Protein misfolding and aggregation contributes to the development of a wide range of diseases. In cancer, over 50% of diagnoses are attributed to p53 malfunction due to missense mutations, many of which result in protein misfolding and accelerated aggregation. p53 mutations also frequently result in alteration or loss of zinc at the DNA-binding site, which increases aggregation via nucleation with zinc-bound p53. Herein, we designed two novel bifunctional ligands, L^I and L^H , to modulate mutant p53 aggregation and restore zinc binding using a metallochaperone approach. Interestingly, only the incorporation of iodine function in L^I resulted in modulation of mutant p53 aggregation, both in recombinant and cellular environments. Native mass spectrometry shows a protein-ligand interaction for L^I , as opposed to L^H , which is hypothesized to lead to the distinct difference in the p53 aggregation profile for the two ligands. Incorporation of a di-2-picolylamine binding unit into the ligand design provided efficient intracellular zinc uptake, resulting in metallochaperone capability for both L^I and L^H . The ability of L^I to reduce mutant p53 aggregation results in increased restoration of p53 transcriptional function and mediates both caspase-dependent and -independent cell death pathways. We further demonstrate that L^I exhibits minimal toxicity in non-cancerous organoids, and that it is well tolerated in mice. These results demonstrate that iodination of our ligand framework restores p53 function by interacting with and inhibiting mutant p53 aggregation and highlights L^I as a suitable candidate for comprehensive in vivo anticancer preclinical evaluations.

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