Modulation of the Surface-Layer Protein of Clostridium difficile through Cwp84 Inhibition.
Cysteine protease Cwp84 is responsible for surface-layer processing in Clostridium difficile and was also shown to cleave several human extracellular matrix components in vitro. To enable the facile identification and characterization of Cwp84 inhibitors, we developed a fluorogenic 10-mer peptide based on the enzyme's natural substrate SlpA that is amenable for use in FRET-based high-throughput screening. The design of substrate-mimetic inhibitors led to epoxysuccinate 8c, which displayed an inactivation efficiency (kinact/KI) of (4.7 ± 0.3) × 10(4) M(-1) min(-1). Further evaluation of 8c demonstrated its ability to inhibit fibronectin cleavage and, more importantly, subvert surface-layer biogenesis in C. difficile.
Departments of Chemistry and Immunology and Microbial Science, The Skaggs Institute for Chemical Biology, and The Worm Institute of Research and Medicine, The Scripps Research Institute , 10550 North Torrey Pines Road, La Jolla, California 92037, United States.
Major D
MD
Gooyit
Departments of Chemistry and Immunology and Microbial Science, The Skaggs Institute for Chemical Biology, and The Worm Institute of Research and Medicine, The Scripps Research Institute , 10550 North Torrey Pines Road, La Jolla, California 92037, United States.
Kim D
KD
Janda
D016360:Clostridioides difficile / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004761:Enterocolitis, Pseudomembranous / Q000382:microbiology*; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry* / Q000494:pharmacology
D004791:Enzyme Inhibitors; D008562:Membrane Glycoproteins; D010455:Peptides; C055764:S-layer proteins; C524258:Cwp84 protein, Clostridium difficile; D003546:Cysteine Endopeptidases
Clostridium difficile; Cwp84; FRET assay; S-layer; substrate mimetics