Thienopyrimidinone Based Sirtuin-2 (SIRT2)-Selective Inhibitors Bind in the Ligand Induced Selectivity Pocket

Abstract

Sirtuins (SIRTs) are NAD-dependent deacylases, known to be involved in a variety of pathophysiological processes and thus remain promising therapeutic targets for further validation. Previously, we reported a novel thienopyrimidinone SIRT2 inhibitor with good potency and excellent selectivity for SIRT2. Herein, we report an extensive SAR study of this chemical series and identify the key pharmacophoric elements and physiochemical properties that underpin the excellent activity observed. New analogues have been identified with submicromolar SIRT2 inhibtory activity and good to excellent SIRT2 subtype-selectivity. Importantly, we report a cocrystal structure of one of our compounds (29c) bound to SIRT2. This reveals our series to induce the formation of a previously reported selectivity pocket but to bind in an inverted fashion to what might be intuitively expected. We believe these findings will contribute significantly to an understanding of the mechanism of action of SIRT2 inhibitors and to the identification of refined, second generation inhibitors.

Figure 1

Structures of representative reported sirtuin inhibitors including their IC₅₀ values. The presented IC₅₀ values should be compared with caution, as differing in vitro assays and assay conditions were used to evaluate these compounds. Comparable structural features of SirReal2 (8) and ICL-SIRT078 (19a) are highlighted (see Discussion in the main text).

Figure 2

Plot of cLogP vs percentage inhibition (at 10 μM) for all analogues. Active molecules have 3/4 aromatic rings and lie within the cLogP range 3.8–5.3 (circled). The analysis was performed in Datawarrior (version 4.4.3)

Figure 3

(a) 29c bound to SIRT2 is represented in green sticks in the Fourier difference omit electron density map (mFo-DFc) contoured at 3 σ. (b) X-ray structure of SIRT2 (green ribbons) bound to 29c (green sticks). Previously reported SIRT2 structure (pink ribbons, PDB 4RMG) co-crystallized with 8 (pink sticks) and NAD⁺ (yellow sticks) is also shown for comparison. The acyl-lysine (orange stick) represents the position of the substrate in the superposed structure of SIRT3 (ribbon not shown) in complex with AceCS2 peptide (PDB 3GLR). (c) Electrostatic surface of SIRT2 illustrating the hydrophobic selectivity pocket induced by 4-methoxy-naphthalen-1-yl ring of 29c. The electrostatic potential was calculated using the APBS plugin of Pymol and represented on the surface of the protein with a scale ranging from −20 (red) to 20 (blue) KbT/Ec. The naphthyl ring of 29c buries more deeply into the selectivity pocket in comparison to the dimethylpyrimidine (DMP) ring of 8. The dimethylisoxazole (DMI) group of 29c occupies a wide channel and extends toward the entry of the substrate binding pocket. (d) Interactions of analogue 29c within the SIRT2 pocket. The naphthyl ring forms π-π interactions (magenta dashed lines) with Tyr139 and Phe190, while the isoxazole moiety forms a π-π stacking interaction with Phe119. The protonated N-7′ amino group maintains π-cation interactions (orange dashed lines) with Phe119 and Phe96. Various hydrophobic interactions are represented by the light pink dashed lines. Non classical C–H hydrogen bonds between 29c and Ala135, Leu138, and Pro94 are shown by yellow dashed lines. For clarity, only interacting amino acid residues are shown. Images were generated using the PyMOL Molecular Graphics System, version 1.7 Schrödinger, LLC.

Figure 4

Effect of SIRT2 inhibitors on tubulin acetylation. MCF-7 cell lines were treated with inhibitors at the doses stated for 24 h in the presence of trichostatin A (TSA) (50 nM). TSA was added to ensure the inhibition of the non-sirtuin histone deacetylase activity. Protein samples were analyzed by Western blotting using antibodies against acetylated (lysine-40) α-tubulin, total α-tubulin, and β-actin. β-Actin was probed as a control for protein loading. (a) Compound 29c increased the α-tubulin acetylation level at 10 μM and retained consistent to 50 μM. (b) Compound 25g, as a negative control, showed no effect on the acetylation of α-tubulin. (c) Compound 19a showed increased acetylated α-tubulin level at 20 μM and decreased level at 50 μM. (d) 6 as a positive control showed significant increase of α-tubulin acetylation at 10 to 50 μM. N = 3.