Mechanistic Insights of a p53-Targeting Small Molecule

Restoring the p53 pathway, particularly by reactivating wild-type (wt) or mutant (mut) p53, is considered a promising approach for cancer treatment. Previously, we identified the tryptophanol-derived oxazoloisoindolinone family as a new scaffold for obtaining wt and mut p53 reactivators. Herein, we report a detailed study on the pharmacokinetic profile and the mechanism of action of RVJB59, an (R)-tryptophanol-derived oxazoloisoindolinone that exhibits six times higher activity and increased selectivity for HCT116 cells with p53 compared to our initial hit, SLMP53-1. In vitro metabolic degradation studies in human liver microsomes and rat liver S9 fractions, assessed by LC/HRMS/MS, showed that RVJB59 is a low-clearance compound. The three main metabolites identified were synthesized, and their antiproliferative activity was evaluated against HCT116 colon cancer cells with and without p53, showing a loss of activity when compared to RVJB59. DSF studies showed that RVJB59 enhances the thermostability of the wt and R273H p53 DNA-binding domain, with this mutant showing melting curves with two melting transitions, distinct from those obtained for the wild-type. The ability of RVJB59 to undergo covalent binding via nucleophilic aromatic substitution was assessed by HRMS/MS, using glutathione and wt p53 as case studies. These assays showed low reactivity toward glutathione and remarkable selectivity toward Cys141 of wt p53. The effect of RVJB59 was also evaluated in 3D spheroids of HCT116 cells and in vivo using chicken embryos, with RVJB59 reducing 3D tumor spheroid growth and exhibiting antiangiogenic potential. This study provides additional evidence of the potential of RVJB59 in activating p53.