A small-molecule inhibitor and degrader of the RNF5 ubiquitin ligase

Abstract

RNF5 E3 ubiquitin ligase has multiple biological roles and has been linked to the development of severe diseases such as cystic fibrosis, acute myeloid leukemia, and certain viral infections, emphasizing the importance of discovering small molecule RNF5 modulators for research and drug development. The present study describes the synthesis of a new benzo[b]thiophene derivative, FX12 that acts as a selective small-molecule inhibitor and degrader of RNF5. We initially identified the previously reported STAT3 inhibitor, Stattic, as an inhibitor of dislocation of misfolded proteins from the endoplasmic reticulum (ER) lumen to the cytosol in ER-associated degradation. A concise structure-activity relationship campaign (SAR) around the Stattic chemotype led to the synthesis of FX12 that has diminished activity in inhibition of STAT3 activation and retains dislocation inhibitory activity. FX12 binds to RNF5 and inhibits its E3 activity in vitro as well as promotes proteasomal degradation of RNF5 in cells. RNF5 as a molecular target for FX12 was supported by the facts that FX12 requires RNF5 to inhibit dislocation and negatively regulates RNF5 function. Thus, this study developed a small molecule inhibitor and degrader of the RNF5 ubiquitin ligase, providing a chemical biology tool for RNF5 research and therapeutic development.

Innovative Strategy toward Mutant CFTR Rescue in Cystic Fibrosis: Design and Synthesis of Thiadiazole Inhibitors of the E3 Ligase RNF5

“…Recently, a small-molecule inhibitor and degrader of RNF5 was discovered based on its ability to inhibit misfolded proteins from the ER lumen to the cytosol and to negatively regulate the RNF5 function . This finding further supports RNF5 druggability.…”

“…A construct coding for human RNF5 protein (aa 1–117) truncated to exclude the transmembrane domains, with an N-terminal GST-tag followed by the recognition site for TEV protease (pET vector backbone), was purchased from CliniSciences. A similar construct was recently described in the work of Ruan et al . The plasmid was first verified by sequencing and then transformed in BL21­(DE3) Competent Cells (EC0114, Thermo Fischer Scientific).…”

“…The solvent was removed under reduced pressure, and the crude product was purified by flash silica gel chromatography (A = PE, B = EtOAc, gradient 0−20% B). Yields: (21) (44). 3-Benzyl-5-methyl-1,3,4thiadiazol-2(3H)-imine 134d (500 mg, 2.43 mmol), N-phenylbenzimidoyl chloride 108b (625 mg, 2.9 mmol), and pyridine (243 μL, 2.9 mmol) were reacted according to general procedure C. The crude product was purified by flash silica gel chromatography (PE/ EtOAc in 98/2 ratio) to achieve the final compound 44 as a yellow solid (222 mg, yield 23%).…”

“…A construct coding for human RNF5 protein (aa 1−117) truncated to exclude the transmembrane domains, with an N-terminal GST-tag followed by the recognition site for TEV protease (pET vector backbone), was purchased from CliniSciences. A similar construct was recently described in the work of Ruan et al 44 The plasmid was first verified by sequencing and then transformed in BL21(DE3) Competent Cells (EC0114, Thermo Fischer Scientific). Overexpression of the protein was achieved in Escherichia coli by growing cells in LB medium at 37 °C to an OD 600 ∼ 0.6 followed by induction with 0.2 mM isopropyl β-D-thiogalactopyranoside (IPTG) for 5 h at 37 °C.…”

Proteomics: Potential techniques for discovering the pathogenesis of connective tissue diseases-interstitial lung disease

“…Similar to the findings of Ma et al. ( 14 ), three differential protein peaks were selected to form a diagnostic model, with a sensitivity of 84% and a specificity of 85.7% ( 59 ). These results suggest that searching differential protein peaks using combinations of proteomics and statistical screening can effectively identify alternative diagnostic models of diseases.…”