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DARPins as pan-reactivators of temperature-sensitive p53 cancer mutants
Philipp Münick1, Dimitrios-Ilias Balourdas2,3, Julianne S Funk4
1Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University, Frankfurt 60438, Germany.
Abstract:
The tumor suppressor p53 is the most frequently mutated protein in tumors and a target for drug development. More than 2000 cancer-associated p53 missense mutations have been reported, most of them located in the DNA-binding domain (DBD). Due to the low intrinsic thermostability of the latter, they often lead to unfolding at physiological temperature. Stabilizing the DBD with small molecules has been shown to be effective in reactivating the cavity-creating cancer mutant Y220C. Unfortunately, the majority of p53 mutants seem to lack druggable binding pockets for small molecules. Here we show that a designed ankyrin repeat protein (DARPin) that binds to the p53 DBD stabilizes temperature-sensitive (TS) p53 cancer mutants, thereby compensating for mutation-induced loss of stability. We determined high-resolution crystal structures of multiple DARPin-mutant p53 complexes, providing mechanistic insights into this mode of stabilization. Reporter gene assays across a comprehensive panel of cancer-associated mutants revealed reactivation of the majority of TS mutants, whereas DNA-contact mutants and those with local misfolding of the DNA-binding surface remained inactive, as expected. We demonstrate that this reactivation induces the transcription of canonical p53 target genes and elicits antiproliferative effects in cancer cell lines. A combination of this DARPin with an mRNA/lipid nanoparticle-based transfection approach may have the potential to reactivate most TS p53 mutants and resensitize cancer cells to chemotherapy.
Insights
A novel designed ankyrin repeat protein (DARPin) stabilizes temperature-sensitive (TS) p53 cancer mutants by binding to the DNA-binding domain (DBD). This approach reactivates mutant p53, inducing anti-cancer effects and potentially resensitizing cells to chemotherapy.
Area of Science:
- Oncology
- Molecular Biology
- Protein Engineering
Background:
- The p53 protein is a critical tumor suppressor frequently mutated in cancer.
- Most p53 mutations occur in the DNA-binding domain (DBD), often leading to instability at physiological temperatures.
- Targeting p53 mutants is a key strategy in cancer drug development, but many mutants lack suitable binding pockets for small molecules.
Purpose of the Study:
- To investigate the potential of a designed ankyrin repeat protein (DARPin) to stabilize temperature-sensitive (TS) p53 cancer mutants.
- To elucidate the mechanism of stabilization and assess the functional reactivation of mutant p53.
- To evaluate the anti-cancer effects of DARPin-mediated p53 reactivation.
Main Methods:
- High-resolution crystal structure determination of DARPin-mutant p53 complexes.
- Reporter gene assays across a panel of cancer-associated p53 mutants.
- Assessment of canonical p53 target gene transcription and antiproliferative effects in cancer cell lines.
Main Results:
- A DARPin effectively stabilized TS p53 mutants by binding to the DBD, compensating for mutation-induced instability.
- Structural analysis provided mechanistic insights into the stabilization process.
- Reactivation of the majority of TS mutants was observed, leading to transcription of p53 target genes and antiproliferative effects.
- DNA-contact mutants and those with local misfolding remained inactive, as expected.
Conclusions:
- DARPin-mediated stabilization is a viable strategy for reactivating a majority of TS p53 cancer mutants.
- This approach offers a potential therapeutic avenue for cancers with specific p53 mutations.
- Combining DARPins with advanced delivery systems like mRNA/lipid nanoparticles could enhance therapeutic potential.
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