Schistosomiasis, caused by a parasitic blood fluke of the genus Schistosoma, is a global health problem for which new chemotherapeutic options are needed. We explored the scaffold of gallinamide A, a natural peptidic metabolite of marine cyanobacteria that has previously been shown to inhibit cathepsin L-type proteases. We screened a library of 19 synthetic gallinamide A analogs and identified nanomolar inhibitors of the cathepsin B-type protease SmCB1, which is a drug target for the treatment of schistosomiasis mansoni. Against cultured S. mansoni schistosomula and adult worms, many of the gallinamides generated a range of deleterious phenotypic responses. Imaging with a fluorescent-activity-based probe derived from gallinamide A demonstrated that SmCB1 is the primary target for gallinamides in the parasite. Furthermore, we solved the high-resolution crystal structures of SmCB1 in complex with gallinamide A and its two analogs and describe the acrylamide covalent warhead and binding mode in the active site. Quantum chemical calculations evaluated the contribution of individual positions in the peptidomimetic scaffold to the inhibition of the target and demonstrated the importance of the P1' and P2 positions. Our study introduces gallinamides as a powerful chemotype that can be exploited for the development of novel antischistosomal chemotherapeutics.

• Keywords
• Schistosoma mansoni, acrylamide inhibitor, cathepsin B, cysteine protease, drug target, parasite,
• MeSH
• Cathepsin B * antagonists & inhibitors   metabolism   MeSH
• Crystallography, X-Ray MeSH
• Models, Molecular MeSH
• Schistosoma mansoni * enzymology   drug effects   MeSH
• Schistosomicides pharmacology   chemistry   MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Cathepsin B * MeSH
• Schistosomicides MeSH

The mature bovine cathepsin C (CC) molecule is composed of four identical monomers, each proteolytically processed into three chains. Five intrachain disulfides and three nonpaired cysteine residues per monomer were identified. Beside catalytic Cys234 in the active site, free-thiol Cys331 and Cys424 were characterized. Cys424 can be classified as inaccessible buried residue. Selective modification of Cys331 results in dissociation of native CC tetramer into dimers. The 3D homology-based model of the CC catalytic core suggests that Cys331 becomes exposed as the activation peptide is removed during procathepsin C activation. The model further shows that exposed Cys331 is surrounded by a surface hydrophobic cluster, unique to CC, forming a dimer-dimer interaction interface. Substrate/inhibitor recognition of the active site in the CC dimer differs significantly from that in the native tetramer. Taken together, a mechanism is proposed that assumes that the CC tetramer formation results in a site-specific occlusion of endopeptidase-like active site cleft of each CC monomeric unit. Thus, tetramerization provides for the structural basis of the dipeptidyl peptidase activity of CC through a substrate access-limiting mechanism different from those found in homologous monomeric exopeptidases cathepsin H and B. In conclusion, the mechanism of tetramer formation as well as specific posttranslational processing segregates CC in the family of papain proteases.

• MeSH
• Enzyme Activation MeSH
• Cysteine chemistry   MeSH
• Disulfides chemistry   MeSH
• Cathepsin C chemistry   isolation & purification   metabolism   MeSH
• Protein Conformation MeSH
• Lysine chemistry   MeSH
• Molecular Sequence Data MeSH
• Molecular Weight MeSH
• Peptide Fragments MeSH
• Protein Processing, Post-Translational MeSH
• Protein Folding MeSH
• Amino Acid Sequence MeSH
• Cattle MeSH
• Spleen enzymology   MeSH
• Chromatography, High Pressure Liquid MeSH
• Animals MeSH
• Check Tag
• Cattle MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cysteine MeSH
• Disulfides MeSH
• Cathepsin C MeSH
• Lysine MeSH
• Peptide Fragments MeSH