A homogeneous time-resolved fluorescence screen to identify SIRT2 deacetylase and defatty-acylase inhibitors
Fig 1
Design and validation of a SIRT2—myristoyl-peptide HTRF binding assay.
(A) Schematic of our HTRF binding assay where His tagged-SIRT2 interacts with a terbium cryptate-labeled antibody and a fluorescein-labeled myristoyl-peptide. Displacement of the peptide from SIRT2 by a competitor then reduces FRET efficiency after terbium cryptate excitation. (B) Fluorescence intensity in HTRF binding assays using different concentrations of SIRT2 and FAM-myristoyl-H4K16 peptide (excitation/emission wavelengths = 330 nm/520 nm, normalized by emission at 620 nm) (see Materials and Methods). (C) Displacement of FAM-myristoyl-H4K16 peptide from SIRT2 in the HTRF binding assay by an identical peptide lacking the FAM-PEG4 group. (D) Displacement of FAM-myristoyl-H4K16 peptide from SIRT2 in the HTRF binding assay by the SIRT2 deacetylase and defatty-acylase inhibitor ascorbyl palmitate. (E) Displacement of FAM-myristoyl-H4K16 peptide from SIRT2 in the HTRF binding assay by the SIRT2 deacetylase inhibitor SirReal2.