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Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling
Published on: September 20, 2016
A highly sensitive fluorescent light-up probe for real-time detection of the endogenous protein target and its
Junlong Geng1, Walter L Goh, Chongjing Zhang
1Institute of Materials Research Engineering, A*STAR, 3 Research Link, Singapore 117602. tanyn@imre.a-star.edu.sg.
Abstract:
Real-time detection and monitoring of cancer-related biomolecular interactions in live cells are of paramount importance for disease diagnostics and drug screening. Herein, we developed a target-specific fluorescent light-up probe for cellular detection of Mdm2, the key negative regulator of the p53 tumour suppressor protein. Conjugation of a uniquely designed fluorogen (TPECM) with aggregation induced-emission properties, to a specific p53-derived peptide (12.1Pep) targeting Mdm2, yielded a cell-permeable probe (TPECM-12.1Pep) with turn-on fluorescence properties for real-time live cell imaging of Mdm2. This specific light-up probe is almost non-fluorescent in its isolated state but is highly emissive upon binding to Mdm2, enabling quantitative detection of both Mdm2 and its antagonism. Using a model compound (Nutlin-3a), we demonstrate that the as-developed probes can be used to screen p53-Mdm2 inhibiting drug candidates, both in vitro and in cells. Furthermore, the probe activity can be accurately monitored in cells using a fluorescently activated cell sorting machine. These features will expedite research in the areas of drug discovery, clinical diagnostics and fundamental cell biology.
Insights
Scientists developed a novel fluorescent probe for real-time detection of Mdm2 protein in live cells. This tool aids in cancer diagnostics and drug discovery by enabling precise monitoring of Mdm2 interactions.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Real-time detection of cancer-related biomolecular interactions in live cells is crucial for diagnostics and drug screening.
- Mdm2 is a key negative regulator of the p53 tumor suppressor protein, making it a significant target in cancer research.
Purpose of the Study:
- To develop a target-specific fluorescent light-up probe for cellular detection of Mdm2.
- To enable real-time live cell imaging and quantitative detection of Mdm2 and its antagonism.
- To demonstrate the probe's utility in screening drug candidates that inhibit the p53-Mdm2 interaction.
Main Methods:
- Conjugation of a fluorogen (TPECM) with aggregation-induced emission properties to a p53-derived peptide (12.1Pep) targeting Mdm2.
- Development of a cell-permeable, turn-on fluorescent probe (TPECM-12.1Pep) with high specificity for Mdm2.
- In vitro and cellular assays using a model compound (Nutlin-3a) to validate probe performance and drug screening capabilities.
- Monitoring probe activity in cells using fluorescently activated cell sorting (FACS).
Main Results:
- The developed probe (TPECM-12.1Pep) exhibits turn-on fluorescence properties, being nearly non-fluorescent in isolation but highly emissive upon binding to Mdm2.
- The probe allows for real-time live cell imaging and quantitative detection of Mdm2.
- The probe successfully facilitated the screening of p53-Mdm2 inhibiting drug candidates both in vitro and in cellular models.
- Probe activity was accurately monitored in cells via FACS.
Conclusions:
- A novel, target-specific fluorescent light-up probe for Mdm2 has been successfully developed.
- The probe enables real-time monitoring and quantitative detection of Mdm2 in live cells.
- This technology holds significant potential for accelerating drug discovery, clinical diagnostics, and fundamental cell biology research.
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