Design of sensitive fluorogenic substrates for human cathepsin D

Abstract

Cathepsin D is a lysosomal aspartic proteinase that has been implicated in several pathological processes such as breast cancer and Alzheimer's disease. We designed and synthesized a number of quenched fluorogenic substrates with P2 variations in the series AcEE(EDANS)KPIXFFRLGK(DA-BCYL)E-NH2, where X = cysteine, methylcysteine, ethylcysteine, fert-butylcysteine, carboxymethylcysteine, methionine, valine or isoleucine. Most of the fluorogenic substrates exhibited greater k cat IK m ratios than the best cathepsin D substrates described so far. Differences in kinetic constants, which were rationalized using structure-based modeling, might make certain substrates useful for particular applications, such as active site titrations or initial velocity determination using a fluorescent plate reader.

Phorbol Ester Activation of a Proteolytic Cascade Capable of Activating Latent Transforming Growth Factor-β

“…Cathepsin L specific activities are reported as fmol of AMC produced per min per 10 3 cells. Cathepsin D was assayed by a published procedure (33) that monitors the cleavage of Ac-GE(Edans)-Z-EVNLDAEF-Z-K(Dabcyl)-G-NH 2 , a highly selective substrate for cathepsin D (33,34). This peptide corresponds to a mutant of the amyloid B protein precursor having an Asn-Leu substitution at amino acids 670 -671 and contains Dabcyl and Edans groups, which act as internal quencher and fluorophore, respectively.…”

Human cathepsin D.

“…Cathepsin D shows a considerably lower affinity as compared to other proteins ( Table 3). The activity of this protease is also determined using micromolecular synthetic substrates [140][141][142][143].…”

Capillary electrophoretic determination of cathepsin D activity using Oregon Green-labeled hemoglobin

“…3). Thus this CE method has limits of detection similar to the most sensitive fluorometric analysis methods [29,30].…”

“…These substrates have K m values in the low mM range and their K cat /K m ratios suggest they would be good substrates for determination of activity in a wide variety of applications. The lower limit of detection using this type of substrate corresponds to approximately 1 nM when assayed with purified enzyme [30].…”

“…Solomayer et al [19] have suggested that both total amount and activity of cathepsin D may be of importance in the metastasis process. Most recently, fluorogenic peptide substrates were developed for assessing the activity of cathepsin D and other important aspartyl proteinases such as HIV protease [29,30]. These substrates are based on fluorophore-labeled peptides (7±12 mers) in which the fluorescence is substantially quenched until the internal PhePhe bond is cleaved by the enzyme.…”