Analytical and Bioanalytical Chemistry

, Volume 408, Issue 3, pp 721–731 | Cite as

Identification of sirtuin 5 inhibitors by ultrafast microchip electrophoresis using nanoliter volume samples

  • Erik D. Guetschow
  • Surinder Kumar
  • David B. Lombard
  • Robert T. Kennedy
Paper in Forefront


Sirtuin 5 (SIRT5) is a member of the sirtuin family of protein deacylases that catalyzes removal of post-translational modifications, such as succinyl and malonyl moieties, on lysine residues. In light of SIRT5’s roles in regulating metabolism, and its reported oncogenic functions, SIRT5 modulators would be valuable tools for basic biological research and perhaps clinically. Several fluorescence assays for sirtuin modulators have been developed; however, the use of fluorogenic substrates has the potential to cause false positive results due to interactions of engineered substrates with enzyme or test compounds. Therefore, development of high-throughput screening (HTS) assays based on other methods is valuable. In this study, we report the development of a SIRT5 assay using microchip electrophoresis (MCE) for identification of SIRT5 modulators. A novel SIRT5 substrate based on succinate dehydrogenase (SDH) was developed to allow rapid and efficient separation of substrate and product peptide. To achieve high throughput, samples were injected onto the microchip using a droplet-based scheme. By coupling this approach to existing HTS sample preparation workflows, 1408 samples were analyzed at 0.5 Hz in 46 min. Using a 250 ms separation time, eight MCE injections could be made from each sample generating >11,000 electropherograms during analysis. Of the 1280 chemicals tested, eight were identified as inhibiting SIRT5 activity by at least 70 % and verified by dose-response analysis.

Graphical Abstract

Overview of high-throughput screening using droplet samples and microchip electrophoresis


Sirtuin Screening Electrophoresis Microfluidics Droplets 



R.T.K and E.D.G were supported by National Institutes of Health grant R01GM102236, the National Institutes of Health Microfluidics in Biomedical Sciences Training Program at University of Michigan T32 EB005582, and the American Chemical Society Division of Analytical Chemistry Graduate Fellowship sponsored by Eli Lilly and Company. D.B.L and S.K. were supported by National Institutes of Health grant R01GM101171, the Glenn Foundation for Medical Research, the National Center for Advancing Translational Sciences of the National Institutes of Health under award UL1TR000433, Department of Defense support for Ovarian Cancer grant OC140123, and the John S. and Suzanne C. Munn Cancer Fund of the University of Michigan Comprehensive Cancer Center.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2015_9206_MOESM1_ESM.pdf (1.1 mb)
ESM 1 (PDF 1145 kb)


  1. 1.
    S-i I, Armstrong CM, Kaeberlein M, Guarente L (2000) Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase. Nature 403(6771):795–800CrossRefGoogle Scholar
  2. 2.
    Imai S-i, Guarente L (2010) Ten years of NAD-dependent SIR2 family deacetylases: implications for metabolic diseases. Trends Pharmacol Sci 31(5):212–220CrossRefGoogle Scholar
  3. 3.
    Jiang H, Khan S, Wang Y, Charron G, He B, Sebastian C, Du J, Kim R, Ge E, Mostoslavsky R, Hang HC, Hao Q, Lin H (2013) SIRT6 regulates TNF-[agr] secretion through hydrolysis of long-chain fatty acyl lysine. Nature 496(7443):110–113CrossRefGoogle Scholar
  4. 4.
    Feldman JL, Baeza J, Denu JM (2013) Activation of the protein deacetylase SIRT6 by long-chain fatty acids and widespread deacylation by mammalian sirtuins. J Biol Chem 288(43):31350–31356CrossRefGoogle Scholar
  5. 5.
