Identification of Lead Compounds Targeting the Cathepsin B-Like Enzyme of Eimeria tenella

Abstract

Cysteine peptidases have been implicated in the development and pathogenesis of Eimeria. We have identified a single-copy cathepsin B-like cysteine peptidase gene in the genome database of Eimeria tenella (EtCatB). Molecular modeling of the predicted protein suggested that it differs significantly from host enzymes and could be a good drug target. EtCatB was expressed and secreted as a soluble, active, glycosylated mature enzyme from Pichia pastoris. Biochemical characterization of the recombinant enzyme confirmed that it is cathepsin B-like. Screening of a focused library against the enzyme identified three inhibitors (a nitrile, a thiosemicarbazone, and an oxazolone) that can be used as leads for novel drug discovery against Eimeria. The oxazolone scaffold is a novel cysteine peptidase inhibitor; it may thus find widespread use.

Localization of Eimeripain, an <i>Eimeria tenella</i> Cathepsin B-Like Cysteine Protease, during Asexual and Sexual Intracellular Development in Chicken Ceca

“…By sequence analysis of RACE products, 1,536 bp of eimeripain was cloned as the complete coding region. All sequences were completely identical with those previously reported [8, 10]. This molecule also contained approximately 410 bp and 1,180 bp non-coding regions at the 5′ and 3′ ends, respectively, consisting of a 512 amino acid protein, with a signal peptide of 21 hydrophobic amino acids, a predicted molecular weight of 54.68 kDa and a pI of 5.53 (excluding signal peptide) using PeptideMass [14].…”

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 ].…”

Identification of Semicarbazones, Thiosemicarbazones and Triazine Nitriles as Inhibitors of Leishmania mexicana Cysteine Protease CPB

“…During hit verification (IC 50 determination from liquid compound stock) BtCatB was used in a counter assay to provide information on the selectivity profile of the inhibitors. We used BtCatB in the counter assay for practical reasons because in parallel to this study we ran a screening on Eimeria tenella Cathepsin B-like enzyme also using BtCatB in the counter screen [23]. …”

“…In Figure 4A the 4-methoxy phenyl portion of CP247129 is oriented toward the deep S2 subsite and the furanyl moiety occupies the shallow S1 subsite. The thiosemicarbazone scaffold was assumed to interact via a 1,2-polar addition of the catalytic C26 to the C=S group of CP247129 (Figure 3A) [23,35]. Thus, the resulting tetrahedral transition state was used as the initial conformation of the thiosemicarbazone scaffold for covalent docking.…”