E-64-d (ethyl (2S, 3S)-3-[(S)-3-methyl-1-(3-methylbutylcarbamoyl) butylcarbamoyl]oxirane-2-carboxylate), a membrane-permeant derivative of the thiol protease-specific inhibitor E-64, was found to arrest human epidermoid carcinoma A431 cells at mitotic metaphase. This effect was dose-dependent with a threshold of 20 micrograms/ml in chemically defined culture medium. Cell cycle analysis by flow cytometry showed that the relative proportion of the G2/M population increased 2.5-fold after treatment of the cells with E-64 (100 micrograms/ml) for 5 hr. In addition, time-lapse video analysis showed that E-64-treated cells remained at metaphase for an extended period after rounding-up, whereas untreated cells completed mitosis within 42.0 +/- 5.7 min. Some treated cells were able to complete mitosis, while others did not do so within the limits of our observation. As an approach to the molecular basis of this phenomenon, we have shown that several cellular proteins can be labeled by incubation of cells with radioactive E-64-d.

Shoji-Kasai, Y., Senshu, M., Iwashita, S., & Imahori, K. (1988). Thiol protease-specific inhibitor E-64 arrests human epidermoid carcinoma A431 cells at mitotic metaphase. Proceedings of the National Academy of Sciences, 85(1), 146–150.