title: Substrate activity screening (SAS): a general procedure for the preparation and screening of a fragment-based non-peptidic protease substrate library for inhibitor discovery.
abstract: Substrate activity screening (SAS) is a fragment-based method for the rapid development of novel substrates and their conversion into non-peptidic inhibitors of Cys and Ser proteases. The method consists of three steps: (i) a library of N-acyl aminocoumarins with diverse, low-molecular-weight N-acyl groups is screened to identify protease substrates using a simple fluorescence-based assay; (ii) the identified N-acyl aminocoumarin substrates are optimized by rapid analog synthesis and evaluation; and (iii) the optimized substrates are converted into inhibitors by direct replacement of the aminocoumarin with known mechanism-based pharmacophores. This protocol describes a general procedure for the solid-phase synthesis of a library of N-acyl aminocoumarin substrates and the screening procedure to identify weak binding substrates.
authors.affiliation: Department of Chemistry, University of California, Berkeley, CA 94720, USA.
authors.forename: Andrew W
authors.initials: AW
authors.lastname: Patterson
authors.forename: Warren J L
authors.initials: WJ
authors.lastname: Wood
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Ellman
mesh_terms: D002851:Chromatography, High Pressure Liquid; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D003374:Coumarins / Q000737:chemistry* / Q000378:metabolism; D005449:Fluorenes; D015394:Molecular Structure; D011480:Protease Inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: C496789:9-fluorenylmethoxycarbonyl; D003374:Coumarins; D005449:Fluorenes; D011480:Protease Inhibitors; C051029:7-amino-4-methylcoumarin-3-acetic acid