Fluorescent Labeling Can Significantly Perturb Measured Binding Affinity and Selectivity of Peptide-Protein...

Area of Science:

• Biochemistry
• Chemical Biology
• Drug Discovery

Background:

• Peptide-based drugs are effective inhibitors of protein-protein interactions.
• Fluorescence-based assays like anisotropy and microarrays are common for assessing drug binding.
• Labeling strategies can potentially introduce artifacts in these assays.

Purpose of the Study:

• To investigate the impact of fluorescent labeling on peptide/protein binding affinity.
• To validate the selectivity of novel peptide inhibitors targeting the SHP2 N-terminal Src homology 2 (SH2) domain.
• To identify and mitigate assay artifacts in drug discovery.

Main Methods:

• Development of peptide inhibitors targeting the SHP2 SH2 domain.
• Assessment of binding affinity using fluorescence anisotropy and quantitative microarrays.
• Comparison of binding affinities using both labeled and unlabeled peptides.

Main Results:

• Fluorescent labeling perturbed peptide/protein binding by over an order of magnitude.
• Anisotropy measurements showed labeling significantly influenced dissociation constants (∼10-fold).
• Displacement assays with unlabeled peptides confirmed inhibitors' affinity for SHP2 is >1,000 times higher than for APS.

Conclusions:

• Fluorescent labeling can significantly impact the measured affinity and selectivity of peptide-based drugs.
• Unlabeled peptide displacement assays are crucial for accurate assessment of drug-target interactions.
• Novel SHP2 inhibitors exhibit high affinity and selectivity when artifacts are controlled.