Abstract
We report here that a number of commonly used small peptide caspase inhibitors consisting of a caspase recognition sequence linked to chloromethylketone, fluoromethylketone or aldehyde reactive group efficiently inhibit other cysteine proteases than caspases. The in vitro studies included cathepsins B, H, L, S, K, F, V, X and C, papain and legumain. Z-DEVD-cmk was shown to be the preferred irreversible inhibitor of most of the cathepsins in vitro, followed by Z-DEVD-fmk, Ac-YVAD-cmk, Z-YVAD-fmk and Z-VAD-fmk. Inactivation of legumain by all the inhibitors investigated was moderate, whereas cathepsins H and C were poorly inhibited or not inhibited at all. Inhibition by aldehydes was not very potent. All the three fluoromethylketones efficiently inhibited cathepsins in Jurkat and human embryonic kidney 293 cells at concentrations of 100 μM. Furthermore, they completely inhibited cathepsins B and X activity in tissue extracts at concentrations as low as 1 μM. These results suggest that data based on the use of these inhibitors should be taken with caution and that other proteases may be implicated in the processes previously ascribed solely to caspases.
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Abbreviations
- Ac:
-
acetyl
- AFC:
-
7-amido-4-trifluoromethylcoumarin
- AMC:
-
7-amido-4-methylcoumarin
- Bz:
-
benzoyl
- CA-074:
-
n-propyl-epoxysuccinyl-Ile-Pro
- Dnp:
-
2,4-dinitrophenyl
- E-64:
-
L-trans-epoxysuccinyl-Leu-amido-(4-guanidino) butane
- E-64d:
-
L-trans-epoxysuccinyl(OEt) -Leu-3-methylbutylamide
- β NA:
-
β -naphthylamide
- pNA:
-
p-nitroanilide
- Z:
-
benzyloxycarbonyl
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Acknowledgements
This work was supported by the Ministry of School, Science and Sports of the Republic of Slovenia (BT, DT, VT), by the Interuniversitaire Attractiepolen V, the Fonds voor Wetenschappelijk Onderzoek Vlaanderen (Grant 3G.0006.01 to PV), the Bijzonder Onderzoeksfonds (PV), and an EC-RTD Grant QLRT-1999-00739 (PV), by Sandler Program in Basic Sciences (MB) and by the NIH Grant AR 46182 (DB).
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Rozman-Pungerčar, J., Kopitar-Jerala, N., Bogyo, M. et al. Inhibition of papain-like cysteine proteases and legumain by caspase-specific inhibitors: when reaction mechanism is more important than specificity. Cell Death Differ 10, 881–888 (2003). https://doi.org/10.1038/sj.cdd.4401247
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DOI: https://doi.org/10.1038/sj.cdd.4401247
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