title: Identification of sirtuin 5 inhibitors by ultrafast microchip electrophoresis using nanoliter volume samples.
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.
authors.affiliation: Department of Chemistry, University of Michigan, 930 N University Ave, Ann Arbor, MI, 48109, USA.
authors.forename: Erik D
authors.initials: ED
authors.lastname: Guetschow
authors.affiliation: Department of Pathology, University of Michigan, 1301 Catherine St, Ann Arbor, MI, 48109, USA.
authors.forename: Surinder
authors.initials: S
authors.lastname: Kumar
authors.affiliation: Department of Pathology, University of Michigan, 1301 Catherine St, Ann Arbor, MI, 48109, USA.|Institute of Gerontology, University of Michigan, 300 N Ingalls St, Ann Arbor, MI, 48109, USA.
authors.forename: David B
authors.initials: DB
authors.lastname: Lombard
authors.affiliation: Department of Chemistry, University of Michigan, 930 N University Ave, Ann Arbor, MI, 48109, USA. rtkenn@umich.edu.|Department of Pharmacology, University of Michigan, 1150 W. Medical Center Dr., Ann Arbor, MI, 48109, USA. rtkenn@umich.edu.
authors.forename: Robert T
authors.initials: RT
authors.lastname: Kennedy
mesh_terms: D046209:Electrophoresis, Microchip / Q000295:instrumentation / Q000379:methods*; D004791:Enzyme Inhibitors / Q000737:chemistry*; D007700:Kinetics; D037761:Sirtuins / Q000037:antagonists & inhibitors* / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D037761:Sirtuins
keywords: Droplets; Electrophoresis; Microfluidics; Screening; Sirtuin