S_NAr Reactive Pyrazine Derivatives as p53-Y220C Cleft Binders with Diverse Binding Modes

The tumor suppressor p53 is most commonly mutated in human cancer. The structural mutant in the β-sandwich of the protein, p53-Y220C, is the ninth most common p53 mutant. The p53-Y220C mutant has a solvent-accessible hydrophobic pocket, leading to thermal destabilization of the protein. Screening of our covalent fragment library (CovLib) revealed the highly reactive pyrazine derivatives SN006 and SN007, which arylate among other cysteines in p53, the mutation-generated Cys220. Herein, comprehensive structure-activity relationship (SAR) studies of these intrinsically reactive CovLib hits were performed, aiming to identify improved stabilizers for p53-Y220C, with a more balanced reactivity profile, diverse binding modes and a better potential for chemical optimization.

The compounds were screened for enhanced stabilization of p53 wild type and its mutants using differential scanning fluorimetry (DSF). To confirm covalent modification, intact mass spectrometry was performed. Thiol reactivity profiles were determined using a standardized Glutathione-modifying (GSH) assay. The binding modes of the identified hits and covalent modification of Cys220 were elucidated by X-ray crystallography. Moreover, the influence of the hits on the DNA-binding affinity of full-length p53 was investigated employing a fluorescence polarization assay (FPA).

The promising pyrazine derivatives SN006/7-3, SN006/7-8, and SN006/7-9 were identified, occupying different subsites of the Y220C binding pocket. The compound SN006/7-8 substantially stabilized the thermosensitive cancer mutant Y220C by up to 5.0 °C, representing a strong enhancement over SN006 (1.8 °C) and SN007 (2.0 °C).