Inhibition by toltrazuril of the recombinant cyclophilin ETCYP21 of Eimeria tenella

Abstract

Toltrazuril is used as a drug against the parasitic protozoan Eimeria tenella, the causative agent of chicken coccidiosis, but its molecular mode of action has remained obscure. In order to identify putative protein targets for toltrazuril in E. tenella, a 12mer peptide phage display library was screened against biotinylated toltrazuril. This resulted in a toltrazuril-binding clone expressing the surface peptide YNVREDIRPVMD, which fitted best with a cDNA encoding a hypothetical 20.5 kDa ABH-like cyclophilin in E. tenella. After cloning the corresponding gene from E. tenella, we recombinantly expressed the cyclophilin EtCyp21, which was enzymatically active with k cat / K m = 8 μM −1 s −1 as determined by peptidyl-prolyl- cis/ trans-isomerase (PPIase) assay and RNase T1 refolding assay. The common cyclophilin inhibitor cyclosporin A (CsA) inhibited PPIase activity (IC 50 = 6.3 nM) as well as RNase T1 refolding activity. In contrast to CsA, toltrazuril did not completely suppress the PPIase activity of EtCyp21 and its IC 50 (1.84 μM) was much higher. Toltrazuril did not affect activity of the CsA-inhibitable EtCyp19, another novel, recombinantly expressed cyclophilin of E. tenella, lacking the toltrazuril-binding 12mer sequence. Our data indicate that the recombinant cyclophilin EtCyp21 of E. tenella is, at least in vitro, a target for the anti-coccidial drug toltrazuril.