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.
Yawei Qi, Laura S. Itzhaki· Clinical Cancer Research· 0 citations
Membrane-embedded bacterial receptors are challenging to express and purify in soluble form, yet their isolated domains are essential tools for structural and ligand-discovery studies. Pseudomonas aeruginosa relies on the TonB-dependent heme receptor HasR for iron acquisition, a process central to its pathogenicity. Here, we report a robust strategy for the recombinant expression, purification, and biophysical characterisation of the two soluble HasR domains directly involved in heme uptake: the N-terminal plug and the Secretin/TonB short N-terminal domain. Each domain was expressed individually in E. coli and purified to homogeneity, adopting well-folded conformations as confirmed by circular dichroism, NMR spectroscopy, and mass spectrometry. We then engineered a fusion construct containing both domains and systematically evaluated multiple solubilisation tags. A GST-His dual-affinity strategy enabled efficient purification of the construct, whereas His-tag alone resulted in insoluble protein and HLT-tag fusions suffered from non-specific proteolysis. Biophysical analyses revealed that the Secretin/TonB short N-terminal domain remains stably folded within the fusion construct, while the N-terminal plug domain becomes partially disordered, a finding further supported by hydrogen/deuterium exchange mass spectrometry. Together, these results establish a generalizable workflow for producing soluble receptor domains from membrane proteins and provide validated HasR constructs suitable for downstream ligand-screening applications, including aptamer and nanobody discovery.
Federico Bosetto, Maria Zacharopoulou, M. Bycroft et al.· bioRxiv· 0 citations
Transcriptional dysregulation is a hallmark of cancer and is frequently driven by oncogenic alterations that rewire downstream gene-expression programmes. Although kinase inhibitors targeting upstream oncogenic drivers can produce clinical benefit, responses are often limited by acquired resistance and pathway adaptation. Targeting transcriptional dependencies therefore represents an alternative therapeutic strategy. The positive transcription elongation factor b complex, composed of CDK9 and Cyclin T1(CCNT1), is a central regulator of RNA polymerase II transcriptional elongation and supports the expression of genes required for tumour cell survival. However, conventional CDK9 inhibitors often lack sufficient selectivity and are associated with dose-limiting toxicities. Here, we report the rational design of a novel CCNT1-targeting binder and its application in biological proteolysis-targeting chimeras designed to selectively degrade Cyclin T1. The degrader platform combines E3 ubiquitin ligase recruitment with a dual-peptide CCNT1-binding module derived from the pTEFb-interacting partners AFF4 and HIV-TAT. This engineered recruitment strategy enabled robust and selective depletion of both tagged and endogenous CCNT1 in cancer cells. CCNT1 degradation was accompanied by destabilisation of its catalytic partner CDK9 and reduced phosphorylation of RNA polymerase II, consistent with suppression of transcriptional elongation. Functionally, CCNT1-targeted degradation produced marked anti-tumour effects in lung cancer models. Degrader-treated cells showed impaired proliferative capacity, reduced colony formation, cell-cycle disruption across multiple checkpoints, and induction of apoptosis. These findings demonstrate that selective degradation of CCNT1 can effectively collapse pTEFb-dependent transcriptional programmes and compromise tumour cell fitness. Together, our study establishes a novel CCNT1 binder-enabled degrader strategy as a translational approach to targeting transcriptional addiction in cancer. By moving beyond catalytic CDK9 inhibition and directly eliminating the Cyclin T1 scaffold, CCNT1-targeted degradation may offer a more selective and durable route for therapeutic intervention in transcriptionally dependent tumours.
Janice Wenzheng Neng, Laura Blenkarn, Laura S. Itzhaki, Catherine H. Wilson. Targeted degradation of cyclin T1 constrains transcription elongation and triggers tumour cell apoptosis [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 B045.
Janice Wenzheng Neng, Laura Blenkarn, Laura S. Itzhaki et al.· Clinical Cancer Research· 0 citations
