Covalent Targeting and Thermostabilization of Oncogenic R280K and R273H Mutants p53 by Small Molecule RVJB59

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Breast cancer is one of the most common cancers worldwide. Approximately 30%-40% of breast cancers harbor mutations in the TP53 gene, leading to structural and functional alterations in the p53 protein. These changes result in mutant proteins that are unable to perform their canonical tumor suppressor functions and, in many cases, exhibit gain-of-function properties, making mutant p53 a highly attractive therapeutic target. Among these mutations, R280K and R273H are two clinically relevant DNA-binding mutations. In this study, we extended our investigation of the (R)-tryptophanol isoindolinone derivative RVJB59 to these two p53 mutants. Differential scanning fluorimetry showed that RVJB59 thermally stabilized the R280K mutant p53 in a dose-dependent manner. Furthermore, the mechanism of action of RVJB59 was investigated by liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-HRMS/MS) using the DNA‑binding domains of the R280K and R273H mutants, confirming covalent binding of the compound to Cys141 in both proteins. Computational studies with both p53 mutants suggested that RVJB59 can stably bind in proximity to Cys141, further reinforcing the results obtained by LC-HRMS/MS. Our findings further underscore the potential of RVJB59 for the development of novel therapies for the treatment of breast cancers harboring these mutations.