Periodic Reporting for period 1 - p53-REACT (Dynamics of p53 mutant reactivation and the anti-carcinogenic action of engineered resveratrol analogues)
Reporting period: 2022-09-01 to 2024-08-31
The p53-REACT project (“named after p53 REACTivation”) tackled a critical challenge in cancer therapy by targeting the mutated p53 protein, implicated in over half of all cancer cases worldwide. While chemotherapy and radiotherapy are common treatments, they often fail in many patients, highlighting the need to understand tumor resistance. Known as the “guardian of the genome,” p53 plays a crucial role in protecting DNA from cancer-inducing mutations. When p53 loses its function, it can form harmful aggregates that contribute to tumor growth and treatment resistance. These p53 clusters share traits with prion diseases and are increasingly linked to cancer progression
The project aimed to develop strategies to inhibit the aggregation of p53. By studying p53’s structure and function, the focus was on restoring its normal activity—a process called reactivation—to advance cancer treatments. Some mutated p53 forms interact with healthy p53 proteins, blocking their function, while others acquire entirely new harmful properties Addressing these diverse dysfunctions is challenging, particularly due to the difficulty of breaking down mature aggregates.
To address these complexities, p53-REACT has identified small molecules, particularly modified structures of resveratrol, an antioxidant polyphenol found in grapes and red wine, that shows potential to interact with p53 and inhibit cancer development. Pre-selected resveratrol analogues were investigated for their ability to enhance the stability of p53 and restore its normal function.
A key aspect of the project was the biosynthesis of resveratrol analogues. Using metabolic engineering, a platform was developed to engineer bacterial strains capable of cyclically producing these variants. The obtained cells served as factories for producing and diversifying target chemicals. In a final controlled study, structural biology and thermodynamics were applied to analyze the interactions between the validated resveratrol analogues and p53 family proteins. The modified chemical groups in resveratrol acted as conformational effectors, influencing p53 by altering the energy landscape and aggregation process based on the size, position, and oxidation properties of the substituents.
The research provided a high-resolution understanding of how resveratrol analogues impact p53 in vitro, offering promising tools to sustainably restore p53 function and combat cancer treatment resistance. This advancement will enable more effective cancer diagnosis and therapeutics, enhancing the visibility of research outcomes to society.
The project aimed to develop strategies to inhibit the aggregation of p53. By studying p53’s structure and function, the focus was on restoring its normal activity—a process called reactivation—to advance cancer treatments. Some mutated p53 forms interact with healthy p53 proteins, blocking their function, while others acquire entirely new harmful properties Addressing these diverse dysfunctions is challenging, particularly due to the difficulty of breaking down mature aggregates.
To address these complexities, p53-REACT has identified small molecules, particularly modified structures of resveratrol, an antioxidant polyphenol found in grapes and red wine, that shows potential to interact with p53 and inhibit cancer development. Pre-selected resveratrol analogues were investigated for their ability to enhance the stability of p53 and restore its normal function.
A key aspect of the project was the biosynthesis of resveratrol analogues. Using metabolic engineering, a platform was developed to engineer bacterial strains capable of cyclically producing these variants. The obtained cells served as factories for producing and diversifying target chemicals. In a final controlled study, structural biology and thermodynamics were applied to analyze the interactions between the validated resveratrol analogues and p53 family proteins. The modified chemical groups in resveratrol acted as conformational effectors, influencing p53 by altering the energy landscape and aggregation process based on the size, position, and oxidation properties of the substituents.
The research provided a high-resolution understanding of how resveratrol analogues impact p53 in vitro, offering promising tools to sustainably restore p53 function and combat cancer treatment resistance. This advancement will enable more effective cancer diagnosis and therapeutics, enhancing the visibility of research outcomes to society.
In the implemented project, a platform was developed to produce and test resveratrol as a promising therapeutic, with progress achieved by partners.
The work initially focused on obtaining the DNA Binding Domain (DBD) of the p53 constructs, a hotspot of the protein, followed by the biosynthesis of resveratrol compounds for subsequent interaction assays and analysis.
Four p53DBD constructs, the wild-type p53, its paralogs p63 and p73, and the mutant M237I, were heterologously synthesized. A sequential purification methodology was developed, increasing protein production and characterization efficiency by 20% compared to the group’s previous workflows. The process included gene subcloning, mutagenesis, protein expression, isolation, validation, and concentration measurements, using advanced biophysical techniques for characterization of all batches. Additionally, isotopically labeled proteins were produced.
During the secondment phase, expertise was gained in applying computational resources and metabolic engineering to biosynthesize resveratrol and four structural variants, including methylated and glycosylated derivatives. A reconstruction algorithm introduced functional groups with minimal genetic modifications, while a novel culture method enhanced product extraction. The compounds were produced and characterized within a DBTL (Design-Build-Test-Learn) cycle, with an estimated platform's potential to biosynthesize and test up to 180 resveratrol-derived compounds in future studies.
Interaction experiments with p53 targets and resveratrol analogues were conducted to evaluate aggregation inhibition and dynamics. Key measurements included molecular masses, kinetic parameters, folding equilibrium, stability profiles, and parameters of energy landscape. Additionally, multidimensional NMR spectroscopy was introduced, yielding a further dataset and analyses.
