Identification of sirtuin 5 inhibitors by ultrafast microchip electrophoresis using nanoliter volume samples
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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.
KeywordsSirtuin 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.
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Conflict of interest
The authors declare that they have no conflict of interest.
- 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.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.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
- 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
- 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
- 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.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
- 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
- 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.Kainkaryam RM, Woolf PJ (2009) Pooling in high-throughput drug screening. Curr Open Drug Discov Dev 12(3):339–350Google Scholar