Botulinum toxin (BoNT) metalloproteases and related proteases are the most selective proteases known. X-ray crystal structures suggest that the native enzymes exist in catalytically incompetent forms that must be activated by substrate binding. In order to characterize the postulated substrate-induced conformational changes, we synthesized a series of transition state analog inhibitors (TSI) in which the dipeptide cleavage site has been replaced by tetrahedral intermediate analogs within the minimal substrate peptide sequence. Reduced amide, alpha-hydroxyamide, alpha-thio-amide, and hydroxyethylamine analogs of -Gln-Phe- were incorporated via solid phase peptide synthesis into 35-mer analogs of the minimal peptide substrate sequence. The synthesis, characterization, and inhibition kinetics for four series of compounds against holotoxin BoNT/B is described. The alpha-thiol amide derivatives of the 35-mer substrate were found to inhibit BONT/B in the low micromolar range.
Copyright 2003 Wiley Periodicals, Inc.