The inhibitory capacity of C-Npys (S-[3-nitro-2-pyridinesulenyl]) derivatives over thiol-containing serine proteases has never been tested. In the present work we used an extracellular serine-thiol protemase activity from the fungal pathogen Paracoccidio ides brasiliensis (PbST) to describe a potent inhibitory capacity of Bzl-C(Npys)KRLTL-NH2, and Bzl-MKRLTLC(Npys)-NH2. The assays were performed with PbST enriched upon affinity chromatography in a p-aminobenzamidine (pABA)-Sepharose column. Although PbST can cleave the fluorescence resonance energy transfer peptide Abz-MKRLTL-EDDnp between L-T, the C(Npys) derivatives were not substrates nor were they toxic in a cell detachment assay, allowing therapeutic use. The best inhibitor was Bzl-C(Npys)KRLTL-NH2 (K-i = 16 nM), suggesting that the peptide sequence promoted a favorable interaction, especially when C(Npys) was placed at a further position from the L-T bond, at the N-terminus. Inhibition was completely reverted with dithioerythritol, indicating that it was due to the reactivity of the C(Npys) moiety with a free SH- group. (c) 2007 Elsevier Inc. All rights reserved.