Highly efficient and versatile synthesis of libraries of constrained beta-strand mimetics.

The general approach of using a bicyclic template to produce inhibitors of the protease superfamily of enzymes has been investigated. The Diels Alder cycloaddition reaction on solid support has been found to be highly efficient for the synthesis of libraries of compounds that mimic the beta-strand secondary structure of proteins. Several potent and selective inhibitors of proteases have been discovered.

Mechanistic studies on the inactivation of papain by epoxysuccinyl inhibitors.

Analogs of the epoxysuccinyl peptide cysteine proteinase inhibitor, EP-475 (2a), in which the free carboxylate has been replaced by hydroxamic acid, amide, methyl ketone, hydroxyl, and ethyl ester functionalities, have been synthesized. Individual rate constants of inhibition of papain were determined for these inhibitors. The results show that a carbonyl-containing functionality is necessary for good activity. The pH dependence of the inhibition of papain was determined for a nonionizable EP-475 (2a) analog; inhibition was found to depend on two acidic ionizations (pKas of 3.93 and 4.09) of papain. Implications for the mechanism of action of epoxysuccinyl peptides with papain are discussed.

Molecular cloning and expression of human alveolar macrophage cathepsin S, an elastinolytic cysteine protease.

Human alveolar macrophages (HAM) express an elastase activity of acidic pH optimum inhibitable by cysteine protease inhibitors. Recent studies indicate that the only known eukaryotic elastinolytic cysteine protease, cathepsin L, cannot completely account for this activity. In order to search for additional cysteine proteases with elastinolytic activity, low degeneracy oligonucleotide primers based on regions of strong homology among the known cysteine proteases were used to screen reverse-transcribed HAM RNA for cysteine proteases by the polymerase chain reaction. Among the cDNA sequences generated was a 493-base pair product highly homologous to bovine cathepsin S. Screening of a HAM cDNA eukaryotic expression library with this cDNA yielded a 1.7-kilobase full-length cDNA highly homologous to bovine cathepsin S (approximately 85% identical). This cDNA predicts a 331-amino acid preprocathepsin. Expression of this cDNA in COS cells revealed the active enzyme to be a single chain 28-kDa protease, as judged by active site labeling with a novel iodinated analogue of N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucylamido-(4-gua nido)butane (E-64). The recombinant enzyme was found to be elastinolytic toward 3H-labeled elastin (bovine ligamentum nuchae) at pH 5.5 but with 25% of this activity retained at pH 7.0. Labeling of HAM with the active site probe revealed these cells express a 28-kDa cysteine protease, and Northern blot analysis revealed the presence of a approximately 1.7-kilobase cathepsin S mRNA. These data establish that human macrophages express at least two cysteine proteases with elastinolytic activity. The relatively broad pH range of human cathepsin S activity suggests this enzyme may contribute to the contact-dependent elastase activity of live human alveolar macrophages.