Abstract A049: Rational grafting of p53TAD onto CTPR scaffolds generates potent p53-MDM2 antagonists Yawei Qi1, Albert Perez-Riba1, Laura S. Itzhaki11Department of Pharmacology, University of Cambridge, Cambridge, UK

Abstract

Pharmacological reactivation of the p53 pathway by disrupting the p53–MDM2 interaction remains an attractive strategy in cancer therapeutics. Although small-molecule MDM2 antagonists have shown clinical promise, their efficacy can be constrained by dose-limiting on-target toxicities arising from systemic p53 activation, incomplete inhibition of the MDM2 paralog MDMX, and pharmacokinetic or resistance issues. In contrast, p53 transactivation domain (p53TAD) mimetic peptides provide a direct means to recapitulate the native hot-spot interactions at the MDM2 binding cleft, but their translational potential is often limited by poor serum stability, rapid clearance, and low cellular uptake. These challenges motivate the development of stable, protein-based mimetics that retain peptide-like recognition while improving biophysical robustness. Here, we report a rational protein-engineering strategy to generate potent p53–MDM2 antagonists by grafting the α-helical p53TAD binding motif onto a consensus tetratricopeptide repeat (CTPR) scaffold. We designed a panel of p53-CTPR variants that present the p53TAD motif from the N terminus, C terminus, or both termini of the modular CTPR framework. Biophysical studies show that CTPR robustly tolerates α-helical motif grafting without compromising overall folding or thermodynamic stability. Importantly, the engineered p53-CTPR variants bind MDM2 with low-nanomolar affinity, comparable to the native p53TAD peptide. Next, we will evaluate the capacity of these p53–CTPR variants to inhibit the p53–MDM2 interaction across a panel of cancer cell lines, induce downstream p53 pathway activation, and assess cellular delivery of the constructs. Together, these results establish CTPR proteins as highly engineerable scaffolds for peptide-motif grafting and expand the toolkit of protein-based antagonists for targeting oncogenic protein–protein interactions, potentially opening up new therapeutic avenues. Yawei Qi, Laura S. Itzhaki. Rational grafting of p53TAD onto CTPR scaffolds generates potent p53-MDM2 antagonists Yawei Qi1, Albert Perez-Riba1, Laura S. Itzhaki11Department of Pharmacology, University of Cambridge, Cambridge, UK [abstract]. In: Proceedings of AACR Drug Discovery and Development (AACR D3) Conference; 2026 Jul 21-24; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(14_Suppl):Abstract nr A049.