Application of a fluorogenic substrate in the assay of proteolytic activity and in the discovery of a potent inhibitor of Candida albicans aspartic proteinase

A fluorescent method for monitoring the activity of the secreted Candida carboxyl (aspartic) proteinase (EC 3.4.23.6) was developed using a fluorogenic substrate based on resonance energy transfer. The fluorescent assay was used to monitor proteinase production, purification, and inhibition. The K m for the fluorogenic substrate, 4-(4-dimethylaminophenylazo)benzoyl-γ-aminobutyryl-Ile-His-Pro-Phe-His-Leu-Val-Ile-His - Thr - [5 - (2 - aminoethyl)amino]naphthalene - 1 -sulfonic acid, was found to be 4.3 μ m at the optimum pH of 4.5. Reaction products were separated by reversephase high-performance liquid chromatography and identified by amino acid analysis or by 252 Cf plasma desorption mass spectrometry. Cleavage of the fluorogenic substrate was between the histidine-threonine residues, releasing the fluorescent product, threonine-[5-(2-aminoethyl)amino]naphthalene-1-sulfonic acid. Proteolytic activity was expressed as nanomoles of fluorescent product released at 22°C/60 min, pH 4.5, and the release of 0.9 nmol product was equivalent to one hemoglobin proteolytic unit (O.D. A700 increase of 0.100) produced at 37°C/60 min, pH 3.5. The aspartic proteinase inhibitor pepstatin had an IC 50 of 27 n m when tested in a dose-response study with the purified enzyme. The apparent K i for pepstatis was 2.9 n m . Several synthetic inhibitors of the enzymes were identified with IC 50 's in the nanomolar range. The most potent compound, A70450, was characterized as a fast, tight-binding inhibitor having an IC 50 of 1.3 n m and apparent K i of 0.17 n m .