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Fluorophore-assisted light inactivation: a high-throughput tool for direct target validation of proteins
Stefan Beck1, Takashi Sakurai, Brenda K Eustace
1Xerion Pharmaceuticals, Martinsried, Germany.
Abstract:
To exploit advances in proteomics for drug discovery, high-throughout methods for target validation that directly address the cellular roles of proteins are required. To do this, we have characterized fluorophore-assisted light inactivation (FALI) which uses coherent or diffuse light targeted by fluorescein-labeled probes to inactivate specific proteins. We have shown that it is spatially restricted and tested its efficacy in living cells. FALI is efficient using conventional antibodies and single chain variable fragment phage display antibodies (that are compatible with high-throughput applications). We have shown that singlet oxygen is one of the major components required for FALI-mediated damage. The half-maximal radius of damage is approximately 40 A. FALI causes the specific loss of function of beta 1 integrin in HT-1080 fibrosarcoma cells resulting in a reduction in invasiveness. The efficacy of diffuse light sources (such as a desk lamp) with FALI to inactivate many samples in parallel provides an inexpensive, high-throughput method of wide general applicability for functional proteomics.
Insights
Fluorophore-assisted light inactivation (FALI) uses targeted light to disable specific proteins, enabling high-throughput functional proteomics for drug discovery. This method is efficient, spatially restricted, and effective in living cells.
Area of Science:
- Proteomics
- Cellular Biology
- Drug Discovery
Background:
- High-throughput methods are needed for protein target validation in drug discovery.
- Fluorophore-assisted light inactivation (FALI) is a technique that uses targeted light to inactivate specific proteins.
Purpose of the Study:
- To characterize FALI for its potential in high-throughput functional proteomics and drug discovery.
- To assess the efficacy and applicability of FALI in living cells.
Main Methods:
- Characterization of FALI using fluorescein-labeled probes and coherent or diffuse light.
- Testing FALI efficacy with conventional antibodies and single chain variable fragment phage display antibodies.
- Investigating the role of singlet oxygen in FALI-mediated damage and determining the radius of damage.
Main Results:
- FALI is spatially restricted with a half-maximal radius of damage of approximately 40 Å.
- FALI is efficient with both conventional antibodies and phage display antibodies, suitable for high-throughput applications.
- FALI specifically inactivated beta 1 integrin in HT-1080 fibrosarcoma cells, reducing invasiveness.
- Singlet oxygen was identified as a key component for FALI-mediated damage.
Conclusions:
- FALI is an effective, high-throughput method for protein inactivation and functional proteomics.
- The use of diffuse light sources makes FALI an inexpensive and widely applicable technique for target validation in drug discovery.