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A FRET-based assay for screening SIRT6 modulators
Yan Li1, Ling You2, Wenfei Huang2
1College of Basic Medical Science, Guiyang Medical University, Guiyang, Guizhou 550004, China.
Abstract:
SIRT6, as one of these seven sirtuins, has been shown to have the therapeutic potentials for treating several human diseases. A fluorogenic assay for SIRT6 has been developed to screen their small molecule modulators based on the recent discovery that SIRT6 is a defatty-acylase (removing long chain fatty acyl groups). However, this assay uses a fluorogenic peptide containing 7-amino-4-methylcoumarin (AMC), which becomes the cause of false positive hits from screenings. To overcome this, we have developed an alternative method called a FRET-based assay suitable for screening SIRT6 modulators, which will be reliable and useful in a high-throughput format since no AMC group present in this assay.
Insights
A new FRET-based assay offers a reliable method for screening SIRT6 modulators, overcoming false positives associated with previous fluorogenic assays for this sirtuin enzyme.
Area of Science:
- Biochemistry
- Enzymology
- Drug Discovery
Background:
- Sirtuins, including SIRT6, are implicated in human disease therapeutics.
- SIRT6 functions as a deacylase, removing long-chain fatty acyl groups.
- Existing fluorogenic assays for SIRT6 screening are prone to false positives due to the use of 7-amino-4-methylcoumarin (AMC).
Purpose of the Study:
- To develop a more reliable assay for screening small molecule modulators of SIRT6.
- To overcome the limitations of existing AMC-based fluorogenic assays.
Main Methods:
- Development of a Förster Resonance Energy Transfer (FRET)-based assay for SIRT6.
- Utilizing the FRET assay in a high-throughput screening format.
Main Results:
- The FRET-based assay provides a reliable method for SIRT6 modulator screening.
- Elimination of the AMC group prevents false positive hits observed in previous assays.
Conclusions:
- The developed FRET assay is a valuable tool for high-throughput screening of SIRT6 modulators.
- This assay offers improved accuracy and reliability for drug discovery efforts targeting SIRT6.
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