title: New strategy for renal fibrosis: Targeting Smad3 proteins for ubiquitination and degradation.
abstract: 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).
  
  
  
  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.
  
  
  
  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.
  
  
  
  The new PROTAC might prevent renal fibrosis by targeting Smad3 for ubiquitination and degradation.
authors.affiliation: 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.
authors.forename: Xin
authors.initials: X
authors.lastname: Wang
authors.affiliation: 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.
authors.forename: Shaozhen
authors.initials: S
authors.lastname: Feng
authors.affiliation: 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.
authors.forename: Jinjin
authors.initials: J
authors.lastname: Fan
authors.affiliation: 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.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Li
authors.affiliation: 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.
authors.forename: Qiong
authors.initials: Q
authors.lastname: Wen
authors.affiliation: 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.
authors.forename: Ning
authors.initials: N
authors.lastname: Luo
mesh_terms: D020816:Amino Acid Motifs; D001572:Benzofurans / Q000737:chemistry / Q000378:metabolism / Q000627:therapeutic use; D001665:Binding Sites; D045744:Cell Line, Tumor; D015195:Drug Design; D005355:Fibrosis; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000737:chemistry / Q000378:metabolism / Q000627:therapeutic use; D007668:Kidney / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D008024:Ligands; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D058990:Molecular Targeted Therapy; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism / Q000627:therapeutic use; D054730:Protein Interaction Domains and Motifs; D059748:Proteolysis / Q000187:drug effects; D011725:Pyridines / Q000737:chemistry / Q000378:metabolism / Q000627:therapeutic use; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism / Q000627:therapeutic use*; D051436:Renal Insufficiency, Chronic / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D051900:Smad3 Protein / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism; D054852:Small Molecule Libraries; D020349:Surface Plasmon Resonance; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000378:metabolism*; D054875:Ubiquitination / Q000187:drug effects
chemical_list: D001572:Benzofurans; C000618339:EN300-72284; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008024:Ligands; D010446:Peptide Fragments; D011725:Pyridines; D011993:Recombinant Fusion Proteins; C497790:SMAD3 protein, human; D051900:Smad3 Protein; D054852:Small Molecule Libraries; D044767:Ubiquitin-Protein Ligases
keywords: Computer-aided drug design; Fibrosis; Proteolysis targeting chimeric molecules; Ubiquitination