(381c) Towards the Rational Design of p53 Stabilizing Drugs: Probing the Surface of the Oncogenic Y220C Mutant

The oncogenic Y220C mutation in the tumour suppressor p53 is among the most frequent genetic alterations in human cancers. The mutation markedly lowers the stability of the protein and, notably, induces formation of a cleft on the protein's surface at the mutation site. This site, which is away from the DNA-binding site of p53, could potentially accommodate a small-molecule drug that, in principle, could reverse the thermodynamic consequences of the mutation. We have combined fragment-based ligand screening and molecular dynamic simulations to explore the Y220C site for potential ligand-binding hotspots. The hit rate of fragments against the destabilized mutant was determined by water-ligand-observed-gradient spectroscopy and thermal denaturation scanning fluorimetry screening with a chemically diverse small-molecule fragment library. Chemical shift mapping by 15N/1H HSQC NMR was subsequently employed to confirm binding of the fragment hits and to identify specific residue involved in the protein-ligand interactions. The binding mode of several of the fragment hits was further elucidated by x-ray crystallography. The structural data found that several residues in the cleft are highly flexible, allowing the cleft to potentially accommodate a larger ligand. Molecular dynamics studies of the protein in a water/isopropanol mixture identified additional ligand-binding hotspots around the cleft and were used to quantify their druggability. Together, these results paint a clear picture of accessible ligand-binding sites on the Y220C mutant of p53 that can be exploited for rational drug design.