SCH529074, a Small Molecule Activator of Mutant p53, Which Binds p53 DNA Binding Domain (DBD), Restores Growth-suppressive Function to Mutant p53 and Interrupts HDM2-mediated Ubiquitination of Wild Type p53

Eugene Maxwell,Randall Rossman,Alan Mallams,W. Robert Bishop,Ronald Doll,David Hesk,Ming Liu,Cheng Li,Cynthia Seidel-Dugan,Mark Demma,Robert Ramos,Bimalendu Dasmahapatra,Lianzhu Liang

doi:10.1074/jbc.m109.083469

Abstract

Abrogation of p53 function occurs in almost all human cancers, with more than 50% of cancers harboring inactivating mutations in p53 itself. Mutation of p53 is indicative of highly aggressive cancers and poor prognosis. The vast majority of mutations in p53 occur in its core DNA binding domain (DBD) and result in inactivation of p53 by reducing its thermodynamic stability at physiological temperature. Here, we report a small molecule, SCH529074, that binds specifically to the p53 DBD in a saturable manner with an affinity of 1-2 microm. Binding restores wild type function to many oncogenic mutant forms of p53. This small molecule reactivates mutant p53 by acting as a chaperone, in a manner similar to that previously reported for the peptide CDB3. Binding of SCH529074 to the p53 DBD is specifically displaced by an oligonucleotide with a sequence derived from the p53-response element. In addition to reactivating mutant p53, SCH529074 binding inhibits ubiquitination of p53 by HDM2. We have also developed a novel variant of p53 by changing a single amino acid in the core domain of p53 (N268R), which abolishes binding of SCH529074. This amino acid change also inhibits HDM2-mediated ubiquitination of p53. Our novel findings indicate that through its interaction with p53 DBD, SCH529074 restores DNA binding activity to mutant p53 and inhibits HDM2-mediated ubiquitination.

Highlights

Aggressive, treatment-refractory cancers [3, 5]
We used recombinantly produced p53 core DNA binding domain of a p53 mutant (R273H) fused to glutathione S-transferase (GST) and measured its ability to bind a biotinylated oligonucleotide that contained a consensus p53 DNA-binding site The biotinylated oligonucleotide was captured by a magnetic streptavidin-coated bead, and bound p53 was detected by an antibody specific to the protein followed by a ruthenium-labeled secondary antibody
We report the identification of a small molecule (SCH529074) that binds to p53 DNA binding domain (DBD) and restores wild type p53 function and conformation to various oncogenic mutants of p53, induces apoptosis in tumor cells harboring mutant p53, and reduces tumor growth in a xenograft model

Summary

Introduction

The majority of inactivating mutations in p53 are missense mutations that reside in the central core DNA binding domain (DBD)4 [3, 5] These mutations can be divided into two main classes as follows: contact point mutations, which alter a residue involved in contact with DNA, and structural mutations, which affect p53 function by distorting the structure of p53 and reducing its thermal stability [6, 7]. Several approaches have been taken to reactivate mutant p53, including treatments with Mab421, carboxyl-terminal peptide of p53, or small molecules such as CP-31398, PRIMA1, and the peptide CDB3 (14 –24) Both CP-31398 and PRIMA1 have been demonstrated to reduce tumor growth in animal models [16, 17]. We have constructed a novel variant of p53 by changing a single amino acid in DBD, and we showed that the variant that maintains wild type DNA binding activity does not bind to SCH529074 and mimics the small molecule in inhibiting ubiquitination of p53 by HDM2

Methods

Results

Conclusion