# Protocol for high-throughput screening of SIRT7 inhibitors using fluorescent peptide technology

## TL;DR

This paper describes a protocol for screening SIRT7 inhibitors using fluorescent peptides to measure enzyme activity and identify potential drug candidates.

## Contribution

A high-throughput screening protocol for SIRT7 inhibitors using fluorescent peptide technology is introduced.

## Key findings

- A protocol for SIRT7 protein purification and enzymatic activity assessment using fluorescent peptides is outlined.
- High-throughput screening and IC50 determination methods for SIRT7 inhibitors are described.
- The protocol enables validation of potential SIRT7 inhibitors using microplate-based assays.

## Abstract

Fluorescent peptides combine the polypeptides with fluorescent groups organically to facilitate the measurement of enzyme activity. Here, we present a protocol to evaluate Sirtuin 7 (SIRT7) enzymatic activity by analyzing changes in luminescent signals from its substrate polypeptides. We outline the steps for SIRT7 protein purification, enzymatic reaction setup, fluorescence spectrum detection, and data analysis. Additionally, we describe two applications targeting SIRT7: high-throughput screening of SIRT7 inhibitors using microplate and the validation of potential SIRT7 inhibitors.

For complete details on the use and execution of this protocol, please refer to Kang et al.1

•Large-scale purification of His-SIRT7 recombinant proteins from E. coli•Assessment of SIRT7 enzymatic activity using fluorescent peptides•High-throughput screening for identifying potential SIRT7 inhibitors•Determination of the IC50 values for tested compounds

Large-scale purification of His-SIRT7 recombinant proteins from E. coli

Assessment of SIRT7 enzymatic activity using fluorescent peptides

High-throughput screening for identifying potential SIRT7 inhibitors

Determination of the IC50 values for tested compounds

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Fluorescent peptides combine the polypeptides with fluorescent groups organically to facilitate the measurement of enzyme activity. Here, we present a protocol to evaluate Sirtuin 7 (SIRT7) enzymatic activity by analyzing changes in luminescent signals from its substrate polypeptides. We outline the steps for SIRT7 protein purification, enzymatic reaction setup, fluorescence spectrum detection, and data analysis. Additionally, we describe two applications targeting SIRT7: high-throughput screening of SIRT7 inhibitors using microplate and the validation of potential SIRT7 inhibitors.