Substrate specificity and novel selective inhibitors of TNF-alpha converting enzyme (TACE) from two-dimensional substrate mapping.

We report a systematic analysis of the P1' and P2' substrate specificity of TNF-alpha converting enzyme (TACE) using a peptide library and a novel analytical method, and we use the substrate specificity information to design novel reverse hydroxamate inhibitors. Initial truncation studies, using the amino acid sequence around the cleavage site in precursor-TNF-alpha, showed that good turnover was obtained with the peptide DNP-LAQAVRSS-NH2. Based on this result, 1000 different peptide substrates of the form Biotin-LAQA-P1'-P2'-SSK(DNP)-NH2 were prepared, with 50 different natural and unnatural amino acids at P1' in combination with 20 different amino acids at P2'.