Protein grafting of p53TAD onto a leucine zipper scaffold generates a potent HDM dual inhibitor

Abstract

Reactivation of the p53 pathway by a potential therapeutic antagonist, which inhibits HDM2 and HDMX, is an attractive strategy for drug development in oncology. Developing blockers towards conserved hydrophobic pockets of both HDMs has mainly focused on small synthetic compounds; however, this approach has proved challenging. Here we describe an approach to generate a potent HDM dual inhibitor, p53LZ2, by rational protein grafting of the p53 transactivation domain onto a homodimeric leucine zipper. p53LZ2 shows tight binding affinity to both HDMs compared with wild-type p53 in vitro. X-ray crystallographic, comparative modelling and small-angle X-ray scattering studies of p53LZ2-HDM complexes show butterfly-shaped structures. A cell-permeable TAT-p53LZ2 effectively inhibits the cancer cell growth in wild-type but not mutant p53 by arresting cell cycle and inducing apoptosis in vitro. Thus, p53LZ2, designed by rational grafting, shows a potential therapeutic approach against cancer.

De novocoiled-coil peptides as scaffolds for disrupting protein–protein interactions

“…In a study by others, 34 p53 hotspot residues have been grafted onto the natural GCN4 leucine zipper and shown to inhibit the hDM2/p53 interaction, including, upon addition of cell-penetrating functionality to the grafted peptide, in cellular models. 34 Using such a naturally derived coiled coil can carry risks. For instance, GCN4 variants show a variety of coiled-coil oligomer states.…”

“… 13 – 15 Considerable work in this area has led to the elaboration of several generic modalities for inhibition, 16 including the use of mini 17 and designed proteins, 18 – 20 stapled peptides 21 – 27 and foldamers, 28 , 29 all of which mimic the topology of the helix; the development of proteomimetics 30 – 32 that mimic the topography of a helix; and protein grafting. 33 , 34 Such constrained peptides, which exploit the functionally optimised specificity and selectivity of natural peptide motifs, are attractive from a recognition perspective as pre-organising such motifs in a recognition competent conformation may enhance affinity; 35 and the constraint may bring benefits such as improved stability to proteolysis 36 and cellular uptake, 37 which can be limiting with linear-peptide-based drugs.…”

“…Others have reported a similar system based on the natural homodimeric leucine zipper, GCN4, from yeast. 34 Here we expand on this theme, employing de novo designed coiled coils over which we have considerable control in directing oligomer state, partner specificity and stability through rational peptide design. 42 , 43 …”

Development of a novel immunoassay to detect interactions with the transactivation domain of p53: application to screening of new drugs

“…Approximately half of these patients bear wild-type p53 in tumor cells, but its function is impaired by negative regulators through degradation or inhibition 6 . Among these negative regulation motifs, binding of the transactivation domain (TAD) of p53, thus blocking its transcriptional activity, is crucial 7 , 8 .…”