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  • Substrate activity screening (SAS): a general procedure for the preparation and screening of a fragment-based non-peptidic protease substrate library for inhibitor discovery.

Substrate activity screening (SAS): a general procedure for the preparation and screening of a fragment-based non-peptidic protease substrate library for inhibitor discovery.

Nature protocols (2007-04-05)
Andrew W Patterson, Warren J L Wood, Jonathan A Ellman
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
7-Amino-4-methyl-3-coumarinylacetic acid, BioReagent, suitable for fluorescence, ~90% (HPLC)
Sigma-Aldrich
2,4,6-Trimethylpyridine, ReagentPlus®, 99%
Sigma-Aldrich
2,6-Dichlorobenzoyl chloride, 99%