potency of initial fragments. Cui et al. [96] used chemical fragments derived from
suramin and nicotinamide and designed several 5-benzamidonaphthalen-1/2-yloxynicotinamide derivatives using the available crystal structure of Sirt2. Among these,
compound 14 (Fig. 5) showed nanomolar inhibitory activity against Sirt2
(IC 50 ¼ 0.0483 μM) and 200–900-fold selectivity over Sirt1 (IC 50 12.0 μM) and
Sirt3 (IC 50 44.2 μM). The docking of inhibitor 14 predicted a binding mode where
the nicotinamide moiety binds into the nicotinamide-binding pocket of Sirt2 and
forms hydrogen bonds with the conserved residues Ile169 and Asp170, whereas the
naphthalene group makes π-π interactions with either Phe96 or Phe119 located in the
acetyllysine tunnel. Interestingly, a structurally similar compound (15, Fig. 5) was
recently crystallized in complex with Sirt2 (PDB ID: 5Y5N) and showed a different
binding mode. Unexpectedly, the benzamide moiety of 15 does not occupy the
C-pocket as the nicotinamide of NAD
+ (Asp170) but undergoes hydrogen bonds
with two cocrystallized water molecules. The hydrophobic aromatic tail of the
inhibitor interacts with the hydrophobic selectivity pocket (Tyr139, Phe143,
Phe190). This hydrophobic pocket was identified as the binding site of the myristoyl
chain, which is also recognized as a substrate modification by Sirt2 [97].
Highly selective Sirt2 inhibitors containing an aminothiazole scaffold were
identified by a focused-library screening of putative kinase inhibitors [90]. These
inhibitors were named SirReals (sirtuin rearranging ligands) because obtained
cocrystal structures of Sirt2 showed that the inhibitors rearrange the Sirt2 binding
pocket and behave as allosteric inhibitors. Due to the allosteric inhibition, SirReals
were found to be highly selective and represented a promising starting point for
structure-based optimization. SirReal2 (16, Figs. 4b and 5) binds to a highly
hydrophobic pocket adjacent to the zinc-binding domain and does not prevent the
binding of NAD
+ in its productive conformation. Guided by the structural insights
obtained from the Sirt2-SirReal2 crystal structure, Schiedel et al. aimed to systematically probe the limits of variation within the scaffold of the SirReals [98]. A
library of designed aminothiazoles was docked into the Sirt2 crystal structure and
was used to guide the structural optimization. About 50 compounds were finally
synthesized and tested in vitro resulting in low nanomolar Sirt2 inhibitors. Binding
free energies of the Sirt2-inhibitor complexes were calculated using the MM-GBSA
approach. The calculated MM-GBSA protein-inhibitor binding energies showed a
good correlation with the observed Sirt2 inhibition values (r
2
¼ 0.67, RMSE ¼ 0.60).
Moreover, the authors were able to rationalize their results with a further crystal
structure of Sirt2 in complex with the most potent inhibitor (17, Fig. 5) from this
series [98].
4 Histone Methyltransferases
Histone methylation is performed by proteins that belong to two different classes of
enzymes called protein arginine methyltransferases (PRMTs) and protein lysine
methyltransferases (PKMTs). The latter class is generally subdivided into SET
Structure-Based Design of Epigenetic Inhibitors
465
suramin and nicotinamide and designed several 5-benzamidonaphthalen-1/2-yloxynicotinamide derivatives using the available crystal structure of Sirt2. Among these,
compound 14 (Fig. 5) showed nanomolar inhibitory activity against Sirt2
(IC 50 ¼ 0.0483 μM) and 200–900-fold selectivity over Sirt1 (IC 50 12.0 μM) and
Sirt3 (IC 50 44.2 μM). The docking of inhibitor 14 predicted a binding mode where
the nicotinamide moiety binds into the nicotinamide-binding pocket of Sirt2 and
forms hydrogen bonds with the conserved residues Ile169 and Asp170, whereas the
naphthalene group makes π-π interactions with either Phe96 or Phe119 located in the
acetyllysine tunnel. Interestingly, a structurally similar compound (15, Fig. 5) was
recently crystallized in complex with Sirt2 (PDB ID: 5Y5N) and showed a different
binding mode. Unexpectedly, the benzamide moiety of 15 does not occupy the
C-pocket as the nicotinamide of NAD
+ (Asp170) but undergoes hydrogen bonds
with two cocrystallized water molecules. The hydrophobic aromatic tail of the
inhibitor interacts with the hydrophobic selectivity pocket (Tyr139, Phe143,
Phe190). This hydrophobic pocket was identified as the binding site of the myristoyl
chain, which is also recognized as a substrate modification by Sirt2 [97].
Highly selective Sirt2 inhibitors containing an aminothiazole scaffold were
identified by a focused-library screening of putative kinase inhibitors [90]. These
inhibitors were named SirReals (sirtuin rearranging ligands) because obtained
cocrystal structures of Sirt2 showed that the inhibitors rearrange the Sirt2 binding
pocket and behave as allosteric inhibitors. Due to the allosteric inhibition, SirReals
were found to be highly selective and represented a promising starting point for
structure-based optimization. SirReal2 (16, Figs. 4b and 5) binds to a highly
hydrophobic pocket adjacent to the zinc-binding domain and does not prevent the
binding of NAD
+ in its productive conformation. Guided by the structural insights
obtained from the Sirt2-SirReal2 crystal structure, Schiedel et al. aimed to systematically probe the limits of variation within the scaffold of the SirReals [98]. A
library of designed aminothiazoles was docked into the Sirt2 crystal structure and
was used to guide the structural optimization. About 50 compounds were finally
synthesized and tested in vitro resulting in low nanomolar Sirt2 inhibitors. Binding
free energies of the Sirt2-inhibitor complexes were calculated using the MM-GBSA
approach. The calculated MM-GBSA protein-inhibitor binding energies showed a
good correlation with the observed Sirt2 inhibition values (r
2
¼ 0.67, RMSE ¼ 0.60).
Moreover, the authors were able to rationalize their results with a further crystal
structure of Sirt2 in complex with the most potent inhibitor (17, Fig. 5) from this
series [98].
4 Histone Methyltransferases
Histone methylation is performed by proteins that belong to two different classes of
enzymes called protein arginine methyltransferases (PRMTs) and protein lysine
methyltransferases (PKMTs). The latter class is generally subdivided into SET
Structure-Based Design of Epigenetic Inhibitors
465