Identification of a cellularly active SIRT6 allosteric activator

Abstract

SIRT6, a member of the SIRT deacetylase family, is responsible for deacetylation of histone H3 Nε-acetyl-lysines 9 (H3K9ac) and 56 (H3K56ac). As a tumor suppressor, SIRT6 has frequently been found to have low expression in various cancers. Here, we report the identification of MDL-800, a selective SIRT6 activator. MDL-800 increased the deacetylase activity of SIRT6 by up to 22-fold via binding to an allosteric site; this interaction led to a global decrease in H3K9ac and H3K56ac levels in human hepatocellular carcinoma (HCC) cells. Consequently, MDL-800 inhibited the proliferation of HCC cells via SIRT6-driven cell-cycle arrest and was effective in a tumor xenograft model. Together, these data demonstrate that pharmacological activation of SIRT6 is a potential therapeutic approach for the treatment of HCC. MDL-800 is a first-in-class small-molecule cellular SIRT6 activator that can be used to physiologically and pathologically investigate the roles of SIRT6 deacetylation.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Discovery and biochemical characterization of MDL-800 and MDL-801 as activators of SIRT6 deacetylation.
Fig. 2: Cocrystal structure of MDL-801 bound to an allosteric site of SIRT6.
Fig. 3: MDL-800 induces H3K9ac and H3K56ac deacetylation and represses proliferation of HCC cells.
Fig. 4: MDL-800 arrests the cell cycle in HCC cells.
Fig. 5: MDL-800 activates SIRT6 deacetylation, thereby regulating the HCC cell cycle.
Fig. 6: MDL-800 inhibits xenograft tumor growth of HCC cells in immunocompromised mice.

Data availability

Crystal structures and diffraction data have been deposited in the Protein Data Bank under accession codes PDB 5X16 (SIRT6-ADPR) and PDB 5Y2F (SIRT6–ADPR–MDL-801). All other data generated or analyzed during the study in this published article (and its supplementary information files) are available from the corresponding author on reasonable request.

References

  1. 1.

    Chalkiadaki, A. & Guarente, L. The multifaceted functions of sirtuins in cancer. Nat. Rev. Cancer 15, 608–624 (2015).

    CAS  Article  Google Scholar 

  2. 2.

    Feldman, J. L., Dittenhafer-Reed, K. E. & Denu, J. M. Sirtuin catalysis and regulation. J. Biol. Chem. 287, 42419–42427 (2012).

    CAS  Article  Google Scholar 

  3. 3.

    Kugel, S. & Mostoslavsky, R. Chromatin and beyond: the multitasking roles for SIRT6. Trends Biochem. Sci. 39, 72–81 (2014).

    CAS  Article  Google Scholar 

  4. 4.

    Tasselli, L., Zheng, W. & Chua, K. F. SIRT6: novel mechanisms and links to aging and disease. Trends Endocrinol. Metab. 28, 168–185 (2017).

    CAS  Article  Google Scholar 

  5. 5.

    Irminger-Finger, I. Science of cancer and aging. J. Clin. Oncol. 25, 1844–1851 (2007).

    CAS  Article  Google Scholar 

  6. 6.

    Sebastián, C. et al. The histone deacetylase SIRT6 is a tumor suppressor that controls cancer metabolism. Cell 151, 1185–1199 (2012).

    Article  Google Scholar 

  7. 7.

    Lin, Z. et al. USP10 antagonizes c-Myc transcriptional activation through SIRT6 stabilization to suppress tumor formation. Cell Rep. 5, 1639–1649 (2013).

    CAS  Article  Google Scholar 

  8. 8.

    Marquardt, J. U. et al. Sirtuin-6-dependent genetic and epigenetic alterations are associated with poor clinical outcome in hepatocellular carcinoma patients. Hepatology 58, 1054–1064 (2013).

    CAS  Article  Google Scholar 

  9. 9.

    Kugel, S. et al. SIRT6 suppresses pancreatic cancer through control of Lin28b. Cell 165, 1401–1415 (2016).

    CAS  Article  Google Scholar 

  10. 10.

    Van Meter, M., Gorbunova, V. & Seluanov, A. SIRT6: a promising target for cancer prevention and therapy. Adv. Exp. Med. Biol. 818, 181–196 (2014).

