title: Characterization of the catalytic properties of the membrane-anchored metalloproteinase ADAM9 in cell-based assays.
abstract: ADAM9 (A Disintegrin And Metalloprotease 9) is a membrane-anchored metalloproteinase that has been implicated in pathological retinal neovascularization and in tumor progression. ADAM9 has constitutive catalytic activity in both biochemical and cell-based assays and can cleave several membrane proteins, including epidermal growth factor and Ephrin receptor B4; yet little is currently known about the catalytic properties of ADAM9 and its post-translational regulation and inhibitor profile in cell-based assays. To address this question, we monitored processing of the membrane-anchored Ephrin receptor B4 (EphB4) by co-expressing ADAM9, with the catalytically inactive ADAM9 E > A mutant serving as a negative control. We found that ADAM9-dependent shedding of EphB4 was not stimulated by three commonly employed activators of ADAM-dependent ectodomain shedding: phorbol esters, pervanadate or calcium ionophores. With respect to the inhibitor profile, we found that ADAM9 was inhibited by the hydroxamate-based metalloprotease inhibitors marimastat, TAPI-2, BB94, GM6001 and GW280264X, and by 10 nM of the tissue inhibitor of metalloproteinases (TIMP)-3, but not by up to 20 nM of TIMP-1 or -2. Additionally, we screened a non-hydroxamate small-molecule library for novel ADAM9 inhibitors and identified four compounds that selectively inhibited ADAM9-dependent proteolysis over ADAM10- or ADAM17-dependent processing. Taken together, the present study provides new information about the molecular fingerprint of ADAM9 in cell-based assays by showing that it is not stimulated by strong activators of ectodomain shedding and by defining a characteristic inhibitor profile. The identification of novel non-hydroxamate inhibitors of ADAM9 could provide the basis for designing more selective compounds that block the contribution of ADAM9 to pathological neovascularization and cancer.
authors.affiliation: Arthritis and Tissue Degeneration Program, Hospital for Special Surgery at Weill Cornell Medicine, New York, NY 10021, U.S.A.
authors.forename: Thorsten
authors.initials: T
authors.lastname: Maretzky
authors.affiliation: Arthritis and Tissue Degeneration Program, Hospital for Special Surgery at Weill Cornell Medicine, New York, NY 10021, U.S.A.|Boston Children's Hospital, Boston, MA 02115, U.S.A.
authors.forename: Steven
authors.initials: S
authors.lastname: Swendeman
authors.affiliation: Arthritis and Tissue Degeneration Program, Hospital for Special Surgery at Weill Cornell Medicine, New York, NY 10021, U.S.A.
authors.forename: Elin
authors.initials: E
authors.lastname: Mogollon
authors.affiliation: Arthritis and Tissue Degeneration Program, Hospital for Special Surgery at Weill Cornell Medicine, New York, NY 10021, U.S.A.
authors.forename: Gisela
authors.initials: G
authors.lastname: Weskamp
authors.affiliation: Arthritis and Tissue Degeneration Program, Hospital for Special Surgery at Weill Cornell Medicine, New York, NY 10021, U.S.A.|Center for Life Sciences and Technologies, Bogazici University, Istanbul 34342, Turkey.
authors.forename: Umut
authors.initials: U
authors.lastname: Sahin
authors.affiliation: Department of Dermatology, University Hospital Schleswig-Holstein, Campus Kiel, Kiel 24105, Germany.
authors.forename: Karina
authors.initials: K
authors.lastname: Reiss
authors.affiliation: Arthritis and Tissue Degeneration Program, Hospital for Special Surgery at Weill Cornell Medicine, New York, NY 10021, U.S.A. blobelc@hss.edu.|Department of Physiology, Biophysics and Systems Biology, Weill Cornell Medicine, New York, NY, U.S.A.|Institute for Advanced Study, Technical University Munich, Garching 85748, Germany.
authors.forename: Carl P
authors.initials: CP
authors.lastname: Blobel
mesh_terms: D051722:ADAM Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D000818:Animals; D019556:COS Cells; D002384:Catalysis; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology*; D002522:Chlorocebus aethiops; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008565:Membrane Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D051379:Mice
chemical_list: D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D051722:ADAM Proteins; C098339:ADAM9 protein, human
keywords: ADAM9 (A Disintegrin And Metalloproteinase 9); EphB4; ectodomain shedding; molecular fingerprint