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, 8 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 percent and verified by dose-response analysis.

Automatic Combination of Microfluidic Nanoliter-Scale Droplet Array with High-Speed Capillary Electrophoresis

“…In the previously reported microfluidic droplet-CE systems with aqueous droplet phase and immiscible, nonvolatile oil phase for the compartmentation of droplets51213141516171819, usually microchip-based continuous flow mode in microchannel network was employed. In these systems, various types of droplet extraction interfaces were commonly required to firstly extract aqueous droplets from segmented flow into aqueous phase channel, and then to inject the aqueous droplet samples into channel for CE separation sequentially.…”

“…In most of the previously reported HSCE systems coupled with droplet-based microfluidic systems, microfabricated chips integrating both continuous-flow droplet module and CE module were frequently adopted5121314151617181920. An interface connecting the droplet channel with the CE channel was usually used to transfer droplets from immiscible phase flow to aqueous phase flow.…”

“…In contrast to continuous-flow droplet systems formed in microchip channels51213141516171819, two-dimensional droplet array systems built on microwells or microregions on microchips, especially those with open or semi-open features212223242526, have more practical benefits of easily handling massive different sample droplets with a capillary probe. In 1998, Whitesides’ group21 reported a simple method to form high-density droplet array using the spreading and discontinuous dewetting of a sample solution on a microwell array chip.…”

Recent advances in protein analysis by capillary and microchip electrophoresis

“…114 The recent application of droplets interfaced to microchip electrophoresis for the analysis of SIRT5 enzymatic screening assay demonstrates subsecond separations using only nanoliter volumes with increased analysis throughout, and this work shows great robustness and reliability with over 11,000 MCE injections from droplets made. 115 Over 1400 samples were screened improving the throughput 3-fold relative to previous MCE-based screens and 25-fold compared to previous MCE/CE screens for sirtuin.…”

Advances in capillary electrophoresis and the implications for drug discovery

“…[12] In general however, commercial CE instruments have limited accessible separation lengths so that it is difficult to reduce separations to shorter than this. Some custom-built CE systems have allowed sub-second separations;[13,14] however, with one exception [15,16], they have not been investigated for screening.…”