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Selective Sirt2 inhibition by ligand-induced rearrangement of the active site.

Rumpf T, Schiedel M, Karaman B, Roessler C, North BJ, Lehotzky A, Oláh J, Ladwein KI, Schmidtkunz K, Gajer M, Pannek M, Steegborn C, Sinclair DA, Gerhardt S, Ovádi J, Schutkowski M, Sippl W, Einsle O, Jung M - Nat Commun (2015)

Bottom Line: Potency and the unprecedented Sirt2 selectivity are based on a ligand-induced structural rearrangement of the active site unveiling a yet-unexploited binding pocket.Application of the most potent Sirtuin-rearranging ligand, termed SirReal2, leads to tubulin hyperacetylation in HeLa cells and induces destabilization of the checkpoint protein BubR1, consistent with Sirt2 inhibition in vivo.Our structural insights into this unique mechanism of selective sirtuin inhibition provide the basis for further inhibitor development and selective tools for sirtuin biology.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmaceutical Sciences, Albert-Ludwigs-University Freiburg, Albertstraße 25, 79104 Freiburg im Breisgau, Germany.

ABSTRACT
Sirtuins are a highly conserved class of NAD(+)-dependent lysine deacylases. The human isotype Sirt2 has been implicated in the pathogenesis of cancer, inflammation and neurodegeneration, which makes the modulation of Sirt2 activity a promising strategy for pharmaceutical intervention. A rational basis for the development of optimized Sirt2 inhibitors is lacking so far. Here we present high-resolution structures of human Sirt2 in complex with highly selective drug-like inhibitors that show a unique inhibitory mechanism. Potency and the unprecedented Sirt2 selectivity are based on a ligand-induced structural rearrangement of the active site unveiling a yet-unexploited binding pocket. Application of the most potent Sirtuin-rearranging ligand, termed SirReal2, leads to tubulin hyperacetylation in HeLa cells and induces destabilization of the checkpoint protein BubR1, consistent with Sirt2 inhibition in vivo. Our structural insights into this unique mechanism of selective sirtuin inhibition provide the basis for further inhibitor development and selective tools for sirtuin biology.

No MeSH data available.


Related in: MedlinePlus

SirReal2 selectively inhibits Sirt2 via a Sirt2-specific amino acid network.(a) Structural sequence alignment of the Sirt1–6 deacylase domain. The residues that presumably interact with SirReal2 are highlighted in yellow if they are equivalent to the residues of Sirt2. If they differ from the residues of Sirt2, they are highlighted in red. The structural sequence alignment was generated using T-Coffee35 and slightly modified. (b–d) Surface representation of the binding pockets of SirReal2 in Sirt2–SirReal2–NAD+ and in the homology models of Sirt1 (Sirt1-HM) and Sirt3 (Sirt3-HM). The residues that differ in the three isotypes are represented as sticks (Sirt2, slate blue; Sirt1, brown; Sirt3, turquoise). SirReal2 is shown as light pink sticks (Sirt2–SirReal2–NAD+), lime sticks (Sirt1-HM) and olive sticks (Sirt3-HM). Despite the highly conserved active site, the binding pockets appear in very different shapes due to differences in the amino acid sequence. In case of Sirt1 the amino acids that contribute to an unfavourable binding of SirReal2 are Ile279, Met296, Phe312, Ile316 and Cys380. In the case of Sirt3, the amino acids are Tyr204, Gly232, Gly265 and Val272.
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f6: SirReal2 selectively inhibits Sirt2 via a Sirt2-specific amino acid network.(a) Structural sequence alignment of the Sirt1–6 deacylase domain. The residues that presumably interact with SirReal2 are highlighted in yellow if they are equivalent to the residues of Sirt2. If they differ from the residues of Sirt2, they are highlighted in red. The structural sequence alignment was generated using T-Coffee35 and slightly modified. (b–d) Surface representation of the binding pockets of SirReal2 in Sirt2–SirReal2–NAD+ and in the homology models of Sirt1 (Sirt1-HM) and Sirt3 (Sirt3-HM). The residues that differ in the three isotypes are represented as sticks (Sirt2, slate blue; Sirt1, brown; Sirt3, turquoise). SirReal2 is shown as light pink sticks (Sirt2–SirReal2–NAD+), lime sticks (Sirt1-HM) and olive sticks (Sirt3-HM). Despite the highly conserved active site, the binding pockets appear in very different shapes due to differences in the amino acid sequence. In case of Sirt1 the amino acids that contribute to an unfavourable binding of SirReal2 are Ile279, Met296, Phe312, Ile316 and Cys380. In the case of Sirt3, the amino acids are Tyr204, Gly232, Gly265 and Val272.

