Abstract
BackgroundRedox cycling compounds have been reported to cause false positive inhibition of proteases in drug discovery studies. This kind of false positives can lead to unusually high hit rates in high-throughput screening campaigns and require further analysis to distinguish true from false positive hits. Such follow-up studies are both time and resource consuming.Methods and FindingsIn this study we show that 5-aminoquinoline-8-ol is a time-dependent inactivator of cathepsin B with a kinact/KI of 36.7±13.6 M−1s−1 using enzyme kinetics. 5-Aminoquinoline-8-ol inhibited cathepsins H, L and B in the same concentration range, implying a non-specific mechanism of inhibition. Further analogues, 4-aminonaphthalene-1-ol and 4-aminophenol, also displayed time-dependent inhibition of cathepsin B with kinact/KI values of 406.4±10.8 and 36.5±1.3 M−1s−1. No inactivation occurred in the absence of either the amino or the hydroxyl group, suggesting that the 4-aminophenol moiety is a prerequisite for enzyme inactivation. Induction of redox oxygen species (ROS) by 4-aminophenols in various redox environments was determined by the fluorescent probe 2′,7′-dichlorodihydrofluorescein diacetate. Addition of catalase to the assay buffer significantly abrogated the ROS signal, indicating that H2O2 is a component of the ROS induced by 4-aminophenols. Furthermore, using mass spectrometry, active site probe DCG-04 and isoelectric focusing we show that redox inactivation of cysteine cathepsins by 5-aminoquinoline-8-ol is active site directed and leads to the formation of sulfinic acid.ConclusionsIn this study we report that compounds containing the 4-aminophenol moiety inactivate cysteine cathepsins through a redox-based mechanism and are thus likely to cause false positive hits in the screening assays for cysteine proteases.
Highlights
Cathepsins are lysosomal cysteine proteases belonging to the papain-subfamily C1A of the clan CA of cysteine proteases [1]
In this study we report that compounds containing the 4-aminophenol moiety inactivate cysteine cathepsins through a redox-based mechanism and are likely to cause false positive hits in the screening assays for cysteine proteases
One of the best studied examples is the causative role of cathepsin B in malignant diseases where it was shown to be involved in tumor formation, growth and invasion, as well as to participate in angiogenesis [2]
Summary
Cathepsins are lysosomal cysteine proteases belonging to the papain-subfamily C1A of the clan CA of cysteine proteases [1]. The group comprises 11 cathepsins (cathepsins B, C, F, H, K, L, O, S, V, W and X) which act predominantly as endopeptidases and are for the most part located intracellularly in endolysosomal vesicles [2]. For this reason it was long believed that their primary function was protein turnover within lysosomes [3]. Redox cycling compounds have been reported to cause false positive inhibition of proteases in drug discovery studies This kind of false positives can lead to unusually high hit rates in high-throughput screening campaigns and require further analysis to distinguish true from false positive hits. Such follow-up studies are both time and resource consuming
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