    Du J, Zhou Y, Su X, Yu JJ, Khan S, Jiang H, Kim J, Woo J, Kim JH, Choi BH, He B, Chen W, Zhang S, Cerione RA, Auwerx J, Hao Q, Lin H (2011) Sirt5 Is a NAD-dependent protein lysine demalonylase and desuccinylase. Science 334(6057):806–809CrossRefGoogle Scholar
  6. 6.
    Zhang Z, Tan M, Xie Z, Dai L, Chen Y, Zhao Y (2011) Identification of lysine succinylation as a new post-translational modification. Nat Chem Biol 7(1):58–63CrossRefGoogle Scholar
  7. 7.
    Peng C, Lu Z, Xie Z, Cheng Z, Chen Y, Tan M, Luo H, Zhang Y, He W, Yang K, Zwaans BMM, Tishkoff D, Ho L, Lombard D, He T-C, Dai J, Verdin E, Ye Y, Zhao Y (2011) The first identification of lysine malonylation substrates and its regulatory enzyme. Mol Cell Proteomics 10 (12):M111.012658Google Scholar
  8. 8.
    Rardin Matthew J, He W, Nishida Y, Newman John C, Carrico C, Danielson Steven R, Guo A, Gut P, Sahu Alexandria K, Li B, Uppala R, Fitch M, Riiff T, Zhu L, Zhou J, Mulhern D, Stevens Robert D, Ilkayeva Olga R, Newgard Christopher B, Jacobson Matthew P, Hellerstein M, Goetzman Eric S, Gibson Bradford W, Verdin E (2013) SIRT5 Regulates the mitochondrial lysine succinylome and metabolic networks. Cell Metab 18(6):920–933CrossRefGoogle Scholar
  9. 9.
    Tan M, Peng C, Anderson Kristin A, Chhoy P, Xie Z, Dai L, Park J, Chen Y, Huang H, Zhang Y, Ro J, Wagner Gregory R, Green Michelle F, Madsen Andreas S, Schmiesing J, Peterson Brett S, Xu G, Ilkayeva Olga R, Muehlbauer Michael J, Braulke T, Mühlhausen C, Backos Donald S, Olsen Christian A, McGuire Peter J, Pletcher Scott D, Lombard David B, Hirschey Matthew D, Zhao Y (2014) Lysine glutarylation is a protein posttranslational modification regulated by SIRT5. Cell Metab 19(4):605–617CrossRefGoogle Scholar
  10. 10.
    Nishida Y, Rardin Matthew J, Carrico C, He W, Sahu Alexandria K, Gut P, Najjar R, Fitch M, Hellerstein M, Gibson Bradford W, Verdin E (2015) SIRT5 regulates both cytosolic and mitochondrial protein malonylation with glycolysis as a major target. Mol Cell 59(2):321–332CrossRefGoogle Scholar
  11. 11.
    Bao X, Wang Y, Li X, Li X-M, Liu Z, Yang T, Wong CF, Zhang J, Hao Q, Li XD (2014) Identification of ‘erasers’ for lysine crotonylated histone marks using a chemical proteomics approach. eLife 3:e02999Google Scholar
  12. 12.
    Park J, Chen Y, Tishkoff Daniel X, Peng C, Tan M, Dai L, Xie Z, Zhang Y, Zwaans Bernadette MM, Skinner Mary E, Lombard David B, Zhao Y (2013) SIRT5-mediated lysine desuccinylation impacts diverse metabolic pathways. Mol Cell 50(6):919–930CrossRefGoogle Scholar
  13. 13.
    Nakagawa T, Lomb DJ, Haigis MC, Guarente L (2009) SIRT5 deacetylates carbamoyl phosphate synthetase 1 and regulates the urea cycle. Cell 137(3):560–570CrossRefGoogle Scholar
  14. 14.
    Ogura M, Nakamura Y, Tanaka D, Zhuang X, Fujita Y, Obara A, Hamasaki A, Hosokawa M, Inagaki N (2010) Overexpression of SIRT5 confirms its involvement in deacetylation and activation of carbamoyl phosphate synthetase 1. Biochem Biophys Res Commun 393(1):73–78CrossRefGoogle Scholar
  15. 15.