    Article  Google Scholar 

  11. 11.

    Pan, P. W. et al. Structure and biochemical functions of SIRT6. J. Biol. Chem. 286, 14575–14587 (2011).

    CAS  Article  Google Scholar 

  12. 12.

    Jiang, H. et al. SIRT6 regulates TNF-α secretion through hydrolysis of long-chain fatty acyl lysine. Nature 496, 110–113 (2013).

    CAS  Article  Google Scholar 

  13. 13.

    Kawahara, T. L. et al. SIRT6 links histone H3 lysine 9 deacetylation to NF-kappaB-dependent gene expression and organismal life span. Cell 136, 62–74 (2009).

    CAS  Article  Google Scholar 

  14. 14.

    Michishita, E. et al. Cell cycle-dependent deacetylation of telomeric histone H3 lysine K56 by human SIRT6. Cell Cycle 8, 2664–2666 (2009).

    CAS  Article  Google Scholar 

  15. 15.

    Gil, R., Barth, S., Kanfi, Y. & Cohen, H. Y. SIRT6 exhibits nucleosome-dependent deacetylase activity. Nucleic Acids Res. 41, 8537–8545 (2013).

    CAS  Article  Google Scholar 

  16. 16.

    Tasselli, L. et al. SIRT6 deacetylates H3K18ac at pericentric chromatin to prevent mitotic errors and cellular senescence. Nat. Struct. Mol. Biol. 23, 434–440 (2016).

    CAS  Article  Google Scholar 

  17. 17.

    Ghosh, S., Liu, B., Wang, Y., Hao, Q. & Zhou, Z. Lamin A is an endogenous SIRT6 activator and promotes SIRT6-mediated DNA repair. Cell Rep. 13, 1396–1406 (2015).

    CAS  Article  Google Scholar 

  18. 18.

    Feldman, J. L., Baeza, J. & Denu, J. M. Activation of the protein deacetylase SIRT6 by long-chain fatty acids and widespread deacylation by mammalian sirtuins. J. Biol. Chem. 288, 31350–31356 (2013).

    CAS  Article  Google Scholar 

  19. 19.

    You, W. et al. Structural basis of sirtuin 6 activation by synthetic small molecules. Angew. Chem. Int. Edn Engl. 56, 1007–1011 (2017).

    CAS  Article  Google Scholar 

  20. 20.

    Parenti, M. D. et al. Discovery of novel and selective SIRT6 inhibitors. J. Med. Chem. 57, 4796–4804 (2014).

    CAS  Article  Google Scholar 

  21. 21.

    He, B., Hu, J., Zhang, X. & Lin, H. Thiomyristoyl peptides as cell-permeable Sirt6 inhibitors. Org. Biomol. Chem. 12, 7498–7502 (2014).

    CAS  Article  Google Scholar 

  22. 22.

    Huang, W. et al. Allosite: a method for predicting allosteric sites. Bioinformatics 29, 2357–2359 (2013).

    CAS  Article  Google Scholar 

  23. 23.

    Kokkonen, P. et al. Studying SIRT6 regulation using H3K56 based substrate and small molecules. Eur. J. Pharm. Sci. 63, 71–76 (2014).

    CAS  Article  Google Scholar 

  24. 24.

    Pacholec, M. et al. SRT1720, SRT2183, SRT1460, and resveratrol are not direct activators of SIRT1. J. Biol. Chem. 285, 8340–8351 (2010).

    CAS  Article  Google Scholar 

  25. 25.

    Sinclair, D. A. & Guarente, L. Small-molecule allosteric activators of sirtuins. Annu. Rev. Pharmacol. Toxicol. 54, 363–380 (2014).

    CAS  Article  Google Scholar 

  26. 26.

    Borra, M. T., Smith, B. C. & Denu, J. M. Mechanism of human SIRT1 activation by resveratrol. J. Biol. Chem. 280, 17187–17195 (2005).

    CAS  Article  Google Scholar 

  27. 27.

    Kaeberlein, M. et al. Substrate-specific activation of sirtuins by resveratrol. J. Biol. Chem. 280, 17038–17045 (2005).

