title: Discovery of novel inhibitors of a disintegrin and metalloprotease 17 (ADAM17) using glycosylated and non-glycosylated substrates.
abstract: A disintegrin and metalloprotease (ADAM) proteases are implicated in multiple diseases, but no drugs based on ADAM inhibition exist. Most of the ADAM inhibitors developed to date feature zinc-binding moieties that target the active site zinc, which leads to a lack of selectivity and off-target toxicity. We hypothesized that secondary binding site (exosite) inhibitors should provide a viable alternative to active site inhibitors. Potential exosites in ADAM structures have been reported, but no studies describing substrate features necessary for exosite interactions exist. Analysis of ADAM cognate substrates revealed that glycosylation is often present in the vicinity of the scissile bond. To study whether glycosylation plays a role in modulating ADAM activity, a tumor necrosis factor α (TNFα) substrate with and without a glycan moiety attached was synthesized and characterized. Glycosylation enhanced ADAM8 and -17 activities and decreased ADAM10 activity. Metalloprotease (MMP) activity was unaffected by TNFα substrate glycosylation. High throughput screening assays were developed using glycosylated and non-glycosylated substrate, and positional scanning was conducted. A novel chemotype of ADAM17-selective probes was discovered from the TPIMS library (Houghten, R. A., Pinilla, C., Giulianotti, M. A., Appel, J. R., Dooley, C. T., Nefzi, A., Ostresh, J. M., Yu, Y., Maggiora, G. M., Medina-Franco, J. L., Brunner, D., and Schneider, J. (2008) Strategies for the use of mixture-based synthetic combinatorial libraries. Scaffold ranking, direct testing in vivo, and enhanced deconvolution by computational methods. J. Comb. Chem. 10, 3-19; Pinilla, C., Appel, J. R., Borràs, E., and Houghten, R. A. (2003) Advances in the use of synthetic combinatorial chemistry. Mixture-based libraries. Nat. Med. 9, 118-122) that preferentially inhibited glycosylated substrate hydrolysis and spared ADAM10, MMP-8, and MMP-14. Kinetic studies revealed that ADAM17 inhibition occurred via a non-zinc-binding mechanism. Thus, modulation of proteolysis via glycosylation may be used for identifying novel, potentially exosite binding compounds. The newly described ADAM17 inhibitors represent research tools to investigate the role of ADAM17 in the progression of various diseases.
authors.affiliation: Torrey Pines Institute for Molecular Studies, Port St. Lucie, Florida 34987, USA. dminond@tpims.org
authors.forename: Dmitriy
authors.initials: D
authors.lastname: Minond
authors.forename: Mare
authors.initials: M
authors.lastname: Cudic
authors.forename: Nina
authors.initials: N
authors.lastname: Bionda
authors.forename: Marc
authors.initials: M
authors.lastname: Giulianotti
authors.forename: Laura
authors.initials: L
authors.lastname: Maida
authors.forename: Richard A
authors.initials: RA
authors.lastname: Houghten
authors.forename: Gregg B
authors.initials: GB
authors.lastname: Fields
mesh_terms: D051722:ADAM Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000072197:ADAM10 Protein; D000072198:ADAM17 Protein; D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D004353:Drug Evaluation, Preclinical / Q000379:methods; D006031:Glycosylation; D006801:Humans; D006868:Hydrolysis; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D019151:Peptide Library; D011480:Protease Inhibitors / Q000737:chemistry*; D013379:Substrate Specificity; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D019151:Peptide Library; D011480:Protease Inhibitors; D014409:Tumor Necrosis Factor-alpha; D053829:Amyloid Precursor Protein Secretases; D051722:ADAM Proteins; C068490:ADAM8 protein, human; D000072197:ADAM10 Protein; C491236:ADAM10 protein, human; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human