2. Academic Validation
  3. Sensitive fluorogenic substrates for sirtuin deacylase inhibitor discovery

Sensitive fluorogenic substrates for sirtuin deacylase inhibitor discovery

  • Eur J Med Chem. 2020 Apr 15;192:112201. doi: 10.1016/j.ejmech.2020.112201.
Ling-Ling Yang 1,  Hua-Li Wang 2,  Yu-Hang Yan 2,  Sha Liu 2,  Zhu-Jun Yu 2,  Meng-Yi Huang 2,  Yubin Luo 3,  Xi Zheng 4,  Yamei Yu 5,  Guo-Bo Li 6
Affiliations

Affiliations

  • 1 College of Food and Bioengineering, Xihua University, Sichuan, 610039, PR China.
  • 2 Key Laboratory of Drug Targeting and Drug Delivery System of Ministry of Education, West China School of Pharmacy, and State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center of Biotherapy, Chengdu, 610041, PR China.
  • 3 Department of Rheumatology and Immunology, West China Hospital, Sichuan University, Chengdu, 610041, PR China.
  • 4 Lung Cancer Center, West China Hospital, Sichuan University, Chengdu, 610041, PR China.
  • 5 Key Laboratory of Drug Targeting and Drug Delivery System of Ministry of Education, West China School of Pharmacy, and State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center of Biotherapy, Chengdu, 610041, PR China. Electronic address: yamei_yu@scu.edu.cn.
  • 6 Key Laboratory of Drug Targeting and Drug Delivery System of Ministry of Education, West China School of Pharmacy, and State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center of Biotherapy, Chengdu, 610041, PR China. Electronic address: liguobo@scu.edu.cn.
PMID: 32163813 DOI: 10.1016/j.ejmech.2020.112201
Abstract

Sirtuins (SIRTs) are NAD+-dependent lysine deacylases, regulating many important biological processes such as metabolism and stress responses. SIRT inhibitors may provide potential benefits against SIRT-driven human diseases. Development of efficient assay platforms based on fluorogenic substrates will facilitate the discovery of high-quality SIRT inhibitors. We here report 16 new fluorogenic peptide substrates (P1-P16) designed with structurally diverse tetrapeptides and acyl modifications. Tests of P1-P16 against SIRT isoforms identified several sensitive substrates for SIRT1, SIRT2, SIRT3 and SIRT5, which manifested lower KM values and higher catalytic efficiency, and particularly had less signal interference in inhibitor screening compared with our previously reported internally quenched fluorescent substrates. Co-crystallization of sensitive substrates P13 and P15 with SIRT5 revealed an unexpected binding mode, involving interactions with residues from active site bordering surfaces, different from that observed for other Peptides derived from natural protein substrates. By using SIRT5 sensitive substrates, we found that TW-37, a Bcl-2 Inhibitor, displayed low micromolar inhibition to SIRT5, which was further validated by isothermal titration calorimetry analyses, offering a new point to develop dual-action SIRT5/Bcl-2 inhibitors against cancers. This work provides assay platform and structural basis for developing new substrates and inhibitors targeting human SIRTs.

Keywords

Bcl-2; Deacylase; Fluorogenic substrate; SIRT5; Sirtuin.

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