    Lin Z-F, Xu H-B, Wang J-Y, Lin Q, Ruan Z, Liu F-B, Jin W, Huang H-H, Chen X (2013) SIRT5 desuccinylates and activates SOD1 to eliminate ROS. Biochem Biophys Res Commun 441(1):191–195CrossRefGoogle Scholar
  16. 16.
    Yu J, Sadhukhan S, Noriega LG, Moullan N, He B, Weiss RS, Lin H, Schoonjans K, Auwerx J (2013) Metabolic characterization of a Sirt5 deficient mouse model. Sci Rep 3:2806Google Scholar
  17. 17.
    Kumar S, Lombard DB (2014) Mitochondrial sirtuins and their relationships with metabolic disease and cancer. Antioxid Redox Signal 22(12):1060–1077CrossRefGoogle Scholar
  18. 18.
    Sebastián C, Mostoslavsky R (2015) The role of mammalian sirtuins in cancer metabolism. Semin Cell Dev Biol. doi: 10.1016/j.semcdb.2015.07.008 Google Scholar
  19. 19.
    Lu W, Zuo Y, Feng Y, Zhang M (2014) SIRT5 facilitates cancer cell growth and drug resistance in non-small cell lung cancer. Tumor Biol 35(11):10699–10705CrossRefGoogle Scholar
  20. 20.
    Borra MT, Smith BC, Denu JM (2005) Mechanism of human SIRT1 activation by resveratrol. J Biol Chem 280(17):17187–17195CrossRefGoogle Scholar
  21. 21.
    Howitz KT, Bitterman KJ, Cohen HY, Lamming DW, Lavu S, Wood JG, Zipkin RE, Chung P, Kisielewski A, Zhang L-L, Scherer B, Sinclair DA (2003) Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan. Nature 425(6954):191–196CrossRefGoogle Scholar
  22. 22.
    Kokkonen P, Rahnasto-Rilla M, Mellini P, Jarho E, Lahtela-Kakkonen M, Kokkola T (2014) Studying SIRT6 regulation using H3K56 based substrate and small molecules. Eur J Pharm Sci 63:71–76CrossRefGoogle Scholar
  23. 23.
    Madsen AS, Olsen CA (2012) Substrates for efficient fluorometric screening employing the NAD-dependent sirtuin 5 lysine deacylase (KDAC) enzyme. J Med Chem 55(11):5582–5590CrossRefGoogle Scholar
  24. 24.
    Parenti MD, Grozio A, Bauer I, Galeno L, Damonte P, Millo E, Sociali G, Franceschi C, Ballestrero A, Bruzzone S, Rio AD, Nencioni A (2014) Discovery of novel and selective SIRT6 inhibitors. J Med Chem 57(11):4796–4804CrossRefGoogle Scholar
  25. 25.
    Wegener D, Hildmann C, Riester D, Schwienhorst A (2003) Improved fluorogenic histone deacetylase assay for high-throughput-screening applications. Anal Biochem 321(2):202–208CrossRefGoogle Scholar
  26. 26.
    Mercken EM, Mitchell SJ, Martin-Montalvo A, Minor RK, Almeida M, Gomes AP, Scheibye-Knudsen M, Palacios HH, Licata JJ, Zhang Y, Becker KG, Khraiwesh H, González-Reyes JA, Villalba JM, Baur JA, Elliott P, Westphal C, Vlasuk GP, Ellis JL, Sinclair DA, Bernier M, de Cabo R (2014) SRT2104 extends survival of male mice on a standard diet and preserves bone and muscle mass. Aging Cell 13(5):787–796CrossRefGoogle Scholar
  27. 27.
