Fragment Screening Yields a Small-Molecule Stabilizer of 14-3-3 Dimers That Modulates Client Protein Interactions

Hendrik J. Brink; Rick Riemens; Stephanie Thee; Berend Beishuizen; Daniel da Costa Pereira; Maikel Wijtmans; Iwan de Esch; Martine J. Smit; Albertus H. de Boer

doi:10.1002/cbic.202200178

Fragment Screening Yields a Small-Molecule Stabilizer of 14-3-3 Dimers That Modulates Client Protein Interactions

Hendrik J. Brink, Rick Riemens, Stephanie Thee, Berend Beishuizen, Daniel da Costa Pereira, Maikel Wijtmans, Iwan de Esch, Martine J. Smit^*, Albertus H. de Boer

^*Corresponding author for this work

Research output: Contribution to Journal › Article › Academic › peer-review

Abstract

The development of protein-protein interaction (PPI) inhibitors has been a successful strategy in drug development. However, the identification of PPI stabilizers has proven much more challenging. Here we report a fragment-based drug screening approach using the regulatory hub-protein 14-3-3 as a platform for identifying PPI stabilizers. A homogenous time-resolved FRET assay was used to monitor stabilization of 14-3-3/peptide binding using the known interaction partner estrogen receptor alpha. Screening of an in-house fragment library identified fragment 2 (VUF15640) as a putative PPI stabilizer capable of cooperatively stabilizing 14-3-3 PPIs in a cooperative fashion with Fusicoccin-A. Mechanistically, fragment 2 appears to enhance 14-3-3 dimerization leading to increased client-protein binding. Functionally, fragment 2 enhanced potency of 14-3-3 in a cell-free system inhibiting the enzyme activity of the nitrate reductase. In conclusion, we identified a general PPI stabilizer targeting 14-3-3, which could be used as a tool compound for investigating 14-3-3 client protein interactions.

Original language	English
Article number	e202200178
Pages (from-to)	1-11
Number of pages	11
Journal	ChemBioChem
Volume	23
Issue number	17
Early online date	29 Jun 2022
DOIs	https://doi.org/10.1002/cbic.202200178
Publication status	Published - 5 Sept 2022

Bibliographical note

Funding Information:
We thank the Amsterdam Institute of Molecular and Life Sciences, Henry Vischer and Saskia Nijmeijer for the experiments carried out at the facilities of the Chemical Biology Platform, and Hans Custers for HRMS measurements. Finally, we would like to thank Reggie Bosma for his insightful comments at all stages of the project.

Publisher Copyright:
© 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH.

Funding

We thank the Amsterdam Institute of Molecular and Life Sciences, Henry Vischer and Saskia Nijmeijer for the experiments carried out at the facilities of the Chemical Biology Platform, and Hans Custers for HRMS measurements. Finally, we would like to thank Reggie Bosma for his insightful comments at all stages of the project.

Keywords

drug discovery
estrogen receptor alpha
fragments
protein-protein interactions
stabilizers

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

Access to Document

10.1002/cbic.202200178

Persistent URL (handle)

Persistent link

Cite this

@article{9cab963a195646598b0ec71ac894a98b,

title = "Fragment Screening Yields a Small-Molecule Stabilizer of 14-3-3 Dimers That Modulates Client Protein Interactions",

abstract = "The development of protein-protein interaction (PPI) inhibitors has been a successful strategy in drug development. However, the identification of PPI stabilizers has proven much more challenging. Here we report a fragment-based drug screening approach using the regulatory hub-protein 14-3-3 as a platform for identifying PPI stabilizers. A homogenous time-resolved FRET assay was used to monitor stabilization of 14-3-3/peptide binding using the known interaction partner estrogen receptor alpha. Screening of an in-house fragment library identified fragment 2 (VUF15640) as a putative PPI stabilizer capable of cooperatively stabilizing 14-3-3 PPIs in a cooperative fashion with Fusicoccin-A. Mechanistically, fragment 2 appears to enhance 14-3-3 dimerization leading to increased client-protein binding. Functionally, fragment 2 enhanced potency of 14-3-3 in a cell-free system inhibiting the enzyme activity of the nitrate reductase. In conclusion, we identified a general PPI stabilizer targeting 14-3-3, which could be used as a tool compound for investigating 14-3-3 client protein interactions.",

keywords = "drug discovery, estrogen receptor alpha, fragments, protein-protein interactions, stabilizers",

author = "Brink, {Hendrik J.} and Rick Riemens and Stephanie Thee and Berend Beishuizen and {da Costa Pereira}, Daniel and Maikel Wijtmans and {de Esch}, Iwan and Smit, {Martine J.} and {de Boer}, {Albertus H.}",

