Inhibitor profiling of the Pseudomonas aeruginosa virulence factor LasB using N-alpha mercaptoamide template-based inhibitors

Comparison of the binding specificity of two bacterial metalloproteases, LasB of Pseudomonas aeruginosa and ZapA of Proteus mirabilis, using N-alpha mercaptoamide template-based inhibitor analogues

“…Table 1 also reveals that in the case of LasB, halogenated inhibitor side chains had a positive effect in inhibitor binding when used in the P2 0 position, in agreement with the preference for bulky hydrophobic groups in the S2 0 binding pocket of LasB reported previously [22]. In this position, the halogenated phenylalanine surpasses the parent compound SH-CH 2 The K i values obtained for the non-aromatic CHA in P1 0 suggest that aromaticity is important for optimal inhibitor binding.…”

“…Each inhibitor was investigated over a concentration range between 1 lM and 50 lM in order to determine accurate kinetic parameters (K i ). Previous observations have revealed that these inhibitors do not fit the standard linear transformation required for determination of K i via Michaelis-Menton kinetics [22,23], therefore K i values were ME-(X)-Tyr ME-Trp-(X) 5). An example of a typical progress curve for the inhibition of protease-mediated substrate hydrolysis is shown in Fig.…”

“…Both metalloproteases exhibited a preference for large aromatic residues in the P1 0 position of the dipeptide inhibitor, and aliphatic residues in P2 0 . However, for ZapA, the ability of the aromatic residue in P1 0 to hydrogen bond appeared important in inhibitor binding, whereas this was not important for the inhibition of LasB [22][23][24].…”

“…Inhibitors were synthesized using a methodology previously described [22,27], using a CEM Liberty microwave assisted peptide synthesizer and standard Fmoc solid-phase peptide synthesis protocols on Rink amide resin. Amino acid analogues were incorporated under standard coupling conditions.…”

“…During the screening of the N-alpha mercaptoamide dipeptide inhibitors against LasB by Cathcart et al [22,24], it was noted that lead compounds possessed non-polar side chains that were often bulky and aromatic. It was proposed that derivatization of these compounds with functionalities lending a high degree of non-polar character, such as halogens, binding to the active site of LasB might be improved.…”