title: Activation and inhibition of Sirt6 by small molecules
Creators: name: You, Weijie
Publisher: name: University of Bayreuth
Year: 2019
Subjects: subject: Sirtuin activators
subject: Sirtuin inhibitors
subject: protein structures
subject: enzyme complex
subject: 570

description: Mammals have 7 Sirtuin isoforms (SIRT1–7) localized in different subcellular compartments and with different functions. Sirt6 is a heterochromatin-associated protein that serves in the regulation of telomere maintenance, DNA repair and gene expression. A Sirt6 knockout causes aging-associated degeneration in mice. Conversely, Sirt6 overexpression has been reported to extend lifespan in male mice. Thus, activators of Sirt6 are considered to be attractive therapeutics to treat cancer and age-related diseases. Sirtuin-activating compounds (STACs) were initially described to activate Sirt1, and bind to the Sirt1’s unique N-terminal segment (SBD domain). In contrast, progress towards the development of activators which modulate the activity of other Sirtuin isoforms that lack the Sirt1’s SBD domain has been significantly taken up speed only recently. The pyrrolo[1,2-a]quinoxaline-derived compound UBCS038 were previously characterized as a very weak Sirt6 activator. We screened a panel of pyrrolo[1,2-a]quinoxaline-derived derivatives and identified several compounds as more potent activators for Sirt6-dependent deacetylation of peptide substrates, histone proteins and complete nucleosomes. Furthermore, these compounds showed no effect on Sirt1, 2 and 3, but promoted the desuccinylase activity of Sirt5. Solving the complex structures by using X ray crystallography revealed that these compounds as the first synthetic Sirt6 activators bind to the Sirt6 specific acyl channel branching from the catalytic core and activate Sirt6 deacetylation by an allosteric mechanism, which can explain their different effects against acyl substrates. Moreover, structure-activity relationship analyses indicate that the presence of a nitrogen atom in the meta position of the pyridine group is crucial for ligand affinity or Sirt6 activation. Our data reveal promising acyl-selective Sirt6 activators and offer clues for the development of Sirt6 drug-like compounds as molecular tools and potential therapeutics. Next, we utilized a robust mass spectrometry-based assay to determine the effect of quercetin on Sirtuins. We confirmed the weak activation effect of quercetin for Sirt6 and found out that quercetin can inhibit other Sirtuin isoforms. We solved crystal structures of a Sirt6/quercetin complex and a Sirt2/quercetin complex to reveal two mutually exclusive binding modes that lead to the dual effects of quercetion among Sirtuins. Based on the structural information, we have identified a novel quercetin derivative, namely isoquercetin, able to activate Sirt6 with improved selectivity. We then determined the crystal structure of Sirt6 and isoquercetin to better understand its activation mechanism. Moreover, we also confirmed the effects of quercetin derivatives catechin gallate (the first potent Sirt6 inhibitor) and cyaniding (the most robust Sirt6 activator) in our mass spectrometry assay and further determined the crystal structures of Sirt6 in complex with these two compounds. Our results demonstrate that the substitution in the C ring of quercetin can significantly alter the effect of compounds from activation to inhibition, and reveal molecular features essential for further drug development. Trichostatin A (TSA) inhibits classical HDACs by chelating the zinc ion in the active site, but in Sirtuins the zinc ion does not play a catalytic role and its binding site is remote from the catalytic site. Surprisingly, TSA is also identified to show promising efficacy towards Sirt6, while demonstrates no appreciable inhibitory effect on Sirt1-3 or Sirt5.We obtained the Sirt6/TSA complex structure by soaking experiment. We found that TSA binds into the C pocket of the NAD+ binding site and inserts into Sirt6's specific acyl binding channel, which thus explains its relative specificity for Sirtuin isoforms. Binding studies further revealed that TSA binds to Sirt6 in a noncompetitive manner with respect to ADP-ribose (fragment of NAD+) or substrate, suggesting that TSA might acts as sirtuin inhibitor via binding to the C pocket upon the dissociation of NAM group and prevent product release by stabilizing the intermediate complex. Our data provides insight into the TSA binding site and the inhibition mechanism to support the development of Sirt6-selective inhibitors.