Scutellarin, one of natural flavonoids, is widely and clinically used for treating many diseases in China. Recently, scutellarin has demonstrated a broad spectrum of anti-proliferative activities against multiple cancer cell lines. However, the molecular mechanism of action remains to be investigated. We herein report the design and synthesis of biotinylated scutellareins as probes, which can be applied to discover scutellarein interacting proteins. Finally, we show that scutellarin directly targets pyruvate kinase M2 (PKM2) and inhibits its cytosolic activity to decrease glycolytic metabolism; on the other hand, scutellarin may also participate in regulating cell cycle and apoptotic proteins by activating MEK/ERK/PIN1 signaling pathway to promote the nuclear translocation of PKM2.Download high-res image (66KB)Download full-size image

•A FRET peptide containing a FRET pair of DABCYL/EDANS have been designed and made for SIRT6 demyristoylation activity.•A FRET assay has been developed for testing SIRT6 demyristoylation activity.•A FRET assay has been developed for screening and evaluating SIRT6 modulators.•A combinational FRET assay has been developed for screening SIRT6 specific modulators.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.

A fluorogenic assay for SIRT5 has been developed to screen their small molecule modulators based on the recent discovery that SIRT5 is a demalonylase and desuccinylase. However, this assay uses a fluorogenic peptide containing 7-amino-4-methylcoumarin (AMC), which becomes the cause of false positive hits from the screening. To overcome this, we have developed an alternative method called a FRET-based assay, which will be reliable and useful for screening SIRT5 modulators in a high-throughput format since no AMC group present in this assay.