Mentions: To analyse the basis of this high isotype selectivity, we created a structural sequence alignment of the deacylase domain of Sirt1–6 and compared the crystal structure of the Sirt2–SirReal2 complex with available crystal structures of sirtuins in their open conformation (Fig. 6a, Supplementary Fig. 7a,b)35. Assuming that SirReal2 binds to the other sirtuin isotypes in a similar fashion as observed for Sirt2, Sirt4–6 exhibit major differences in their amino acid sequence. The structural differences are also very pronounced (Supplementary Fig. 7a) rationalizing the observed lacking in vitro inhibition of Sirt4–6 by SirReal2. Sirt1 and Sirt3, on the other hand, are phylogenetically more closely related to Sirt2 and show only minor sequence variations36. Their conformation is more similar to the Sirt2–SirReal2–NAD+ complex than to the conformation of the isotypes Sirt5/6 (Supplementary Fig. 7b). But they still show major structural differences (r.m.s.d. (Cα atoms)=1.6 Å). As it was not possible to dock SirReal2 in any of the available Sirt1 and Sirt3 X-ray crystal structures (Supplementary Methods), we wanted to probe whether Sirt1 and Sirt3 were able to adopt a similar conformation as observed in the Sirt2–SirReal2 structures that would allow binding of SirReal2. This would enable us to see whether the minor sequence variations within the deacylase domain of Sirt1–3 would have an influence on SirReal2 binding. Therefore, we generated homology models of Sirt1 (Sirt1-HM) and Sirt3 (Sirt3-HM) based on our Sirt2–SirReal2 structures (Supplementary Methods). Stereochemical analyses as well as molecular dynamics simulations indicated high-quality model structures, and it was indeed possible to dock SirReal2 into these homology models (Supplementary Fig. 7c–h). However, the docking poses of SirReal2 in Sirt1-HM and Sirt3-HM gave less favourable docking scores compared with the requisite scores for the docking poses of SirReal2 in Sirt2–SirReal2 structures. Here the position and the conformation of SirReal2 were correctly predicted (Fig. 6b). In case of Sirt1, residues Leu103, Ile118, Leu134, Leu138 and Leu206 of Sirt2 are substituted with Ile279, Met296, Phe312, Ile316 and Cys380 (Fig. 6c). Cys380 gives the hypothetical selectivity pocket of SirReal2 in Sirt1-HM a very different shape and changes its surface characteristics. The bulky Phe312 and Ile316 as well as Met296 and Ile279 also tighten the EC-site, resulting in an unfavourable orientation of the aminothiazole and naphthyl moieties in possible docking poses. In the case of Sirt3, the differences are mainly located at the selectivity pocket. Here Phe143, Thr171, Leu206 and Ile213 of Sirt2 are substituted by Tyr204, Gly232, Gly265 and Val272 (Fig. 6d). The less bulky Gly232, Gly265, Val272 of Sirt3 form a much wider and also more solvent-accessible selectivity pocket as compared with the Sirt2–SirReal2 structures. In contrast to the SirReal2-binding pockets of the homology models of Sirt1 and Sirt3, SirReal2 bound to Sirt2 can adopt a conformation that is in almost perfect complementarity with the protein, which is stabilized by the intramolecular hydrogen bond between the DMP substituent and the amide. This is not possible in Sirt1 and Sirt3 and also rationalizes the observed isotype selectivity.


