No abstract

The transcription factor p53 has a tumor suppressor role in leading damaged cells to apoptosis. Its activity is regulated/inhibited in healthy cells by the proteins MDM2 and MDMX. Overexpression of MDM2 and/or MDMX in cancer cells inactivates p53, facilitating tumor development. A 12-mer dual inhibitor peptide (pDI) was previously reported to be able to target and inhibit MDMX:p53 and MDM2:p53 interactions with nanomolar potency in vitro. With the aim of improving its cellular inhibitory activity, we produced a series of constrained pDI analogs featuring lactam staples that stabilize the bioactive helical conformation and fused them with a cell-penetrating peptide to increase cytosol delivery. We compared pDI and its analogs on their inhibitory potency, toxicity, and ability to enter cancer cells. Overall, the results show that these analogs keep their nanomolar affinity for MDM2 and MDMX and are highly active against cancer cells. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 853-863, 2016.

The tumor suppressor protein p53 is inactive in a large number of cancers, including some forms of sarcoma, 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 toward proteases, cell-penetrating properties, and toxicity toward cancer cells. This peptide uses a stable cyclic helix−loop−helix (cHLH) scaffold, which includes two helices connected with a Gly loop and cyclized to improve stability. In the current study, we were interested in examining the cell selectivity of cHLH-p53-R, its cellular internalization, and ability to reactivate the p53 pathway. 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 exploit their potential to develop targeted therapies.