New strategy for renal fibrosis: Targeting Smad3 proteins for ubiquitination and degradation

• Published in: Biochemical pharmacology Vol. 116; pp. 200 - 209
• Main Authors: Wang, Xin, Feng, Shaozhen, Fan, Jinjin, Li, Xiaoyan, Wen, Qiong, Luo, Ning
• Format: Journal Article
• Language: English
• Published: England Elsevier Inc 15.09.2016
• Subjects: Amino Acid Motifs; Benzofurans - chemistry; Benzofurans - metabolism; Benzofurans - therapeutic use; Binding Sites; Cell Line, Tumor; Computer-aided drug design; Drug Design; Fibrosis; Humans; Hypoxia-Inducible Factor 1, alpha Subunit - chemistry; Hypoxia-Inducible Factor 1, alpha Subunit - metabolism; Hypoxia-Inducible Factor 1, alpha Subunit - therapeutic use; Kidney - drug effects; Kidney - metabolism; Kidney - pathology; Ligands; Models, Molecular; Molecular Docking Simulation; Molecular Targeted Therapy; Peptide Fragments - chemistry; Peptide Fragments - metabolism; Peptide Fragments - therapeutic use; Protein Interaction Domains and Motifs; Proteolysis - drug effects; Proteolysis targeting chimeric molecules; Pyridines - chemistry; Pyridines - metabolism; Pyridines - therapeutic use; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - metabolism; Recombinant Fusion Proteins - therapeutic use; Renal Insufficiency, Chronic - drug therapy; Renal Insufficiency, Chronic - metabolism; Renal Insufficiency, Chronic - pathology; Smad3 Protein - antagonists & inhibitors; Smad3 Protein - chemistry; Smad3 Protein - metabolism; Small Molecule Libraries; Surface Plasmon Resonance; Ubiquitin-Protein Ligases - chemistry; Ubiquitin-Protein Ligases - metabolism; Ubiquitination; Ubiquitination - drug effects; Computer-aided drug design; Ubiquitination; Proteolysis targeting chimeric molecules; Fibrosis
• ISSN: 0006-2952; 1873-2968; 1873-2968
• DOI: 10.1016/j.bcp.2016.07.017

[Display omitted] 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=4.547×10−5M). 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.