Discovery and validation of SIRT2 inhibitors based on tenovin-6: use of a ¹H-NMR method to assess deacetylase activity

Abstract

The search for potent and selective sirtuin inhibitors continues as chemical tools of this type are of use in helping to assign the function of this interesting class of deacetylases. Here we describe SAR studies starting from the unselective sirtuin inhibitor tenovin-6. These studies identify a sub-micromolar inhibitor that has increased selectivity for SIRT2 over SIRT1 compared to tenovin-6. In addition, a ¹H-NMR-based method is developed and used to validate further this class of sirtuin inhibitors. A thermal shift analysis of SIRT2 in the presence of tenovin-6, -43, a control tenovin and the known SIRT2 inhibitor AGK2 is also presented.

Scheme 1

Synthesis of tenovin analogues.

Figure 1

Commercially available sirtuin assay uses a fluorescently labelled peptide substrate containing an N-acetylated lysine residue [20]. Removal of the N-acetyl group is coupled with conversion of NAD⁺ to nicotinamide (in red), 2′-O-acetyl ADP-ribose (in blue) and the deacetylated substrate. Subsequent reaction with trypsin releases the quenched fluorophore.

Figure 2

1D ¹H-NMR with double solvent suppression (for both H₂O and Tris buffer) recorded at 37 °C. Sample contained 1 mM NAD⁺, 200 µM peptide, 10 µM SIRT2 in buffer (pH 8). (a) Before addition of enzyme. (b) After addition of enzyme and 15 mins. incubation. (c) Sample containing 200 μM tenovin-6 after incubation with enzyme for 15 min. Throughout this work, the H4 substrate was used at a final concentration of 200 μM as this enabled monitoring using a reasonable number of scans.

Figure 3

1D ¹H-NMR with double solvent suppression (H₂O and Tris buffer) recorded at 37 °C. (a) before addition of enzyme; (b) after incubation with SIRT2 for 20 mins at 37 °C. (c) after addition of authentic nicotinamide. Circles: signals from NAD⁺; Triangles signals from nicotinamide.

Figure 4

¹H-NMR spectrum obtained: (a) before addition of SIRT2; (b) 8 min after addition of SIRT2; the N-acetyl signal had disappeared consistent with substrate turnover by SIRT2. (c–g) 8 min after addition of enzyme with tenovin-6 at a final concentration of: (c) 25 µM; (d) 50 µM. (e) 100 µM; (f) 200 µM; (g) 500 µM.

Figure 5

The ¹H-NMR spectrum obtained: (a) before addition of enzyme; (b–f) 20 min after addition of: (b) SIRT2; (c) SIRT2 and tenovin-43 (200 µM); (d) SIRT2 with tenovin-6 (25 µM); (e) SIRT2 with tenovin-43 (25 µM); (f) SIRT2 with AGK2 (25 µM).

Figure 6

Ligand dosing curves showing the T_m shift dependence of SIRT2 on ligand concentration. Datapoints are experimental data obtained from curves as in Figure S6 while the lines are simulated according to the model as previously explained [26,27,28,29,30,31,32]. The dissociation constants at 37 °C, for the three ligands were: 0.67 µM for tenovin-43, 15 µM for tenovin-6 and 50 µM for tenovin-30f [9].