*2.2.1. Intracellular pathway*

cytotoxic mechanism, which differs from that of RNases that exert its action on the cell's cytosol, ND‐RNases are very interesting antitumor agents that can cope with the complexity of cancer cell phenotype, and their multiple effects allow anticipating synergism with many currently clinically used antitumor agents. In *in vivo* studies with animal models, the ND‐RNases have shown very low toxicity (it has not been possible to calculate the maximum tolerated doses (MTD) but the maximum feasible dose (MFD) which is of 80 mg/kg) (Castro et al., results not

**2.2. Engineered RNases that might saturate the intracellular RI and/or gain selectivity**

The efficiency of cell internalization is another important determinant of the cytotoxicity of the RNases because an RI‐sensitive RNase is still a potential danger provided that enough enzyme molecules reach the cell cytosol. The most basic strategy to increase the internalization of RNases is their cationization by chemical or genetic modification, that is, to make the RNases even more basic to increase their interaction with the anionic membranes of tumor cells. As stated above, this fact may also increase their selectivity for cancer cells [133]. Several examples of this approach can be found in the literature. The chemical modification of the carbonyl groups of RC‐RNase with a water‐soluble carbodiimide in the presence of nucleophiles or the amidation with ethylenediamine, 2‐aminoethanol, taurine, or ethylenediamine of HP‐RNase and RNase A increases their cytotoxicity [134–136]. The preparation of RNase A and noncy‐ totoxic cross‐linked dimers of RNase A, both covalently linked to polyspermine to increase their basicity, slightly increased their cytotoxicity [137]. In general, the higher cationic variants were more efficiently internalized into the cells. However, in some cases, the chemical modifications seriously compromised the ribonucleolytic activity of the modified enzymes [134, 135] and generated heterogeneous products difficult to use as antitumor drugs. RC‐RNase and RNase Sa variants were engineered substituting acidic residues by Asn, Gln, or Arg [138] or by positively charged residues [139, 140], respectively, showing antitumor activity and enhanced internalization. Gly38Lys‐BS‐RNase that bears an enforced cluster of positive charges at the N‐terminal surface also presented an increased cytotoxicity relative to its parental RNase and a higher membrane interaction capability [141]. Fuchs et al. [142] replaced two residues of a cytotoxic variant of RNase A to create a patch of Arg residues on its surface that rendered a threefold increase in cytotoxicity and added a protein translocation domain (nona‐arginine) to a previously cytotoxic RNase variant that increased their cytotoxicity [142, 143]. However, the same group has proposed that the internalization of pancreatic RNases by cationization can be counterbalanced by an increased affinity for the anionic RI in the cytosol [92]. Like for RI evasion, one has to be very cautious in the design of these variants in order to not counterbalance the increased internalization by the loss of other important characteristics responsible for the RNase cytotoxicity. In the same line but with a different approach, co‐ treating cells with a cationic 2 poly(amidoamine) dendrimer [144] increase the cytotoxicity of the RNase probably by increasing its translocation from the endosomes without affecting its ribonucleolytic activity or conformational stability observed upon cationization of some

published).

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RNases.

The engineering of RNases to amend the intracellular pathway, apart from the ND‐RNases described in Section 2.1.3 that have a different goal, is the less explored approach. This is likely because the productive intracellular route followed by cytotoxic RNases is the part of the intoxication process that looks like more as a black box. Nevertheless, there are some examples to deliver RNases to cellular compartments other than the cytosol. The KDEL consensus sequence, which drives proteins to the endoplasmic reticulum (ER), was added to the C‐ terminal end of BS‐RNase resulting in a loss of cytotoxicity compared to the parental enzyme [65] indicating that this compartment is not an essential intracellular station for the arrival of BS‐RNase to the cell cytosol. In addition, it has been tried to decrease the lysosomal degradation of one of the cytotoxic Evade™ RNases, the Gly88Arg variant, by introducing the change Lys7Arg in the Lys‐Phe‐Glu‐Arg‐Gln sequence that targets proteins to the lysosomes. In this case, Lys7Arg/Gly88Arg RNase A is nearly tenfold more cytotoxic than Gly88Arg RNase A variant but has more than tenfold less affinity for RI [90]. Once again, the changes introduced to enhance one of the aspects of RNase cytotoxicity affect other important features of this process precluding a clear conclusion.
