cb9b00593_si_005.mp4 (1.03 MB)
Cell Membrane Composition Drives Selectivity and Toxicity of Designed Cyclic Helix–Loop–Helix Peptides with Cell Penetrating and Tumor Suppressor Properties
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posted on 2019-08-21, 13:57 authored by Grégoire
J.-B. Philippe, Diana Gaspar, Caibin Sheng, Yen-Hua Huang, Aurélie
H. Benfield, Nicholas D. Condon, Joachim Weidmann, Nicole Lawrence, Alexander Löwer, Miguel A. R. B. Castanho, David J. Craik, Sónia Troeira HenriquesThe
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.