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Yeast As a Chassis for Developing Functional Assays to Study Human P53
Published on: August 4, 2019
Multifunctional metallochaperone modifications for targeting subsite cavities in mutant p53-Y220C
Jessica J Miller1, Kalvin Kwan1, Anaïs Blanchet2
1Department of Chemistry, Simon Fraser University, 8888 University Drive, Burnaby, Canada.
Abstract:
The p53 protein, known as the 'guardian of the genome', plays an important role in cancer prevention. Unfortunately, p53 mutations result in compromised activity with over 50% of cancers resulting from point mutations to p53. There is considerable interest in mutant p53 reactivation, with the development of small-molecule reactivators showing promise. We have focused our efforts on the common p53 mutation Y220C, which causes protein unfolding, aggregation, and can result in the loss of a structural Zn from the DNA-binding domain. In addition, the Y220C mutant creates a surface pocket that can be stabilized using small molecules. We previously reported the bifunctional ligand L5 as a Zn metallochaperone and reactivator of the p53-Y220C mutant. Herein we report two new ligands L5-P and L5-O that are designed to act as Zn metallochaperones and non-covalent binders in the Y220C mutant pocket. For L5-P the distance between the Zn-binding di-(2-picolyl)amine function and the pocket-binding diiodophenol was extended in comparison to L5, while for L5-O we extended the pocket-binding moiety via attachment of an alkyne function. While both new ligands displayed similar Zn-binding affinity to L5, neither acted as efficient Zn-metallochaperones. However, the new ligands exhibited significant cytotoxicity in the NCI-60 cell line screen as well as in the NUGC3 Y220C mutant cell line. We identified that the primary mode of cytotoxicity is likely reactive oxygen species (ROS) generation for L5-P and L5-O, in comparison to mutant p53 reactivation for L5, demonstrating that subtle changes to the ligand scaffold can change the toxicity pathway.
Insights
New small molecules targeting the p53 Y220C mutation show promise. While not reactivating the mutant protein, these compounds induce cancer cell death through reactive oxygen species generation.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- The p53 protein is crucial for cancer prevention, but mutations, like Y220C, compromise its function.
- Over 50% of cancers involve p53 point mutations, creating a need for therapeutic strategies.
- The Y220C mutation unfolds the protein and creates a druggable pocket.
Purpose of the Study:
- To design and synthesize novel small molecules (L5-P, L5-O) as potential zinc metallochaperones and binders for the p53 Y220C mutant.
- To evaluate the efficacy of these new ligands in reactivating mutant p53 and their cytotoxic effects.
- To investigate the mechanism of action and toxicity pathways of the novel ligands.
Main Methods:
- Synthesis of two new bifunctional ligands, L5-P and L5-O, with modified structures compared to a previously reported ligand (L5).
- Assessment of zinc-binding affinity and metallochaperone activity.
- Evaluation of cytotoxicity using NCI-60 cell line screening and a specific Y220C mutant cell line (NUGC3).
- Determination of the primary mechanism of cytotoxicity, including reactive oxygen species (ROS) generation.
Main Results:
- L5-P and L5-O exhibited similar zinc-binding affinity to L5 but were not efficient zinc metallochaperones.
- Both L5-P and L5-O demonstrated significant cytotoxicity in various cancer cell lines, including the Y220C mutant line.
- The primary mechanism for L5-P and L5-O cytotoxicity was identified as reactive oxygen species (ROS) generation, distinct from L5's mechanism of mutant p53 reactivation.
Conclusions:
- Subtle modifications in ligand structure can significantly alter the mechanism of action and therapeutic pathway.
- L5-P and L5-O represent a new class of compounds that induce cancer cell death via ROS generation, offering an alternative strategy to p53 reactivation.
- Further investigation into ROS-generating compounds targeting p53 mutants is warranted for cancer therapy development.
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