Cell membrane composition drives selectivity and toxicity of designed cyclic helix-loop-helix peptides with cell penetrating and tumor suppressor properties

The tumor suppressor protein p53 is inactive in a large number of cancers, including some forms of sar-coma, breast cancer and leukemia, due to overexpression of its intrinsic inhibitors MDM2 and MDMX. Reactivation of p53 tumor suppressor activity, via disruption of interactions between MDM2/X and p53 in the cytosol, is a promising strategy to treat cancer. Peptides able to bind MDM2 and/or MDMX were shown to prevent MDM2/X:p53 interactions, but most possess low cell penetrability, low stability, and/or high toxicity to healthy cells. Recently, the designed peptide cHLH-p53-R was reported to possess high affinity for MDM2, resistance towards proteases, cell-penetrating properties, and toxicity towards cancer cells. This peptide uses a stable cyclic helix-loop-helix (cHLH) scaffold, which includes two helices con-nected with a Gly loop and cyclized to improve stability. In the current study, we were interested in exam-ining the cell selectivity of cHLH-p53-R, its cellular internalization and ability to reactivate the p53 path-way. We designed analogues of cHLH-p53-R and employed biochemical and biophysical methodologies using in vitro model membranes and cell-based assays to compare their structure, activity and mode-of-action. Our studies show that cHLH is an excellent scaffold to stabilize and constrain p53-mimetic peptides with helical conformation, and reveal that anticancer properties of cHLH-p53-R are mediated by its ability to selectively target, cross and disrupt cancer cell membranes, and not by activation of the p53 pathway. These findings highlight the importance of examining the mode-of-action of designed peptides to fully ex-ploit their potential to develop targeted therapies.