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A FRET-Based Fluorescent Probe to Screen Anticancer Drugs, Inhibiting p73 Binding to MDM2
A Ro Han1, Trishaladevi Durgannavar1, Dohee Ahn1
1School of Pharmacy, Sungkyunkwan University, Suwon, 16419 (Republic of, Korea.
Abstract:
The protein p73 acts as a transcription factor, resulting in tumour suppression. MDM2, an oncogenic protein, can negatively influence p73-mediated apoptosis by binding to p73 transactivation domains (TAD). Inhibition of the protein-protein interaction between p73 and oncogenic proteins is an attractive strategy for promoting p73-mediated apoptosis. Herein, we describe the use of a modified p73-TAD peptide for the FRET-based assay of the binding of p73-TAD to MDM2. The FRET probe, equipped with 1-naphthylamine (λex =330 nm, λem =445 nm), serves as a FRET acceptor, and the tryptophan of the protein acts as FRET donor (λex =280 nm, λem =340 nm). Sensitized emission from the FRET probe was observed upon excitation of the protein-FRET-probe complex at the excitation wavelength of Trp. Furthermore, addition of the MDM2 inhibitor Nutiln-3 drastically reduced the FRET signal, thus indicating that the FRET probe competes with Nutiln-3 for MDM2 binding. The developed FRET binding assay might be applicable in high-throughput screening of novel drugs that inhibit interactions between p73 and MDM2.
Insights
A new FRET assay uses a modified peptide to detect interactions between the tumor suppressor p73 and oncogenic MDM2. This assay can screen for drugs that disrupt this interaction, promoting cancer cell death.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- The tumor suppressor protein p73 is crucial for preventing cancer.
- The oncogenic protein MDM2 inhibits p73's function by binding to its transactivation domain (TAD).
- Inhibiting the p73-MDM2 interaction is a promising strategy to restore p73's tumor-suppressive activity.
Purpose of the Study:
- To develop a FRET-based assay to measure the binding affinity between p73-TAD and MDM2.
- To validate the assay's ability to detect inhibitors of the p73-MDM2 interaction.
Main Methods:
- A modified p73-TAD peptide was used as a FRET probe.
- 1-naphthylamine served as the FRET acceptor, and tryptophan residues in p73 acted as the FRET donor.
- The assay measured changes in FRET signals upon binding events and in the presence of an MDM2 inhibitor.
Main Results:
- The FRET probe successfully detected binding between p73-TAD and MDM2.
- The addition of MDM2 inhibitor Nutiln-3 significantly decreased the FRET signal, confirming competitive binding.
- The developed assay demonstrated its utility in identifying compounds that disrupt the p73-MDM2 interaction.
Conclusions:
- A novel FRET binding assay for the p73-MDM2 interaction has been successfully developed.
- This assay provides a valuable tool for high-throughput screening of potential anti-cancer drugs.
- The findings support targeting the p73-MDM2 interaction as a therapeutic strategy in cancer treatment.
