Xin Wang1, Shaozhen Feng2, Jinjin Fan2, Xiaoyan Li2, Qiong Wen2, Ning Luo2. 1. Key Nephrology Laboratory, Ministry of Health, China; Department of Nephrology, The First Affiliated Hospital, Sun Yat-sen University, 58 Zhongshan Road II, Guangzhou 510080, China. Electronic address: wangxin8@mail.sysu.edu.cn. 2. Key Nephrology Laboratory, Ministry of Health, China; Department of Nephrology, The First Affiliated Hospital, Sun Yat-sen University, 58 Zhongshan Road II, Guangzhou 510080, China.
Abstract
PURPOSE: Smad3 is a critical signaling protein in renal fibrosis. Proteolysis targeting chimeric molecules (PROTACs) are small molecules designed to degrade target proteins via ubiquitination. They have three components: (1) a recognition motif for E3 ligase; (2) a linker; and (3) a ligand for the target protein. We aimed to design a new PROTAC to prevent renal fibrosis by targeting Smad3 proteins and using hydroxylated pentapeptide of hypoxia-inducible factor-1α as the recognition motif for von Hippel-Lindau (VHL) ubiquitin ligase (E3). METHODS: Computer-aided drug design was used to find a specific ligand targeting Smad3. Surface plasmon resonance (SPR) was used to verify and optimize screening results. Synthesized PROTAC was validated by two-stage mass spectrometry. The PROTAC's specificity for VHL (E3 ligase) was proved with two human renal carcinoma cell lines, 786-0 (VHL(-)) and ACHN (VHL(+)), and its anti-fibrosis effect was tested in renal fibrosis cell models. RESULTS: Thirteen small molecular compounds (SMCs) were obtained from the Enamine library using GLIDE molecular docking program. SPR results showed that #8 SMC (EN300-72284) combined best with Smad3 (KD=4.547×10(-5)M). Mass spectrometry showed that synthesized PROTAC had the correct peptide molecular weights. Western blot showed Smad3 was degraded by PROTAC with whole-cell lysate of ACHN but not 786-0. Degradation, but not ubiquitination, of Smad3 was inhibited by proteasome inhibitor MG132. The upregulation of fibronectin and Collagen I induced by TGF-β1 in both renal fibroblast and mesangial cells were inhibited by PROTAC. CONCLUSION: The new PROTAC might prevent renal fibrosis by targeting Smad3 for ubiquitination and degradation.
PURPOSE:Smad3 is a critical signaling protein in renal fibrosis. Proteolysis targeting chimeric molecules (PROTACs) are small molecules designed to degrade target proteins via ubiquitination. They have three components: (1) a recognition motif for E3 ligase; (2) a linker; and (3) a ligand for the target protein. We aimed to design a new PROTAC to prevent renal fibrosis by targeting Smad3 proteins and using hydroxylated pentapeptide of hypoxia-inducible factor-1α as the recognition motif for von Hippel-Lindau (VHL) ubiquitin ligase (E3). METHODS: Computer-aided drug design was used to find a specific ligand targeting Smad3. Surface plasmon resonance (SPR) was used to verify and optimize screening results. Synthesized PROTAC was validated by two-stage mass spectrometry. The PROTAC's specificity for VHL (E3 ligase) was proved with two humanrenal carcinoma cell lines, 786-0 (VHL(-)) and ACHN (VHL(+)), and its anti-fibrosis effect was tested in renal fibrosis cell models. RESULTS: Thirteen small molecular compounds (SMCs) were obtained from the Enamine library using GLIDE molecular docking program. SPR results showed that #8 SMC (EN300-72284) combined best with Smad3 (KD=4.547×10(-5)M). Mass spectrometry showed that synthesized PROTAC had the correct peptide molecular weights. Western blot showed Smad3 was degraded by PROTAC with whole-cell lysate of ACHN but not 786-0. Degradation, but not ubiquitination, of Smad3 was inhibited by proteasome inhibitor MG132. The upregulation of fibronectin and Collagen I induced by TGF-β1 in both renal fibroblast and mesangial cells were inhibited by PROTAC. CONCLUSION: The new PROTAC might prevent renal fibrosis by targeting Smad3 for ubiquitination and degradation.
Authors: Afua A Akuffo; Aileen Y Alontaga; Rainer Metcalf; Matthew S Beatty; Andreas Becker; Jessica M McDaniel; Rebecca S Hesterberg; William E Goodheart; Steven Gunawan; Muhammad Ayaz; Yan Yang; Md Rezaul Karim; Morgan E Orobello; Kenyon Daniel; Wayne Guida; Jeffrey A Yoder; Anjali M Rajadhyaksha; Ernst Schönbrunn; Harshani R Lawrence; Nicholas J Lawrence; Pearlie K Epling-Burnette Journal: J Biol Chem Date: 2018-02-15 Impact factor: 5.157