    CAS  Article  Google Scholar 

  28. 28.

    Mao, Z. et al. SIRT6 promotes DNA repair under stress by activating PARP1. Science 332, 1443–1446 (2011).

    CAS  Article  Google Scholar 

  29. 29.

    Hu, J., He, B., Bhargava, S. & Lin, H. A fluorogenic assay for screening Sirt6 modulators. Org. Biomol. Chem. 11, 5213–5216 (2013).

    CAS  Article  Google Scholar 

  30. 30.

    Tan, Y. et al. A SIRT4-like auto ADP-ribosyltransferase is essential for the environmental growth of Mycobacterium smegmatis. Acta Biochim. Biophys. Sin. (Shanghai) 48, 145–152 (2016).

    CAS  Article  Google Scholar 

  31. 31.

    Dai, H., Ellis, J. L., Sinclair, D. A. & Hubbard, B. P. Synthesis and assay of SIRT1-activating compounds. Methods Enzymol. 574, 213–244 (2016).

    CAS  Article  Google Scholar 

  32. 32.

    Davenport, A. M., Huber, F. M. & Hoelz, A. Structural and functional analysis of human SIRT1. J. Mol. Biol. 426, 526–541 (2014).

    CAS  Article  Google Scholar 

  33. 33.

    Moniot, S., Schutkowski, M. & Steegborn, C. Crystal structure analysis of human Sirt2 and its ADP-ribose complex. J. Struct. Biol. 182, 136–143 (2013).

    CAS  Article  Google Scholar 

  34. 34.

    Nguyen, G. T., Schaefer, S., Gertz, M., Weyand, M. & Steegborn, C. Structures of human sirtuin 3 complexes with ADP-ribose and with carba-NAD+ and SRT1720: binding details and inhibition mechanism. Acta Crystallogr. D Biol. Crystallogr 69, 1423–1432 (2013).

    CAS  Article  Google Scholar 

  35. 35.

    Du, J. et al. Sirt5 is a NAD-dependent protein lysine demalonylase and desuccinylase. Science 334, 806–809 (2011).

    CAS  Article  Google Scholar 

  36. 36.

    Hubatsch, I., Ragnarsson, E. G. & Artursson, P. Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers. Nat. Protoc. 2, 2111–2119 (2007).

    CAS  Article  Google Scholar 

  37. 37.

    Min, L. et al. Liver cancer initiation is controlled by AP-1 through SIRT6-dependent inhibition of survivin. Nat. Cell Biol. 14, 1203–1211 (2012).

    CAS  Article  Google Scholar 

  38. 38.

    Elhanati, S. et al. Reciprocal regulation between SIRT6 and miR-122 controls liver metabolism and predicts hepatocarcinoma prognosis. Cell Rep. 14, 234–242 (2016).

    CAS  Article  Google Scholar 

  39. 39.

    Suter, M. A. et al. A maternal high-fat diet modulates fetal SIRT1 histone and protein deacetylase activity in nonhuman primates. FASEB J. 26, 5106–5114 (2012).

    CAS  Article  Google Scholar 

  40. 40.

    Gu, Y., Turck, C. W. & Morgan, D. O. Inhibition of CDK2 activity in vivo by an associated 20K regulatory subunit. Nature 366, 707–710 (1993).

    CAS  Article  Google Scholar 

  41. 41.

    el-Deiry, W. S. et al. WAF1, a potential mediator of p53 tumor suppression. Cell 75, 817–825 (1993).

    CAS  Article  Google Scholar 

  42. 42.

    Harper, J. W., Adami, G. R., Wei, N., Keyomarsi, K. & Elledge, S. J. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 75, 805–816 (1993).

    CAS  Article  Google Scholar 

  43. 43.

    Hengst, L., Dulic, V., Slingerland, J. M., Lees, E. & Reed, S. I. A cell cycle-regulated inhibitor of cyclin-dependent kinases. Proc. Natl Acad. Sci. USA 91, 5291–5295 (1994).

    CAS  Article  Google Scholar 

  44. 44.

    Polyak, K. et al. Cloning of p27Kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals. Cell 78, 59–66 (1994).

    CAS  Article  Google Scholar 

  45. 45.

