Stabilization of mutant p53 via alkylation of cysteines and effects on DNA binding

Oncogenic mutations inactivate the tumor suppressor p53 by lowering its stability or by weakening its binding to DNA. Alkylating agents that reactivate mutant p53 are currently being explored for cancer therapy. We have discovered ligands containing an α,β-unsaturated double bond, characteristic of Michael acceptors, that bind covalently to generic cysteine sites in the p53 core domain. They raised the melting temperature of the core domain of wild-type p53 and the hotspot mutants R175H, Y220C, G245S, R249S, and R282 by up to 3°C. Analysis of the relative reactivity of the cysteines in p53 by mass spectrometry found that C124 and C141 react first, followed by C135, C182, and C277, and eventually C176 and C275. Post-translational modifications of cysteines are known to be involved in regulation of other transcription factors. Modification of C277, which sits on the DNA-binding surface, may, for example, play a role in regulating p53 activity in cells in response to environmental cues. We found that the modifications progressively reduced DNA-binding activity of full-length p53. In light of these results, it is likely that the anticancer activity of the alkylating drugs works via a nontranscriptional activity of p53.