A Structure-Guided Molecular Chaperone Approach for Restoring the Transcriptional Activity of the p53 Cancer Mutant Y220C

Abstract

Aim:The p53 cancer mutation Y220C creates a conformationally unstable protein with a unique elongated surface crevice that can be targeted by molecular chaperones. We report the structure-guided optimization of the carbazole-based stabilizer PK083.Materials & methods:Biophysical, cellular and x-ray crystallographic techniques have been employed to elucidate the mode of action of the carbazole scaffolds.Results:Targeting an unoccupied subsite of the surface crevice with heterocycle-substituted PK083 analogs resulted in a 70-fold affinity increase to single-digit micromolar levels, increased thermal stability and decreased rate of aggregation of the mutant protein. PK9318, one of the most potent binders, restored p53 signaling in the liver cancer cell line HUH-7 with homozygous Y220C mutation.Conclusion:The p53-Y220C mutant is an excellent paradigm for the development of mutant p53 rescue drugs via protein stabilization. Similar rescue strategies may be applicable to other cavity-creating p53 cancer mutations.

Zinc shapes the folding landscape of p53 and establishes a pathway for reactivating structurally diverse cancer mutants

“…One of the primary predictions of the model is that increasing zinc concentration will refold stability class as well as zinc-binding class mutants. The archetypical example of small-molecule-induced p53 stabilization is binding of PhiKan and similar compounds to the surface cavity on Y220C left by the Tyr220→Cys alteration ( Boeckler et al, 2008 ; Liu et al, 2013 ; Baud et al, 2018 ; Bauer et al, 2019 ). These drugs offer the potential of bio-orthogonality but bind to only a single p53 mutant and do so with (thus far) weak affinity (K d = 1–100 μM).…”

Zinc shapes the folding landscape of p53 and establishes a new pathway for reactivating structurally diverse p53 mutants

“…One of the primary predictions of the model is that increasing zinc concentration will refold stability class as well as zinc-binding class mutants. The archetypical example of small molecule-induced p53 stabilization is binding of PhiKan and similar compounds to the surface cavity on Y220C left by the Tyr220→Cys alteration [28][29][30][31] . These drugs offer the potential of bioorthogonality but bind to only a single p53 mutant and do so with (thus far) weak affinity (Kd = Previous work by our laboratory as well as that of Fersht established that the stability of DBD is low at 37 °C.…”

Markov state models and NMR uncover an overlooked allosteric loop in p53

“…in a recessed conformation similar to that observed experimentally (Figure 4b-c, pink R state). This organization of the loop allows for the formation of a crevice in between loops L6 and S3/S4 upon the substitution of the bulky tyrosine for the much smaller cysteine residue in the Y220C mutant, which results in the pocket currently targeted for p53 rescue [29][30][31][32][33][34] .…”

“…One such mutant targeted for small molecule reactivation is Y220C, a structural mutant responsible for about 100,000 new cancer cases every year 14 and the most frequent p53 cancer mutation observed outside the DNA-binding interface of the protein. The mutation of the bulky tyrosine to the smaller cysteine induces the formation of a crevice in the protein surface that is amenable to small molecule binding [29][30][31] , but so far current efforts have failed to yield very high affinity binders [32][33][34] .…”