    Toyoshima, H. & Hunter, T. p27, a novel inhibitor of G1 cyclin-Cdk protein kinase activity, is related to p21. Cell 78, 67–74 (1994).

    CAS  Article  Google Scholar 

  46. 46.

    Morgan, D. O. Principles of CDK regulation. Nature 374, 131–134 (1995).

    CAS  Article  Google Scholar 

  47. 47.

    Hubbard, B. P. et al. Evidence for a common mechanism of SIRT1 regulation by allosteric activators. Science 339, 1216–1219 (2013).

    CAS  Article  Google Scholar 

  48. 48.

    Dai, H. et al. Crystallographic structure of a small molecule SIRT1 activator-enzyme complex. Nat. Commun. 6, 7645 (2015).

    Article  Google Scholar 

  49. 49.

    Huang, M. et al. Screening and biological evaluation of a novel STAT3 signaling pathway inhibitor against cancer. Bioorg. Med. Chem. Lett. 26, 5172–5176 (2016).

    CAS  Article  Google Scholar 

  50. 50.

    Zhao, Y. et al. Crystal structures of PI3Kα complexed with PI103 and its derivatives: new directions for inhibitors design. ACS Med. Chem. Lett. 5, 138–142 (2013).

    Article  Google Scholar 

  51. 51.

    Jiang, H. et al. Peptidomimetic inhibitors of APC-Asef interaction block colorectal cancer migration. Nat. Chem. Biol. 13, 994–1001 (2017).

    CAS  Article  Google Scholar 

  52. 52.

    Ran, F. A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We sincerely thank D. Sinclair for discussion on activator application, and C. Steegborn and A. Mai for discussion on crystallization. We thank Z.-G. Han (Key Laboratory of Systems Biomedicine, Ministry of Education, and Collaborative Innovation Center of Systems Biomedicine of Rui-Jin Hospital, Shanghai Jiao Tong University School of Medicine) for providing cell lines. This work was supported in part by grants from the National Basic Research Program of China (973 Program) (2015CB910403 to J.Z. and G.C.), the National Natural Science Foundation of China (81721004 to G.C. and J.Z.; 91753117 to J.Z.; 81322046 to J.Z.; 81302698 to S.L.; 31671459 to Y.X.; U1605221 to H.L.; and 2181001006 to H.J.), the Program for Changjiang Scholars and the Innovative Research Team of the University of the Ministry Education of China (2017 to J.Z.), the CAS Interdisciplinary Innovation Team (2017 to J.Z.), the Innovation Program of the Shanghai Municipal Education Commission (2019 to J.Z.), the State Key Laboratory of Luminescence and application (SKLA-2016-12 to Y.X.), and the Strategic Priority Research Program of the Chinese Academy of Sciences, ‘Personalized Medicines—Molecular Signature-based Drug Discovery and Development’ (XDA12040100 to H.J.).

Author information

Affiliations

Authors

Contributions

J. Zhang conceived and supervised the project. J. Zhang and Z.H. designed the experiments. Z.H., J. Zhao,Y.C., J.S., L.Z., C.W., H.H., J.X., J. Zhong, Q.Z., X.S., and Y.X. performed the biological experiments. J. Zhang, G.C.,Y.X., Z.H., S.L., and H.L. analyzed data. Z.H. and K.S. performed the crystallography. Y.C. and X.Y. carried out synthesis, purification, and characterization of compounds. W.D., S.L., B.H., J.M., H.H., M.T., X.S., Z.M., M.X., Y.E.C., and H.J. generated key protein reagents. K.S. solved the crystal structures. J. Zhang wrote the manuscript, and all other authors contributed specific parts of the manuscript; J. Zhang, G.C., and Y.X. assume responsibility for the manuscript in its entirety.

Corresponding authors

Correspondence to Ying Xu or Guoqiang Chen or Jian Zhang.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–69 and Supplementary Tables 1–20

Reporting Summary

Supplementary Note

Synthetic Procedures

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Huang, Z., Zhao, J., Deng, W. et al. Identification of a cellularly active SIRT6 allosteric activator. Nat Chem Biol 14, 1118–1126 (2018). https://doi.org/10.1038/s41589-018-0150-0

Download citation

Search

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing