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.
