title: Design and Synthesis of Activity-Based Probes and Inhibitors for Bleomycin Hydrolase.
abstract: Bleomycin hydrolase (BLMH) is a neutral cysteine aminopeptidase that has been ascribed roles in many physiological and pathological processes, yet its primary biological function remains enigmatic. In this work, we describe the results of screening of a library of fluorogenic substrates to identify non-natural amino acids that are optimally recognized by BLMH. This screen identified several substrates with kcat/KM values that are substantially improved over the previously reported fluorogenic substrates for this enzyme. The substrate sequences were used to design activity-based probes that showed potent labeling of recombinant BLMH as well as endogenously expressed BLMH in cell extracts, and in intact cells. Importantly, we identify potent BLMH inhibitors that are able to fully inhibit endogenous BLMH activity in intact cells. These probes and inhibitors will be valuable new reagents to study BLMH function in cellular and animal models of human diseases where BLMH is likely to be involved.
authors.affiliation: Departments of Pathology and Microbiology and Immunology, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305, USA.
authors.forename: Wouter A
authors.initials: WA
authors.lastname: van der Linden
authors.affiliation: Departments of Pathology and Microbiology and Immunology, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305, USA.
authors.forename: Ehud
authors.initials: E
authors.lastname: Segal
authors.affiliation: Departments of Pathology and Microbiology and Immunology, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305, USA.
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Child
authors.affiliation: Division of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology, Wyb. Wyspianskiego 27, 50-370 Wroclaw, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Byzia
authors.affiliation: Division of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Technology, Wyb. Wyspianskiego 27, 50-370 Wroclaw, Poland.
authors.forename: Marcin
authors.initials: M
authors.lastname: Drąg
authors.affiliation: Departments of Pathology and Microbiology and Immunology, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, CA 94305, USA. Electronic address: mbogyo@stanford.edu.
authors.forename: Matthew
authors.initials: M
authors.lastname: Bogyo
mesh_terms: D000818:Animals; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical; D006801:Humans; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D015335:Molecular Probes / Q000138:chemical synthesis / Q000737:chemistry; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D015853:Cysteine Proteinase Inhibitors; D015335:Molecular Probes; D003546:Cysteine Endopeptidases; C031262:bleomycin hydrolase