The project revealed a reversible equilibrium among reaction species, providing deeper information into how resveratrol analogues influence specificity at p53 binding sites. Biophysical studies characterized p53-Resveratrol interactions through aggregation kinetics and fluorescence spectroscopy, revealing parameters like fluorescence quenching, wavelength shifts, and molecular weight. Calorimetry quantified binding affinities and variation in enthalpy and entropy during steady-state interactions. Multidimensional NMR analyses resolved p53 structures, mapped binding sites via chemical shift perturbations, and examined conformational changes. An integrated platform tested p53DBD stability and aggregation, highlighting the distinct effects of biosynthesized resveratrol analogues.
The modified chemical groups introduced were shown to modulate the p53 aggregation process. The understanding of pre-amyloidogenic intermediates established a foundation for developing new therapeutics targeting early-stage p53 aggregation.
The work initially focused on obtaining the DNA Binding Domain (DBD) of the p53 constructs, a hotspot of the protein, followed by the biosynthesis of resveratrol compounds for subsequent interaction assays and analysis.
Four p53DBD constructs, the wild-type p53, its paralogs p63 and p73, and the mutant M237I, were heterologously synthesized. A sequential purification methodology was developed, increasing protein production and characterization efficiency by 20% compared to the group’s previous workflows. The process included gene subcloning, mutagenesis, protein expression, isolation, validation, and concentration measurements, using advanced biophysical techniques for characterization of all batches. Additionally, isotopically labeled proteins were produced.
During the secondment phase, expertise was gained in applying computational resources and metabolic engineering to biosynthesize resveratrol and four structural variants, including methylated and glycosylated derivatives. A reconstruction algorithm introduced functional groups with minimal genetic modifications, while a novel culture method enhanced product extraction. The compounds were produced and characterized within a DBTL (Design-Build-Test-Learn) cycle, with an estimated platform's potential to biosynthesize and test up to 180 resveratrol-derived compounds in future studies.
Interaction experiments with p53 targets and resveratrol analogues were conducted to evaluate aggregation inhibition and dynamics. Key measurements included molecular masses, kinetic parameters, folding equilibrium, stability profiles, and parameters of energy landscape. Additionally, multidimensional NMR spectroscopy was introduced, yielding a further dataset and analyses.
The project revealed a reversible equilibrium among reaction species, providing deeper information into how resveratrol analogues influence specificity at p53 binding sites. Biophysical studies characterized p53-Resveratrol interactions through aggregation kinetics and fluorescence spectroscopy, revealing parameters like fluorescence quenching, wavelength shifts, and molecular weight. Calorimetry quantified binding affinities and variation in enthalpy and entropy during steady-state interactions. Multidimensional NMR analyses resolved p53 structures, mapped binding sites via chemical shift perturbations, and examined conformational changes. An integrated platform tested p53DBD stability and aggregation, highlighting the distinct effects of biosynthesized resveratrol analogues.
The modified chemical groups introduced were shown to modulate the p53 aggregation process. The understanding of pre-amyloidogenic intermediates established a foundation for developing new therapeutics targeting early-stage p53 aggregation.
The p53-REACT project made advancements by integrating biophysical and structural biology techniques with breakthroughs in metabolic engineering. It generated a wealth of information about the biochemical properties of resveratrol variants interacting with the p53 protein family. Exploitable results included the identification of promising therapeutic agents capable of disrupting p53 aggregation and potentially slowing cancer proliferation, the development of a microbial host platform for producing plant-based polyphenol compounds, the incorporation of safe and efficient gene modification methods into cell factories, and the characterization of thermodynamic signatures of processes that restore wild-type function to mutant p53. These findings also pave the way for enzyme exploration and modifications to enhance the therapeutic potential of numerous unexplored compounds.
To ensure continued acceptance and impact, future research should further analyze the dynamic interactions between the studied compounds and p53, investigate additional protein mutations, tailor compound screenings for specific conformational states, and evaluate their effects on cancer cell viability. Leading academic and non-academic research groups studying p53 in living cells—many with a history of discovering TP53 mutations and characterizing p53 isoforms—have shown strong interest in testing the project’s promising anti-aggregative compounds in live-cell p53 assays. Potential collaborations aim to demonstrate these compounds’ ability to modulate p53-dependent responses, evaluate p53 as a clinical marker, and explore strategies for targeting mutant p53 in tumors and cancer subtypes.
To ensure continued acceptance and impact, future research should further analyze the dynamic interactions between the studied compounds and p53, investigate additional protein mutations, tailor compound screenings for specific conformational states, and evaluate their effects on cancer cell viability. Leading academic and non-academic research groups studying p53 in living cells—many with a history of discovering TP53 mutations and characterizing p53 isoforms—have shown strong interest in testing the project’s promising anti-aggregative compounds in live-cell p53 assays. Potential collaborations aim to demonstrate these compounds’ ability to modulate p53-dependent responses, evaluate p53 as a clinical marker, and explore strategies for targeting mutant p53 in tumors and cancer subtypes.