Selective Sirt2 inhibition by ligand-induced rearrangement of the active site.

Rumpf T, Schiedel M, Karaman B, Roessler C, North BJ, Lehotzky A, Oláh J, Ladwein KI, Schmidtkunz K, Gajer M, Pannek M, Steegborn C, Sinclair DA, Gerhardt S, Ovádi J, Schutkowski M, Sippl W, Einsle O, Jung M - Nat Commun (2015)

SirReal2 selectively inhibits Sirt2 via a Sirt2-specific amino acid network.(a) Structural sequence alignment of the Sirt1–6 deacylase domain. The residues that presumably interact with SirReal2 are highlighted in yellow if they are equivalent to the residues of Sirt2. If they differ from the residues of Sirt2, they are highlighted in red. The structural sequence alignment was generated using T-Coffee35 and slightly modified. (b–d) Surface representation of the binding pockets of SirReal2 in Sirt2–SirReal2–NAD+ and in the homology models of Sirt1 (Sirt1-HM) and Sirt3 (Sirt3-HM). The residues that differ in the three isotypes are represented as sticks (Sirt2, slate blue; Sirt1, brown; Sirt3, turquoise). SirReal2 is shown as light pink sticks (Sirt2–SirReal2–NAD+), lime sticks (Sirt1-HM) and olive sticks (Sirt3-HM). Despite the highly conserved active site, the binding pockets appear in very different shapes due to differences in the amino acid sequence. In case of Sirt1 the amino acids that contribute to an unfavourable binding of SirReal2 are Ile279, Met296, Phe312, Ile316 and Cys380. In the case of Sirt3, the amino acids are Tyr204, Gly232, Gly265 and Val272.
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Related In: Results  -  Collection

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f6: SirReal2 selectively inhibits Sirt2 via a Sirt2-specific amino acid network.(a) Structural sequence alignment of the Sirt1–6 deacylase domain. The residues that presumably interact with SirReal2 are highlighted in yellow if they are equivalent to the residues of Sirt2. If they differ from the residues of Sirt2, they are highlighted in red. The structural sequence alignment was generated using T-Coffee35 and slightly modified. (b–d) Surface representation of the binding pockets of SirReal2 in Sirt2–SirReal2–NAD+ and in the homology models of Sirt1 (Sirt1-HM) and Sirt3 (Sirt3-HM). The residues that differ in the three isotypes are represented as sticks (Sirt2, slate blue; Sirt1, brown; Sirt3, turquoise). SirReal2 is shown as light pink sticks (Sirt2–SirReal2–NAD+), lime sticks (Sirt1-HM) and olive sticks (Sirt3-HM). Despite the highly conserved active site, the binding pockets appear in very different shapes due to differences in the amino acid sequence. In case of Sirt1 the amino acids that contribute to an unfavourable binding of SirReal2 are Ile279, Met296, Phe312, Ile316 and Cys380. In the case of Sirt3, the amino acids are Tyr204, Gly232, Gly265 and Val272.
Mentions: To analyse the basis of this high isotype selectivity, we created a structural sequence alignment of the deacylase domain of Sirt1–6 and compared the crystal structure of the Sirt2–SirReal2 complex with available crystal structures of sirtuins in their open conformation (Fig. 6a, Supplementary Fig. 7a,b)35. Assuming that SirReal2 binds to the other sirtuin isotypes in a similar fashion as observed for Sirt2, Sirt4–6 exhibit major differences in their amino acid sequence. The structural differences are also very pronounced (Supplementary Fig. 7a) rationalizing the observed lacking in vitro inhibition of Sirt4–6 by SirReal2. Sirt1 and Sirt3, on the other hand, are phylogenetically more closely related to Sirt2 and show only minor sequence variations36. Their conformation is more similar to the Sirt2–SirReal2–NAD+ complex than to the conformation of the isotypes Sirt5/6 (Supplementary Fig. 7b). But they still show major structural differences (r.m.s.d. (Cα atoms)=1.6 Å). As it was not possible to dock SirReal2 in any of the available Sirt1 and Sirt3 X-ray crystal structures (Supplementary Methods), we wanted to probe whether Sirt1 and Sirt3 were able to adopt a similar conformation as observed in the Sirt2–SirReal2 structures that would allow binding of SirReal2. This would enable us to see whether the minor sequence variations within the deacylase domain of Sirt1–3 would have an influence on SirReal2 binding. Therefore, we generated homology models of Sirt1 (Sirt1-HM) and Sirt3 (Sirt3-HM) based on our Sirt2–SirReal2 structures (Supplementary Methods). Stereochemical analyses as well as molecular dynamics simulations indicated high-quality model structures, and it was indeed possible to dock SirReal2 into these homology models (Supplementary Fig. 7c–h). However, the docking poses of SirReal2 in Sirt1-HM and Sirt3-HM gave less favourable docking scores compared with the requisite scores for the docking poses of SirReal2 in Sirt2–SirReal2 structures. Here the position and the conformation of SirReal2 were correctly predicted (Fig. 6b). In case of Sirt1, residues Leu103, Ile118, Leu134, Leu138 and Leu206 of Sirt2 are substituted with Ile279, Met296, Phe312, Ile316 and Cys380 (Fig. 6c). Cys380 gives the hypothetical selectivity pocket of SirReal2 in Sirt1-HM a very different shape and changes its surface characteristics. The bulky Phe312 and Ile316 as well as Met296 and Ile279 also tighten the EC-site, resulting in an unfavourable orientation of the aminothiazole and naphthyl moieties in possible docking poses. In the case of Sirt3, the differences are mainly located at the selectivity pocket. Here Phe143, Thr171, Leu206 and Ile213 of Sirt2 are substituted by Tyr204, Gly232, Gly265 and Val272 (Fig. 6d). The less bulky Gly232, Gly265, Val272 of Sirt3 form a much wider and also more solvent-accessible selectivity pocket as compared with the Sirt2–SirReal2 structures. In contrast to the SirReal2-binding pockets of the homology models of Sirt1 and Sirt3, SirReal2 bound to Sirt2 can adopt a conformation that is in almost perfect complementarity with the protein, which is stabilized by the intramolecular hydrogen bond between the DMP substituent and the amide. This is not possible in Sirt1 and Sirt3 and also rationalizes the observed isotype selectivity.

