The bacterial protein tetanus toxin (TeNt), which belongs to the family of zinc endopeptidases, cleaves synaptobrevin, an essential synaptic protein component of the neurotransmitter exocytosis apparatus, at a single peptide bond (Gln(76)-Phe(77)). This protease activity is a particularly attractive target for designing potent and selective synthetic inhibitors as a possible drug therapy for tetanus. beta-Aminothiols mimicking Gln(76) of synaptobrevin have been previously shown to inhibit the tetanus neurotoxin enzymatic activity in the 35-250 mu M range. These compounds have now been modified to interact with S' subsites of the TeNt active site, with the aim of increasing their inhibitory potencies. Combinatorial libraries of pseudotripeptides, containing an ethylene sulfonamide or an m-sulfonamidophenyl moiety as the P-1 side chain and natural amino acids in P-1' and P-2' positions, were synthesized. The best inhibitory activity was observed with Tyr and His as P-1' and P-2' components, respectively. This led to new inhibitors of TeNt with K-i values in the 3-4 mu M range. These molecules are the most potent inhibitors of TeNt described so far.