    Mitchell Sarah J, Martin-Montalvo A, Mercken Evi M, Palacios Hector H, Ward Theresa M, Abulwerdi G, Minor Robin K, Vlasuk George P, Ellis James L, Sinclair David A, Dawson J, Allison David B, Zhang Y, Becker Kevin G, Bernier M, de Cabo R (2014) The SIRT1 activator SRT1720 extends lifespan and improves health of mice fed a standard diet. Cell Rep 6(5):836–843CrossRefGoogle Scholar
  28. 28.
    Beher D, Wu J, Cumine S, Kim KW, Lu S-C, Atangan L, Wang M (2009) Resveratrol is not a direct activator of SIRT1 enzyme activity. Chem Biol Drug Des 74(6):619–624CrossRefGoogle Scholar
  29. 29.
    Pacholec M, Bleasdale JE, Chrunyk B, Cunningham D, Flynn D, Garofalo RS, Griffith D, Griffor M, Loulakis P, Pabst B, Qiu X, Stockman B, Thanabal V, Varghese A, Ward J, Withka J, Ahn K (2010) SRT1720, SRT2183, SRT1460, and Resveratrol are not direct activators of SIRT1. J Biol Chem 285(11):8340–8351CrossRefGoogle Scholar
  30. 30.
    Smith BC, Hallows WC, Denu JM (2009) A continuous microplate assay for sirtuins and nicotinamide-producing enzymes. Anal Biochem 394(1):101–109CrossRefGoogle Scholar
  31. 31.
    Roessler C, Tüting C, Meleshin M, Steegborn C, Schutkowski M (2015) A novel continuous assay for the deacylase sirtuin 5 and other deacetylases. J Med Chem 58(18):7217–7223CrossRefGoogle Scholar
  32. 32.
    Fischer F, Gertz M, Suenkel B, Lakshminarasimhan M, Schutkowski M, Steegborn C (2012) Sirt5 deacylation activities show differential sensitivities to nicotinamide inhibition. PLoS ONE 7(9), e45098CrossRefGoogle Scholar
  33. 33.
    Roessler C, Nowak T, Pannek M, Gertz M, Nguyen GTT, Scharfe M, Born I, Sippl W, Steegborn C, Schutkowski M (2014) Chemical probing of the human sirtuin 5 active site reveals its substrate acyl specificity and peptide-based inhibitors. Angew Chem Int Ed 53(40):10728–10732CrossRefGoogle Scholar
  34. 34.
    Li Y, Huang W, You L, Xie T, He B (2015) A FRET-based assay for screening SIRT5 specific modulators. Bioorg Med Chem Lett 25(8):1671–1674CrossRefGoogle Scholar
  35. 35.
    Fan Y, Ludewig R, Imhof D, Scriba GKE (2008) Development of a capillary electrophoresis-based assay of sirtuin enzymes. Electrophoresis 29(18):3717–3723CrossRefGoogle Scholar
  36. 36.
    Fan Y, Ludewig R, Scriba GKE (2009) 9-Fluorenylmethoxycarbonyl-labeled peptides as substrates in a capillary electrophoresis-based assay for sirtuin enzymes. Anal Biochem 387(2):243–248CrossRefGoogle Scholar
  37. 37.
    Liu Y, Gerber R, Wu J, Tsuruda T, McCarter JD (2008) High-throughput assays for sirtuin enzymes: a microfluidic mobility shift assay and a bioluminescence assay. Anal Biochem 378(1):53–59CrossRefGoogle Scholar
  38. 38.
    Ohla S, Beyreiss R, Scriba GKE, Fan Y, Belder D (2010) An integrated on-chip sirtuin assay. Electrophoresis 31(19):3263–3267CrossRefGoogle Scholar
  39. 39.
    Abromeit H, Kannan S, Sippl W, Scriba GKE (2012) A new nonpeptide substrate of human sirtuin in a capillary electrophoresis-based assay. Investigation of the binding mode by docking experiments. Electrophoresis 33(11):1652–1659CrossRefGoogle Scholar
  40. 40.
