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Updated: Dec 13, 2025
Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b
Published on: November 11, 2016
A FRET-based screening method to detect potential inhibitors of the binding of CNNM3 to PRL2
Faji Cai1, Yichen Huang1, Mengqi Wang1
1State Key Laboratory of Genetic Engineering, Collaborative Innovation Center of Genetics and Development, Department of Physiology and Biophysics, School of Life Sciences, Fudan University, 2005 Songhu Road, Yangpu District, Shanghai, 200438, China.
Abstract:
The cyclin M (CNNM) family of Mg2+ transporters is reported to promote tumour progression by binding to phosphatase of regenerating liver (PRL) proteins. Here, we established an assay for detection of the binding between the cystathionine-beta-synthase (CBS) domain of human CNNM3 (a region responsible for PRL binding) and human PRL2 using fluorescence resonance energy transfer (FRET) techniques. By fusing YPet to the C-terminus of the CNNM3 CBS domain and CyPet to the N-terminus of PRL2, we performed a FRET-based binding assay with purified proteins in multiwell plates and successfully detected the changes in fluorescence intensity derived from FRET with a reasonable Kd. We then confirmed that the addition of non-YPet-tagged CNNM3 and non-CyPet-tagged PRL proteins inhibited the changes in FRET intensity, whereas non-YPet-tagged CNNM3 with a mutation at the PRL2-binding site did not exhibit such inhibition. Furthermore, newly synthesized peptides derived from the CNNM loop region, with the PRL-binding sequences of the CNNM3 CBS domain, inhibited the interactions between CNNM3 and PRL2. Overall, these results showed that this method can be used for screening to identify inhibitors of CNNM-PRL interactions, potentially for novel anticancer therapy.
Insights
Researchers developed a new assay to detect interactions between CNNM3 and PRL2 proteins, crucial for tumor progression. This method can screen for novel anticancer therapies targeting these CNNM-PRL interactions.
Area of Science:
- Biochemistry
- Molecular Biology
- Cancer Research
Background:
- The cyclin M (CNNM) family of Mg2+ transporters is implicated in tumor progression.
- CNNM proteins interact with phosphatase of regenerating liver (PRL) proteins, influencing cellular processes.
Purpose of the Study:
- To establish a fluorescence resonance energy transfer (FRET)-based assay for detecting CNNM3-PRL2 binding.
- To validate the assay's ability to screen for inhibitors of CNNM-PRL interactions.
Main Methods:
- Utilized FRET by fusing fluorescent proteins (YPet and CyPet) to human CNNM3 CBS domain and human PRL2.
- Performed binding assays with purified proteins in multiwell plates.
- Tested inhibition using unlabeled proteins and synthesized peptides.
Main Results:
- Successfully detected CNNM3-PRL2 binding using FRET with a quantifiable dissociation constant (Kd).
- Demonstrated inhibition of FRET signal by unlabeled CNNM3 and PRL2 proteins.
- Showed that peptides mimicking CNNM3's PRL-binding site inhibit CNNM3-PRL2 interaction.
Conclusions:
- The developed FRET assay reliably detects CNNM3-PRL2 interactions.
- This assay is a valuable tool for screening potential inhibitors of CNNM-PRL interactions.
- Identifying such inhibitors could lead to novel anticancer therapies.
