Two-colored fluorescence correlation spectroscopy screening for LC3-P62 interaction inhibitors.

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Protein-protein interactions are vital in cellular processes, including selective autophagy.
• Inhibiting these interactions is a key therapeutic strategy.
• Conventional screening methods like fluorescent polarization assays have limitations.

Purpose of the Study:

• To develop and validate a highly sensitive, two-colored fluorescence correlation spectroscopy (FCS) assay for screening protein-protein interaction inhibitors.
• To identify inhibitors of the LC3-p62 interaction critical for selective autophagy.
• To overcome the challenge of false positives in FCS-based screening.

Main Methods:

• A two-colored FCS competitive binding assay was developed.
• Approximately 10,000 compounds were screened using a TAMRA-labeled p62 probe.
• Hits were re-evaluated using a second FCS assay with an Alexa647-labeled p62 probe.
• Surface plasmon resonance was used for final validation.

Main Results:

• The initial FCS screen identified 29 hit compounds.
• The orthogonal two-colored FCS assay reduced false positives, yielding six inhibitors.
• Surface plasmon resonance confirmed two potent inhibitors with IC50 < 2 µM.

Conclusions:

• The developed two-colored FCS screening is an effective and sensitive method for identifying protein-protein interaction inhibitors.
• This approach successfully identified inhibitors of the LC3-p62 interaction involved in selective autophagy.
• The method offers improved accuracy by mitigating false-positive results.