Fluorescent Labeling Can Significantly Perturb Measured Binding Affinity and Selectivity of Peptide–Protein Interactions

Peptide-based drugs are powerful inhibitors of therapeutically relevant protein-protein interactions. Their affinity and selectivity for target proteins are commonly assessed using fluorescence-based assays such as anisotropy/polarization or quantitative microarrays. This study reveals that labeling can perturb peptide/protein binding by more than 1 order of magnitude. We have recently developed inhibitors targeted to the N-terminal Src homology 2 (SH2) domain of oncogenic phosphatase SHP2. Despite their high activity and selectivity, these molecules demonstrated an undesired interaction with the SH2 domain of another protein, known as APS, in a fluorescence microarray assay. Fluorescence anisotropy measurement in solution showed that the dissociation constant was significantly influenced by labeling (∼10 times), and the effect depended on the specific fluorophore and SH2 domain. Notably, displacement assays performed with unlabeled peptides were successfully used to eliminate these artifacts, demonstrating that the inhibitors' affinity for their target is over 1,000 times higher than for APS.

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Publication Details

Journal
The Journal of Physical Chemistry Letters
Published
2024-10-03
DOI
https://doi.org/10.1021/acs.jpclett.4c01767
Citations
3
Primary Topic
Chemical Synthesis and Analysis
Type
article
Field-Weighted Citation Impact
0.70

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Fluorescent Labeling Can Significantly Perturb Measured Binding Affinity and Selectivity of Peptide–Protein Interactions

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Fluorescent Labeling Can Significantly Perturb Measured Binding Affinity and Selectivity of Peptide–Protein Interactions

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article en
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Abstract

Peptide-based drugs are powerful inhibitors of therapeutically relevant protein-protein interactions. Their affinity and selectivity for target proteins are commonly assessed using fluorescence-based assays such as anisotropy/polarization or quantitative microarrays. This study reveals that labeling can perturb peptide/protein binding by more than 1 order of magnitude. We have recently developed inhibitors targeted to the N-terminal Src homology 2 (SH2) domain of oncogenic phosphatase SHP2. Despite their high activity and selectivity, these molecules demonstrated an undesired interaction with the SH2 domain of another protein, known as APS, in a fluorescence microarray assay. Fluorescence anisotropy measurement in solution showed that the dissociation constant was significantly influenced by labeling (∼10 times), and the effect depended on the specific fluorophore and SH2 domain. Notably, displacement assays performed with unlabeled peptides were successfully used to eliminate these artifacts, demonstrating that the inhibitors' affinity for their target is over 1,000 times higher than for APS.

The Journal of Physical Chemistry LettersVol. 15(40)
University of Rome Tor Vergata (IT), University of Padua (IT), Istituto Superiore di Sanità (IT), Bambino Gesù Children's Hospital (IT), Istituti di Ricovero e Cura a Carattere Scientifico (IT)
European Commission, Ministero dell’Istruzione, dell’Università e della Ricerca, Regione Lazio
Openalex Percentile: Top 29%
Chemical Synthesis and Analysis
0.70
DOISourceOpenAlex
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