    Fan Y, Hense M, Ludewig R, Weisgerber C, Scriba GKE (2011) Capillary electrophoresis-based sirtuin assay using non-peptide substrates. J Pharm Biomed Anal 54(4):772–778CrossRefGoogle Scholar
  41. 41.
    Pei J, Nie J, Kennedy RT (2010) Parallel electrophoretic analysis of segmented samples on chip for high-throughput determination of enzyme activities. Anal Chem 82(22):9261–9267CrossRefGoogle Scholar
  42. 42.
    Perrin D, Frémaux C, Shutes A (2009) Capillary microfluidic electrophoretic mobility shift assays: application to enzymatic assays in drug discovery. Expert Opin Drug Discovery 5(1):51–63CrossRefGoogle Scholar
  43. 43.
    Shanmuganathan M, Britz-McKibbin P (2013) High quality drug screening by capillary electrophoresis: a review. Anal Chim Acta 773:24–36CrossRefGoogle Scholar
  44. 44.
    Guetschow ED, Steyer DJ, Kennedy RT (2014) Subsecond electrophoretic separations from droplet samples for screening of enzyme modulators. Anal Chem 86(20):10373–10379CrossRefGoogle Scholar
  45. 45.
    DeLaMarre MF, Shippy SA (2014) Development of a simplified microfluidic injector for analysis of droplet content via capillary electrophoresis. Anal Chem 86(20):10193–10200CrossRefGoogle Scholar
  46. 46.
    Niu X, Pereira F, Edel JB, de Mello AJ (2013) Droplet-interfaced microchip and capillary electrophoretic separations. Anal Chem 85(18):8654–8660CrossRefGoogle Scholar
  47. 47.
    Niu XZ, Zhang B, Marszalek RT, Ces O, Edel JB, Klug DR, deMello AJ (2009) Droplet-based compartmentalization of chemically separated components in two-dimensional separations. Chem Commun 41:6159–6161CrossRefGoogle Scholar
  48. 48.
    Roman GT, Wang M, Shultz KN, Jennings C, Kennedy RT (2008) Sampling and electrophoretic analysis of segmented flow streams using virtual walls in a microfluidic device. Anal Chem 80(21):8231–8238CrossRefGoogle Scholar
  49. 49.
    Wang M, Roman GT, Perry ML, Kennedy RT (2009) Microfluidic chip for high efficiency electrophoretic analysis of segmented flow from a microdialysis probe and in vivo chemical monitoring. Anal Chem 81(21):9072–9078CrossRefGoogle Scholar
  50. 50.
    Harrison DJ, Fluri K, Seiler K, Fan Z, Effenhauser CS, Manz A (1993) Micromachining a miniaturized capillary electrophoresis-based chemical analysis system on a chip. Science 261(5123):895–897CrossRefGoogle Scholar
  51. 51.
    Roper MG, Shackman JG, Dahlgren GM, Kennedy RT (2003) Microfluidic chip for continuous monitoring of hormone secretion from live cells using an electrophoresis-based immunoassay. Anal Chem 75(18):4711–4717CrossRefGoogle Scholar
  52. 52.
    Simpson PC, Roach D, Woolley AT, Thorsen T, Johnston R, Sensabaugh GF, Mathies RA (1998) High-throughput genetic analysis using microfabricated 96-sample capillary array electrophoresis microplates. Proc Natl Acad Sci U S A 95(5):2256–2261CrossRefGoogle Scholar
  53. 53.
    Chabert M, Dorfman KD, de Cremoux P, Roeraade J, Viovy J-L (2006) Automated microdroplet platform for sample manipulation and polymerase chain reaction. Anal Chem 78(22):7722–7728CrossRefGoogle Scholar
  54. 54.
    Pei J, Li Q, Lee MS, Valaskovic GA, Kennedy RT (2009) Analysis of samples stored as individual plugs in a capillary by electrospray ionization mass spectrometry. Anal Chem 81(15):6558–6561CrossRefGoogle Scholar
  55. 55.
