Onconase cytotoxicity relies on the distribution of its positive charge

Abstract

Onconase® (ONC) is a member of the ribonuclease A superfamily that is toxic to cancer cells in vitro and in vivo. ONC is now in Phase IIIb clinical trials for the treatment of malignant mesothelioma. Internalization of ONC to the cytosol of cancer cells is essential for its cytotoxic activity, despite the apparent absence of a cell‐surface receptor protein. Endocytosis and cytotoxicity do, however, appear to correlate with the net positive charge of ribonucleases. To dissect the contribution made by the endogenous arginine and lysine residues of ONC to its cytotoxicity, 22 variants were created in which cationic residues were replaced with alanine. Variants with the same net charge (+2 to +5) as well as equivalent catalytic activity and conformational stability were found to exhibit large (> 10‐fold) differences in toxicity for the cells of a human leukemia line. In addition, a more cationic ONC variant could be either much more or much less cytotoxic than a less cationic variant, again depending on the distribution of its cationic residues. The endocytosis of variants with widely divergent cytotoxic activity was quantified by flow cytometry using a small‐molecule fluorogenic label, and was found to vary by twofold or less. This small difference in endocytosis did not account for the large difference in cytotoxicity, implicating the distribution of cationic residues as being critical for lipid‐bilayer translocation subsequent to endocytosis. This finding has fundamental implications for understanding the interaction of ribonucleases and other proteins with mammalian cells.

Pullulan-based nanoparticles as carriers for transmucosal protein delivery

“…In this context, positively charged materials have been frequently found to be more cytotoxic than neutral or negatively charged counterparts, because positive charges provide a means for stronger interaction with cell surfaces, in many cases associated with internalisation of the material (Bhattacharjee et al, 2010;Ilinskaya et al, 2002;Turcotte et al, 2009). Curiously, in this case, although chitosan is also positively charged, it does not evidence any cytotoxic effect, cell viability remaining around 100% irrespective of the used dose or time of exposure to the polymer solution.…”

Cytosolic Delivery of Proteins by Bioreversible Esterification

“…For example, mammalian ribonucleases are capable of cytosolic entry that is mediated by clusters of positively charged residues. 11 Cellular uptake can also be enhanced by exogenous hydrophobic moieties. 12 For example, noncovalent complexation with pyrene butyrate enables the cytosolic delivery of a green fluorescent protein (GFP) conjugate to a cationic CPP.…”

Boronic Acid for the Traceless Delivery of Proteins into Cells

“…The number of labels in the B-TML–GFP conjugate did not decrease after a month of storage in PBS (Figure S2), consistent with the stability observed for other TML conjugates. 38–40 Labeling was, however, “bioreversible”. Incubation with a lysate from Chinese hamster ovary (CHO) K1 cells removed all of the labels from B-TML–GFP (Figure 1B).…”