A cell-based high-throughput screening method based on a ubiquitin-reference technique for identifying modulators of...

Area of Science:

• Biochemistry
• Molecular Biology
• Drug Discovery

Background:

• E3 ubiquitin ligases play critical roles in human diseases.
• Targeting E3 ubiquitin ligases with small molecules is a promising therapeutic strategy.
• SMAD ubiquitin regulatory factor 1 (SMURF1) is implicated in tumorigenesis.

Purpose of the Study:

• To develop a cell-based high-throughput screening (HTS) method for identifying E3 ubiquitin ligase modulators.
• To screen for small-molecule modulators of SMURF1, a key E3 ubiquitin ligase in cancer.
• To validate the identified modulators' effects on SMURF1 substrates and cellular functions.

Main Methods:

• Integration of the ubiquitin-reference technique (URT) with a Dual-Luciferase system for HTS.
• Screening for SMURF1 modulators using RAS homolog family member B (RHOB) as a substrate.
• In vitro auto-ubiquitination assays and cell-based functional assays (e.g., TGFβ-induced EMT).

Main Results:

• A potent SMURF1 inhibitor was identified.
• The inhibitor blocked SMURF1-dependent degradation of SMAD1 and RHOA.
• The compound inhibited SMURF1 and SMURF2 in vitro and demonstrated functional effects in cell assays, including inhibition of protrusive activity and TGFβ-induced EMT.

Conclusions:

• The developed URT-Dual-Luciferase HTS method is effective for identifying E3 ubiquitin ligase modulators.
• The identified SMURF1 inhibitor shows therapeutic potential for diseases involving SMURF1.
• This screening approach holds significant promise for future drug discovery efforts targeting E3 ubiquitin ligases.