nm show no inhibitory effects, and host cell toxicity is observed at concentrations above 600 nM. Therefore, E. tenella CDKs are considered to be chemically validated drug targets (Engels et al , 2010). …”

Enzyme Activity Assays for Protein Kinases: Strategies to Identify Active Substrates

“…Engels et al validated EtCRK2 as a drug target using the cyclin-dependent kinase-specific inhibitor flavopiridol, (IC 50 of 33 nM and Ki of 11 nM) which was similar to the human isoform (IC 50 36 nM, Ki 19 nM). They also identified four chemically diverse compounds with Ki values in the low micromolar range [68]. …”