A fluorescence correlation spectroscopy-based assay for fragment screening of slowly inhibiting protein–peptide interaction inhibitors

https://doi.org/10.1016/j.ab.2010.03.019Get rights and content

Abstract

A fluorescence correlation spectroscopy (FCS)-based competitive binding assay to screen fragment-size compounds that weakly and slowly inhibit protein–peptide interactions was established. The interactions were detected by the increased diffusion time of a fluorescently labeled peptide probe after binding to its interacting protein. We analyzed the interactions between the c-Cbl TKB domain and phosphopeptides derived from ZAP-70, APS, and EGFR with the FCS assay and obtained 6 hit fragments that bound to the c-Cbl interaction sites. The binding amounts of the fragments were measured by direct binding measurements using surface plasmon resonance, and 5 fragments were found to bind selectively. The effect of 2 of the 5 fragments on the interaction with c-Cbl and the peptide exhibited strong time dependency. Furthermore, the inhibition by the selected 5 fragments on the protein–peptide interaction was confirmed by their effect on pull-down assays of c-Cbl with the biotin-conjugated interaction peptides. These results indicate the advantage of our FCS-based assay to study the time-dependent binding of compounds to their target protein.

Section snippets

Preparation of c-Cbl TKB domain (24–349)

The DNA fragment encoding the TKB domain of mouse c-Cbl (24–349) (P22682.2) was cloned into the TOPO vector (Life Technologies) as a fusion fragment with an N-terminal native His affinity tag and a TEV protease cleavage site [14]. The protein was synthesized by a cell-free expression system [14]. The obtained crude protein sample was applied to a HisTrap HP column (GE Healthcare) equilibrated with 20 mM Tris–HCl buffer (pH 8.0) containing 100 mM NaCl and 20 mM imidazole on an ÄKTA explorer system

FCS-based binding assay

First, we measured the binding of APSt to c-Cbl by FCS. Final 1 nM APSt and 0–30 μM c-Cbl were mixed, and FCS data of the binding mixtures were obtained. Fig. 1A shows FCS data measured with samples of the free form of the probe (left curve), the c-Cbl-bound form of the probe (right curve), and their mixture (middle curve). Fig. 1B shows the fit of the FCS data. After the fit, percentage ratios of the c-Cbl binding form were plotted (Fig. 2). As shown in Fig. 2, the amounts of the binding form of

Acknowledgments

We thank Noriko Kato and Mitsushiro Yamaguchi (Olympus) and Koji Kuruma (Fujifilm) for their contributions. This work was supported in part by the Target Protein Research Programs of the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

References (16)

There are more references available in the full text version of this article.

Cited by (13)

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

    2013, Journal of Biomolecular Screening
    Citation Excerpt :

    Thus, specific inhibitors of the interaction are desired to clarify the detailed mechanisms of autophagy. We previously reported the fluorescence correlation spectroscopy (FCS)–based competitive binding assay, for efficient screening of protein-protein interaction inhibitors.16 The FCS-based assay is a highly sensitive method as compared with the fluorescent polarization assay used conventionally, although the FCS assay identifies many false-positive compounds, which requires specifically designed orthogonal screenings.16,17

  • A novel Pim-1 kinase inhibitor targeting residues that bind the substrate peptide

    2012, Journal of Molecular Biology
    Citation Excerpt :

    In our strategy, the primary screening was performed using a conventional ATP-depleting kinase assay.15 In the second screening, we applied our competitive FCS method16 to select compounds that inhibit binding of a fluorescently labeled substrate and thus target the Pim-1 residues involved in substrate binding. We also report the activities of a newly identified Pim-1 inhibitor and its complex structure with Pim-1.

View all citing articles on Scopus
View full text