Inhibitors directed to binding domains in neutrophil elastase

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Abstract
Human neutrophil elastase (HNE) can be inhibited by unsaturated fatty acids, including oleic acid [Ashe, B. M., and Zimmerman, M. (1977) Biochem. Biophys. Res. Commun. 75, 194-199; Cook, L., and Ternai, B. (1988) Biol. Chem. Hoppe-Seyler 369, 627-631], but it not affected by saturated fatty acids. We have shown that the interaction of oleic acid with HNE can be characterized by two apparent inhibitory modes; a high-affinity mode (Ki = 48 .+-. 3 nM), resulting in partial noncompetitive inhibition (87% residual activity), and a competitive inhibitory mode of lower affinity (Ki = 16 .+-. 1 .mu.M). Binding of oleate in the high-affinity mode induces a blue shift in the endogenous fluorescence arising from the tryptophan residues in HNE. The shift is maximal in the presence of 1 .mu.M oleate; higher concentrations of fatty acid have no further effect on the fluorescence spectrum. The negatively charged fluorescent ester of oleic acid and hydroxypyrenetrisulfonate (HPTSoleate) interacts with HNE at an apparent single site (Ki = 44 .+-. 3 nM), resulting in competitive inhibition. A blue shift in the emission maximum of the pyrene fluorescence at 410 nm and a decrease in the ratio of the intensities of the maximum at 388 and 410 nm indicate that upon binding to HNE the environment of the pyrene ring in HPTSoleate becomes more hydrophobic. In order to probe further the roles of nonpolar and electrostatic interactions in binding of negatively charged hydrophobic inhibitors to HNE, the enzyme was modified with the arginine-specific reagent pyreneglyoxal (PYG). Under conditions in which only two arginines are modified by PYG, the catalytic activity of HNE is eliminated. Upon reaction with HNE, the two maxima in the emission spectrum of PYG are both blue-shifted, and the ratio of the intensities around 378 and 395 nm is decreased, indicating increased hydrophobicity of the environment surrounding the pyrene ring. An additional blue shift of both maxima and a further change in intensity ratio are seen in the presence of oleic acid, but only at high concentrations (200 .mu.M), suggesting that the apparent high-affinity mode of binding for oleate may no longer be accessible after reaction of HNE with PYG. These results suggest a role for at least one arginine residue in a hydrophobic environment in regulating substrate binding and catalysis by HNE. Inhibitors which interact with both this positively charged center and the neighboring hydrophobic environment should be especially potent and selectively for HNE.