Research Article: Structure-based development of novel sirtuin inhibitors

Date Published: September 20, 2011

Publisher: Impact Journals LLC

Author(s): Christine Schlicker, Gina Boanca, Mahadevan Lakshminarasimhan, Clemens Steegborn.

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Abstract

Sirtuins are NAD+-dependent protein deacetylases regulating metabolism, stress responses, and aging processes. Mammalia possess seven Sirtuin isoforms, Sirt1-7, which differ in their subcellular localization and in the substrate proteins they deacetylate. The physiological roles of Sirtuins and their potential use as therapeutic targets for metabolic and aging-related diseases have spurred interest in the development of small-molecule Sirtuin modulators. Here, we describe an approach exploiting the structures available for four human Sirtuins for the development of isoform-specific inhibitors. Virtual docking of a compound library into the peptide binding pockets of crystal structures of Sirt2, 3, 5 and 6 yielded compounds potentially discriminating between these isoforms. Further characterization in activity assays revealed several inhibitory compounds with little isoform specificity, but also two compounds with micromolar potency and high specificity for Sirt2. Structure comparison and the predicted, shared binding mode of the Sirt2-specific compounds indicate a pocket extending from the peptide-binding groove as target side enabling isoform specificity. Our family-wide structure-based approach thus identified potent, Sirt2-specific inhibitors as well as lead structures and a target site for the development of compounds specific for other Sirtuin isoform, constituting an important step toward the identification of a complete panel of isoform-specific Sirtuin inhibitors.

Partial Text

Sirtuin proteins are protein deacetylases that contribute to the regulation of metabolism, stress responses, and aging processes [1-3]. They form class III of the protein deacetylase superfamily and hydrolyze one nico-tinamide adenine dinucleotide (NAD+) cosubstrate for each protein lysine side chain they deacetylate [4]. The seven mammalian Sirtuins (Sirt1-7) show different intracellular localization [5] and deacetylate different sets of substrate proteins. Sirt1 locates to the nucleus and regulates, e.g., transcription factors such as p53 and PGC-1α[1, 6]. Sirt6 and Sirt7 are also nuclear isoforms; Sirt7 regulates RNA polymerase I [7] and can deacetylate p53 [8], whereas Sirt6 deacetylates histones and regulates DNA stability and repair [9-11]. Sirt2 mainly resides in the cytosol where it can deacetylate α-tubulin [12]. Sirt3, 4, and 5 are located in mitochondria[5,13]. Sirt3 appears to regulate a large set of metabolic enzymes, whereas only one physiological Sirt5 substrate is known, carbamoylphosphate synthetase I [13-19]. Sirt4 is the only mammalian Sirtuin without known deacetylation substrate. Instead, Sirt4 was shown to ADP-ribosylate – a second type of reaction that can be catalyzed by Sirtuins – glutamate dehydrogenase [20].

The roles of Sirtuins in central physiological processes and as drug targets have led to great demand for specific inhibitors for research and therapy [22, 23]. Available compounds often feature limited or unknown specificities and mostly high micromolar potencies, and surprisingly little structural information is available for Sirtuin/inhibitor complexes, which could be used for rational improvement. However, we show here that the increasing number of Sirtuin isoform structures in non-inhibited state [31] allow the structure-based identification of novel, isoform-specific inhibitor classes. A previous docking study on Sirt2 [44], the only structurally characterized mammalian isoform at that time, yielded only two Sirt2 inhibitors with IC50 values below 100 μM (57 and 74 μM, respectively), and their isoform specificities were not evaluated. The lower hit rate and affinities than in our study might have several reasons. Tervo et al. used a structure as docking receptor that had undergone a molecular dynamics (MD) simulation, resulting in major deviations from the experimental structure, which might not well represent the major conformations of the protein. To take protein flexibility into account, using several structures – representing different conformers – simultaneously, or simulating flexible side-chains during docking are now often used approaches [45]. The first option is not yet possible for most Sirtuins due to the lack of such multiple structures, and it appears that ignoring receptor flexibility is in fact a viable strategy for identifying Sirtuin inhibitors, at least for Sirt2. To take side chain flexibility into account, however, might be an approach for identifying compounds specific for the other isoforms studied here, Sirt3, 5, and 6 [40]. A further difference to our approach was that several potential inhibitors were not considered for experimental testing due to missing features assumed to be important for inhibition, which might have removed potent compounds. Also, a different docking software (GOLD) and compound database were used (Maybridge). The Mabridge database, being much larger than the NCI diversity set used here, is unlikely to be a bottleneck, but it has been observed repeatedly that docking programs differ in their performance depending on the interaction type, e.g. small versus large and hydrophobicversus hydrophilic ligands [40]. Binding of the most potent inhibitors identified appears to be dominated by hydrophobic interactions with a large cavity, which was previously observed not to give best results with GOLD [40]. Finally, we used a receptor with partially occupied NAD+ binding site. Thereby, we tried to avoid to obtain compounds binding to the NAD+ binding site present in similar form in all Sirtuins, as well as other NAD-dependent enzymes, likely resulting in little specificity. This approach further takes into account that interactions with the bound NAD+ can contribute to inhibitor affinity. The docking models obtained indeed indicate that NAD+ forms part of the bottom of the pocket likely occupied by the inhibitors. However, instead of mainly occupying the peptide binding cleft, the compounds were preferentially docked perpendicular to this cleft, extending into a hydrophobic pocket in the Zn2+ domain. If this binding orientation is confirmed by structural studies, then fusing to compounds such as 6 smaller substituents exploiting the isoforms specific features of the peptide binding grooves might be an attractive approach for further improvement of inhibitor potency and specificity.

 

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