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 <b>RVJB59</b>, an (<i>R</i>)-tryptophanol-derived oxazoloisoindolinone that exhibits six times higher activity and increased selectivity for HCT116 cells with p53 compared to our initial hit, <b>SLMP53-1</b>. <i>In vitro</i> metabolic degradation studies in human liver microsomes and rat liver S9 fractions, assessed by LC/HRMS/MS, showed that <b>RVJB59</b> 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 <b>RVJB59</b>. DSF studies showed that <b>RVJB59</b> 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 <b>RVJB59</b> 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 <b>RVJB59</b> was also evaluated in 3D spheroids of HCT116 cells and <i>in vivo</i> using chicken embryos, with <b>RVJB59</b> reducing 3D tumor spheroid growth and exhibiting antiangiogenic potential. This study provides additional evidence of the potential of <b>RVJB59</b> in activating p53.