In Vitro Inhibition of Pseudomonas aeruginosa Elastase by Metal-Chelating Peptide Derivatives

Abstract

Pseudomonas aeruginosa elastase is a zinc metalloendopeptidase, probably responsible for the tissue destruction observed during infections with this organism. The elastase of a virulent Pseudomonas aeruginosa strain (Habs serotype 1) was isolated and found to have a molecular weight of 35,000; it readily degraded elastin and cartilage proteoglycans. A series of amino acid and peptide derivatives containing the metal-chelating moieties hydroxamate, phosphoryl, or thiol were synthesized and tested as potential inhibitors of the enzyme. Inhibition constants ( K i s) for the compounds were determined with the chromophoric substrate furylacryloyl-glycyl- l -leucyl- l -alanine. The hydroxamic acid derivatives of benzyloxycarbonyl-glycine, benzyloxycarbonyl- l -leucine and benzyloxycarbonyl- l -phenylalanine had inhibition constants in the range of 11 to 28 μM. The 2-mercaptoacetyl derivatives of l -leucyl- d -phenylalanine and l -leucyl- l -phenylalanine had K i values of 34 and 1.5 μM, respectively, demonstrating the stereospecificity of the inhibition. The most potent inhibitors tested were 2- mercaptoacetyl- l -phenylalanyl- l -leucine and phosphoryl- l -leucyl- l -phenylala-nine ( K i = 0.2 μM). Similar compounds lacking the metal-chelating moiety were about 3 orders of magnitude poorer inhibitors. When the inhibition of the enzyme activity towards azocasein, elastin, or cartilage was examined, inhibitor concentrations approximately 50-fold higher than the respective K i s were required to obtain 60 to 90% inhibition. Virtually complete inhibition was achieved with these substrates at inhibitor concentrations 500-fold higher than the respective K i s (0.1 to 14 mM). Although, 2-mercaptoacetyl- l -phenylalanyl- l -leucine and phosphoryl- l -leucyl- l -phenylalanine exhibited the same affinity to the enzyme, the latter was inferior in inhibiting cartilage proteoglycan degradation. 2-Mercaptoacetyl- l -phenylalanyl- l -leucine represents a class of potent elastase inhibitors that might prove useful in the management of P. aeruginosa infections.