note = "Funding Information: We thank the Amsterdam Institute of Molecular and Life Sciences, Henry Vischer and Saskia Nijmeijer for the experiments carried out at the facilities of the Chemical Biology Platform, and Hans Custers for HRMS measurements. Finally, we would like to thank Reggie Bosma for his insightful comments at all stages of the project. Publisher Copyright: {\textcopyright} 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH.",

year = "2022",

month = sep,

day = "5",

doi = "10.1002/cbic.202200178",

language = "English",

volume = "23",

pages = "1--11",

journal = "ChemBioChem",

issn = "1439-4227",

publisher = "John Wiley and Sons Inc.",

number = "17",

}

TY - JOUR

T1 - Fragment Screening Yields a Small-Molecule Stabilizer of 14-3-3 Dimers That Modulates Client Protein Interactions

AU - Brink, Hendrik J.

AU - Riemens, Rick

AU - Thee, Stephanie

AU - Beishuizen, Berend

AU - da Costa Pereira, Daniel

AU - Wijtmans, Maikel

AU - de Esch, Iwan

AU - Smit, Martine J.

AU - de Boer, Albertus H.

N1 - Funding Information: We thank the Amsterdam Institute of Molecular and Life Sciences, Henry Vischer and Saskia Nijmeijer for the experiments carried out at the facilities of the Chemical Biology Platform, and Hans Custers for HRMS measurements. Finally, we would like to thank Reggie Bosma for his insightful comments at all stages of the project. Publisher Copyright: © 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH.

PY - 2022/9/5

Y1 - 2022/9/5

N2 - The development of protein-protein interaction (PPI) inhibitors has been a successful strategy in drug development. However, the identification of PPI stabilizers has proven much more challenging. Here we report a fragment-based drug screening approach using the regulatory hub-protein 14-3-3 as a platform for identifying PPI stabilizers. A homogenous time-resolved FRET assay was used to monitor stabilization of 14-3-3/peptide binding using the known interaction partner estrogen receptor alpha. Screening of an in-house fragment library identified fragment 2 (VUF15640) as a putative PPI stabilizer capable of cooperatively stabilizing 14-3-3 PPIs in a cooperative fashion with Fusicoccin-A. Mechanistically, fragment 2 appears to enhance 14-3-3 dimerization leading to increased client-protein binding. Functionally, fragment 2 enhanced potency of 14-3-3 in a cell-free system inhibiting the enzyme activity of the nitrate reductase. In conclusion, we identified a general PPI stabilizer targeting 14-3-3, which could be used as a tool compound for investigating 14-3-3 client protein interactions.

AB - The development of protein-protein interaction (PPI) inhibitors has been a successful strategy in drug development. However, the identification of PPI stabilizers has proven much more challenging. Here we report a fragment-based drug screening approach using the regulatory hub-protein 14-3-3 as a platform for identifying PPI stabilizers. A homogenous time-resolved FRET assay was used to monitor stabilization of 14-3-3/peptide binding using the known interaction partner estrogen receptor alpha. Screening of an in-house fragment library identified fragment 2 (VUF15640) as a putative PPI stabilizer capable of cooperatively stabilizing 14-3-3 PPIs in a cooperative fashion with Fusicoccin-A. Mechanistically, fragment 2 appears to enhance 14-3-3 dimerization leading to increased client-protein binding. Functionally, fragment 2 enhanced potency of 14-3-3 in a cell-free system inhibiting the enzyme activity of the nitrate reductase. In conclusion, we identified a general PPI stabilizer targeting 14-3-3, which could be used as a tool compound for investigating 14-3-3 client protein interactions.

KW - drug discovery

KW - estrogen receptor alpha

KW - fragments

KW - protein-protein interactions

KW - stabilizers

UR - http://www.scopus.com/inward/record.url?scp=85134183978&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85134183978&partnerID=8YFLogxK

U2 - 10.1002/cbic.202200178

DO - 10.1002/cbic.202200178

M3 - Article

C2 - 35767695

AN - SCOPUS:85134183978

SN - 1439-4227

VL - 23

SP - 1

EP - 11

JO - ChemBioChem

JF - ChemBioChem

IS - 17

M1 - e202200178

ER -

Fragment Screening Yields a Small-Molecule Stabilizer of 14-3-3 Dimers That Modulates Client Protein Interactions

Abstract

Bibliographical note

Funding

Keywords

UN SDGs

Access to Document

Persistent URL (handle)

Other files and links

Fingerprint

Cite this