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.
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Acknowledgements
The authors would like to thank the National Institutes of Health (GM54051) for support of this work. A.W.P. greatly appreciates an American Chemical Society Medicinal Chemistry Graduate Student Fellowship sponsored by Bristol-Myers Squibb.
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Patterson, A., Wood, W. & Ellman, J. Substrate activity screening (SAS): a general procedure for the preparation and screening of a fragment-based non-peptidic protease substrate library for inhibitor discovery. Nat Protoc 2, 424–433 (2007). https://doi.org/10.1038/nprot.2007.28
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DOI: https://doi.org/10.1038/nprot.2007.28
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