Bottom Line: Potency and the unprecedented Sirt2 selectivity are based on a ligand-induced structural rearrangement of the active site unveiling a yet-unexploited binding pocket.Application of the most potent Sirtuin-rearranging ligand, termed SirReal2, leads to tubulin hyperacetylation in HeLa cells and induces destabilization of the checkpoint protein BubR1, consistent with Sirt2 inhibition in vivo.Our structural insights into this unique mechanism of selective sirtuin inhibition provide the basis for further inhibitor development and selective tools for sirtuin biology.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pharmaceutical Sciences, Albert-Ludwigs-University Freiburg, Albertstraße 25, 79104 Freiburg im Breisgau, Germany.

ABSTRACT
Sirtuins are a highly conserved class of NAD(+)-dependent lysine deacylases. The human isotype Sirt2 has been implicated in the pathogenesis of cancer, inflammation and neurodegeneration, which makes the modulation of Sirt2 activity a promising strategy for pharmaceutical intervention. A rational basis for the development of optimized Sirt2 inhibitors is lacking so far. Here we present high-resolution structures of human Sirt2 in complex with highly selective drug-like inhibitors that show a unique inhibitory mechanism. Potency and the unprecedented Sirt2 selectivity are based on a ligand-induced structural rearrangement of the active site unveiling a yet-unexploited binding pocket. Application of the most potent Sirtuin-rearranging ligand, termed SirReal2, leads to tubulin hyperacetylation in HeLa cells and induces destabilization of the checkpoint protein BubR1, consistent with Sirt2 inhibition in vivo. Our structural insights into this unique mechanism of selective sirtuin inhibition provide the basis for further inhibitor development and selective tools for sirtuin biology.

No MeSH data available.


Related in: MedlinePlus