Irreversible inhibition of the bacterial cysteine protease-transpeptidase sortase (SrtA) by substrate-derived affinity labels

We report on the first synthesis, kinetic evaluation and application of novel substrate-derived inhibitors against the Staphylococcus aureus cysteine protease-transpeptidase, sortase (staphylococcal surface protein sorting A, SrtA). The peptidyl-diazomethane and peptidyl-chloromethane analogues, Cbz (benzyloxycarbonyl)-Leu-Pro-Ala-Thr-CHN₂ (I) and Cbz-Leu-Pro-Ala-Thr-CH₂Cl (II) respectively were found to act as time-dependent irreversible inhibitors of recombinant sortase (SrtA_ΔN). The peptidyl-chloromethane analogue (II) was the most powerful with an inhibitor specificity constant (k_i/K_i) of 5.3×10⁴M^-1·min^-1, approx. 2-fold greater than that determined for the peptidyl-diazomethane (I). Additionally, using Western-blot analysis, we have been able to demonstrate that a biotinylated version of the peptidyl-diazomethane analogue, biotin-Ahx (aminohexanoyl)-Leu-Pro-Ala-Thr-CHN₂ (III), can be used as an affinity label to detect the presence of wild-type SrtA in crude cell lysates prepared from S. aureus.