Targeting Cavity-Creating p53 Cancer Mutations with Small-Molecule Stabilizers: the Y220X Paradigm

Abstract

We have previously shown that the thermolabile, cavity-creating p53 cancer mutant Y220C can be reactivated by small-molecule stabilizers. In our ongoing efforts to unearth druggable variants of the p53 mutome, we have now analyzed the effects of other cancer-associated mutations at codon 220 on the structure, stability, and dynamics of the p53 DNA-binding domain (DBD). We found that the oncogenic Y220H, Y220N, and Y220S mutations are also highly destabilizing, suggesting that they are largely unfolded under physiological conditions. A high-resolution crystal structure of the Y220S mutant DBD revealed a mutation-induced surface crevice similar to that of Y220C, whereas the corresponding pocket's accessibility to small molecules was blocked in the structure of the Y220H mutant. Accordingly, a series of carbazole-based small molecules, designed for stabilizing the Y220C mutant, also bound to and stabilized the folded state of the Y220S mutant, albeit with varying affinities due to structural differences in the binding pocket of the two mutants. Some of the compounds also bound to and stabilized the Y220N mutant, but not the Y220H mutant. Our data validate the Y220S and Y220N mutants as druggable targets and provide a framework for the design of Y220S or Y220N-specific compounds as well as compounds with dual Y220C/Y220S specificity for use in personalized cancer therapy.

Pathogenic genetic variants from highly connected cancer susceptibility genes confer the loss of structural stability

“…Several carbazole derivatives such as PK083, PK9284, PK9295, PK9318, PK9320 etc. were tested to stabilize Y220S and Y220N p53 mutants and their binding constant (Kd) were also determined 42 . Among them, PK083 (PhiKan083), was found to bind to the cavity formed due to mutation with a dissociation constant of ≈ 150 μM50 42 .…”

“…were tested to stabilize Y220S and Y220N p53 mutants and their binding constant (Kd) were also determined 42 . Among them, PK083 (PhiKan083), was found to bind to the cavity formed due to mutation with a dissociation constant of ≈ 150 μM50 42 . It was also found to raise the melting temperature of the mutant and to slow down its rate of thermal denaturation.…”

Genotoxicity and Epigenotoxicity of Carbazole-Derived Molecules on MCF-7 Breast Cancer Cells

“…Here, we have analyzed the DNA damage-inducing potential and methylation-changing capability of three carbazoles, PK083, PK9320, and PK9323, in human breast adenocarcinoma cells (MCF-7) to evaluate their potential use as “anticancer (epi)drugs” in a combinatorial molecular therapy [ 26 ], in addition to their reported action as Y220C mutant p53 reactivators [ 15 , 16 ].…”

“…Structure-guided design then led to second-generation analogues, including PK9320 and PK9323 , which bound the mutant with higher affinity and were more potent in stabilizing and reactivating the mutant than the parent compound ( Figure 1 ). Importantly, those second-generation carbazoles restored p53 signaling in the liver cancer cell line HUH-7 bearing a homozygous Y220C mutation, with potential future application in personalized cancer therapy [ 15 , 16 ].…”

Pathogenic Genetic Variants From Highly Connected Cancer Susceptibility Genes Confer the Loss of Structural Stability

“…A carbazole derivative, PK083 (1-(9-ethylcarbazol-3-yl)-N-methylmethanamine) was used as ligand. Carbazole based small-molecules were tested to act as stabilizers in restoring the function of several mutants of p53 DBD 50,51 . So, we tested the stabilizing capability of PK083 to the highly damaging mutants of our study.…”