    Jacobson SC, Hergenroder R, Moore AW Jr, Ramsey JM (1994) Precolumn reactions with electrophoretic analysis integrated on a microchip. Anal Chem 66(23):4127–4132CrossRefGoogle Scholar
  56. 56.
    Jacobson SC, Koutny LB, Hergenroeder R, Moore AW, Ramsey JM (1994) Microchip capillary electrophoresis with an integrated postcolumn reactor. Anal Chem 66(20):3472–3476CrossRefGoogle Scholar
  57. 57.
    Shackman JG, Watson CJ, Kennedy RT (2004) High-throughput automated post-processing of separation data. J Chromatogr A 1040(2):273–282CrossRefGoogle Scholar
  58. 58.
    Cosgrove MS, Bever K, Avalos JL, Muhammad S, Zhang X, Wolberger C (2006) The structural basis of sirtuin substrate affinity. Biochemistry 45(24):7511–7521CrossRefGoogle Scholar
  59. 59.
    Inglese J, Johnson RL, Simeonov A, Xia M, Zheng W, Austin CP, Auld DS (2007) High-throughput screening assays for the identification of chemical probes. Nat Chem Biol 3(8):466–479CrossRefGoogle Scholar
  60. 60.
    von Ahsen O, Bömer U (2005) High-throughput screening for kinase inhibitors. ChemBioChem 6(3):481–490CrossRefGoogle Scholar
  61. 61.
    Schuetz A, Min J, Antoshenko T, Wang C-L, Allali-Hassani A, Dong A, Loppnau P, Vedadi M, Bochkarev A, Sternglanz R, Plotnikov AN (2007) Structural basis of inhibition of the human NAD + -dependent deacetylase SIRT5 by suramin. Structure 15(3):377–389CrossRefGoogle Scholar
  62. 62.
    Maurer B, Rumpf T, Scharfe M, Stolfa DA, Schmitt ML, He W, Verdin E, Sippl W, Jung M (2012) Inhibitors of the NAD + -dependent protein desuccinylase and demalonylase Sirt5. ACS Med Chem Lett 3(12):1050–1053CrossRefGoogle Scholar
  63. 63.
    He B, Du J, Lin H (2012) Thiosuccinyl peptides as sirt5-specific inhibitors. J Am Chem Soc 134(4):1922–1925CrossRefGoogle Scholar
  64. 64.
    Sun S, Kennedy RT (2014) Droplet electrospray ionization mass spectrometry for high throughput screening for enzyme inhibitors. Anal Chem 86(18):9309–9314CrossRefGoogle Scholar
  65. 65.
    Suenkel B, Fischer F, Steegborn C (2013) Inhibition of the human deacylase sirtuin 5 by the indole GW5074. Bioorg Med Chem Lett 23(1):143–146CrossRefGoogle Scholar
  66. 66.
    Su J, Chang C, Xiang Q, Zhou Z-W, Luo R, Yang L, He Z-X, Yang H, Li J, Bei Y, Xu J, Zhang M, Zhang Q, Su Z, Huang Y, Pang J, Zhou S-F (2014) Xyloketal B, a marine compound, acts on a network of molecular proteins and regulates the activity and expression of rat cytochrome P450 3a: a bioinformatic and animal study. Drug Des Devel Ther 8:2555–2602Google Scholar
  67. 67.
    Kainkaryam RM, Woolf PJ (2009) Pooling in high-throughput drug screening. Curr Open Drug Discov Dev 12(3):339–350Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Erik D. Guetschow
    • 1
  • Surinder Kumar
    • 2
  • David B. Lombard
    • 2
    • 3
  • Robert T. Kennedy
    • 1
    • 4
  1. 1.Department of ChemistryUniversity of MichiganAnn ArborUSA
  2. 2.Department of PathologyUniversity of MichiganAnn ArborUSA
  3. 3.Institute of GerontologyUniversity of MichiganAnn ArborUSA
  4. 4.Department of PharmacologyUniversity of MichiganAnn ArborUSA

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