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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

Abstract

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

Keywords

Sirtuin Screening Electrophoresis Microfluidics Droplets 

Notes

Acknowledgments

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)

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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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