Inhibition of Eimeria tenella CDK-related kinase 2: From target identification to lead compounds.

Apicomplexan parasites encompass several human- and animal-pathogenic protozoans such as Plasmodium falciparum, Toxoplasma gondii, and Eimeria tenella. E. tenella causes coccidiosis, a disease that afflicts chickens, leading to tremendous economic losses to the global poultry industry. The considerable increase in drug resistance makes it necessary to develop new therapeutic strategies against this parasite. Cyclin-dependent kinases (CDKs) are key molecules in cell-cycle regulation and are therefore prominent target proteins in parasitic diseases. Bioinformatics analysis revealed four potential CDK-like proteins, of which one-E. tenella CDK-related kinase 2 (EtCRK2)-has already been characterized by gene cloning and expression.1 By using the CDK-specific inhibitor flavopiridol in EtCRK2 enzyme assays and schizont maturation assays (SMA), we could chemically validate CDK-like proteins as potential drug targets. An X-ray crystal structure of human CDK2 (HsCDK2) served as a template to build protein models of EtCRK2 by comparative homology modeling. Structural differences in the ATP binding site between EtCRK2 and HsCDK2, as well as chicken CDK3, were addressed for the optimization of selective ATP-competitive 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 for development as EtCRK2 inhibitors.

The impact of tautomer forms on pharmacophore-based virtual screening.

In the field of in silico screening, many applications do not automatically consider possible tautomeric states of molecules. However, the detection of new compound candidates might rely on correct structural description, which is important for the perfect fit toward the biologically relevant interactions. In this paper, we present a new exhaustive tautomer enumeration approach implemented by means of the CACTVS software package. The approach contains a set of 21 predefined SMIRKS-based transforms and a powerful transformation engine that is capable of generating most tautomers described comprehensively in the literature or found in databases in the field of medicinal chemistry. User-defined tautomer rules applied to specific structural databases or scientific issues can be implemented easily and used instead of the predefined rules. In addition, we describe the impact of tautomer-enriched databases on pharmacophore screening approaches for human matrix metalloproteinase 8 as an example of a protein-based pharmacophore screening scenario and for human cyclin-dependent kinases as an example of a ligand-based pharmacophore screening approach. In both test cases, as a preprocessing step, we have used our new tautomer enumerator tool for the tautomer enrichment of the screening data sets and have used it as a postprocessing step to remove tautomeric duplicates from the results. We could demonstrate that the tautomer-enriched screening data sets show significant advantages compared to their non-enhanced counterparts. The discrimination between hits and nonhits was significantly better in the case of tautomer-enriched databases. Moreover, it has been proved that tautomer-enhanced databases will lead to a higher number of potential hits.

Highly efficient transport of carboxyfluorescein diacetate succinimidyl ester into COS7 cells using human papillomavirus-like particles.

Human papillomavirus virus-like particles (VLPs) have recently been used to deliver genes into mammalian cells in vitro and in vivo. Here, we investigated whether VLPs may serve as an efficient carrier of low molecular weight compounds (e.g. hormones, vitamins, peptides etc.) into cells. COS7 cells were incubated with recombinant HPV-16L1/L2 VLPs labelled with the fluorescence dye carboxyfluorescein diacetate succinimidyl ester. Using flow cytometry, we demonstrate that labelled VLPs can specifically bind to the cell surface followed by their complete internalisation. Our results indicate that VLPs are promising vehicles for highly efficient delivery of low molecular weight compounds into cells.