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Evaluation of dual-function molecules containing both Zn-Ionophore and aggregation inhibition moieties for mutant p53
Kalvin Kwan1, Ashkan Saffari1, Lauryn Grcic1
1Department of Chemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada.
Abstract:
The p53 protein plays an important role in preventing cancer and is critical in inducing an antiproliferative response. Unfortunately, the p53 pathway is compromised in almost all cancers, and mutations to p53 lead to loss of function due to protein unfolding, compromised Zn2+ binding, aggregation and amyloid formation. Herein, two new multidentate N,O donor ligands LI-A and LH-A were tested to potentially restore Zn2+ binding to mutant p53, while also inhibiting protein aggregation. The design of these ligands centered on combining an iminodiacetate (IDA) metal binding moiety which was previously determined to exhibit favourable Zn2+ binding affinity and Cu2+/Zn2+ selectivity ratio, with a benzothiazole unit that has been demonstrated to limit mutant p53 protein aggregation. The new ligands were shown to exhibit Zn2+ Kd values in the low nM range based on a fluorophore competition assay, while also inhibiting mutant p53 aggregation via a Thioflavin-T (ThT) assay. In cell-based assays and NCI-60 screening, neither of the new ligands displayed significant cytotoxicity. A reactive oxygen species (ROS) assay showed that neither of the new ligands increased intracellular ROS, however, the previously studied LI compound did show an increase in ROS, providing further information on the mechanism of action of this class of compounds. The current results highlight that the dipicolylamine (DPA) unit is important for anticancer activity, and that phenolate substitution has an important role in dictating the mechanism of cytotoxicity.
Insights
New ligands show promise in restoring function to mutant p53 protein, a key factor in cancer. These compounds bind zinc and inhibit protein aggregation without significant toxicity, offering a potential new avenue for cancer therapy.
Area of Science:
- Biochemistry
- Medicinal Chemistry
- Cancer Biology
Background:
- The p53 protein is crucial for cancer prevention, but its pathway is often compromised in cancer cells.
- Mutations in p53 lead to loss of function, protein unfolding, impaired zinc binding, aggregation, and amyloid formation.
Purpose of the Study:
- To design and evaluate novel multidentate ligands for restoring Zn2+ binding to mutant p53.
- To inhibit mutant p53 protein aggregation using these new ligands.
Main Methods:
- Design of ligands combining iminodiacetate (IDA) and benzothiazole moieties.
- Fluorophore competition assay to determine Zn2+ binding affinity (Kd).
- Thioflavin-T (ThT) assay to assess inhibition of protein aggregation.
- Cell-based assays, NCI-60 screening, and reactive oxygen species (ROS) assays to evaluate cytotoxicity and mechanism of action.
Main Results:
- The new ligands demonstrated low nM Zn2+ binding affinity.
- Ligands effectively inhibited mutant p53 aggregation.
- No significant cytotoxicity was observed in cell-based assays or NCI-60 screening.
- Unlike a previously studied compound, the new ligands did not increase intracellular ROS.
Conclusions:
- The dipicolylamine (DPA) unit is vital for the anticancer activity of these compounds.
- Phenolate substitution influences the mechanism of cytotoxicity.
- These ligands show potential for restoring p53 function and inhibiting cancer progression with a favorable safety profile.
