We report the development of a 384-well formatted NanoBRET assay to characterize molecular glues of 14-3-3/client interactions in living cells. The seven isoforms of 14-3-3 are dimeric hub proteins with diverse roles including transcription factor regulation and signal transduction. 14-3-3 interacts with hundreds of client proteins to regulate their function and is therefore an ideal therapeutic target when client selectivity can be achieved. We have developed the NanoBRET system for three 14-3-3σ client proteins CRAF, TAZ, and estrogen receptor α (ERα), which represent three specific binding modes. We have measured stabilization of 14-3-3σ/client complexes by molecular glues with EC values between 100 nM and 1 μM in cells, which align with the EC values calculated by fluorescence anisotropy in vitro. Developing this NanoBRET system for the hub protein 14-3-3σ allows for a streamlined approach, bypassing multiple optimization steps in the assay development process for other 14-3-3σ clients. The NanoBRET system allows for an assessment of PPI stabilization in a more physiologically relevant, cell-based environment using full-length proteins. The method is applicable to diverse protein-protein interactions (PPIs) and offers a robust platform to explore libraries of compounds for both PPI stabilizers and inhibitors.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1016/j.slasd.2024.100165 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Visible-light-switchable Molecular Glues for Reversible Control of Protein Function.
Chemistry
January 2025
Umeå Universitet: Umea Universitet, Department of Chemistry, Department of Chemistry, 90187, Umeå, SWEDEN.
Chemically induced dimerization/proximity (CID/CIP) systems controlled by chemical dimerizers (also known as molecular glues) provide valuable means for understanding and manipulating complex, dynamic biological systems. In this study, we present the development of versatile chemo-optogenetic systems utilizing azobenzene-based photoswitchable molecular glues (sMGs) for reversible protein dimerization controlled by visible light. These systems allow multiple cycles of light-induced dimerization, overcoming the limitations of irreversible photolysis in previous systems.
View Article and Find Full Text PDFBioadhesives and bioactive hydrogels for wound management.
J Control Release
January 2025
Department of Bioengineering and Nano-Bioengineering, College of Life Sciences and Bioengineering, Incheon National University, 119 Academy-ro, Yeonsu-gu, Incheon 22012, Republic of Korea; Research Center for Bio Materials & Process Development, Incheon National University, 119 Academy-ro, Yeonsu-gu, Incheon 22012, Republic of Korea. Electronic address:
Delayed wound healing remains a major challenge in biomedical research, often leading to complications such as scarring, acute trauma, and chronic diseases. Effective wound management is crucial for enhancing treatment outcomes, preventing complications, and promoting tissue regeneration. In response to this need, a variety of polymeric biomaterials have been developed.
View Article and Find Full Text PDFDynamic Peptide Nanoframework-Guided Protein Coassembly: Advancing Adhesion Performance with Hierarchical Structures.
J Am Chem Soc
January 2025
Engineering Research Center of Advanced Rare Earth Materials (Ministry of Education), Department of Chemistry, Tsinghua University, Beijing 100084, China.
Hierarchical structures are essential in natural adhesion systems. Replicating these in synthetic adhesives is challenging due to intricate molecular mechanisms and multiscale processes. Here, we report three phosphorylated peptides featuring a hydrophobic self-assembly motif linked to a hydrophilic phosphorylated sequence (pSGSS), forming peptide fibril nanoframeworks.
View Article and Find Full Text PDFMechanistic Insights Underlying the Drug Release and Skin Permeation of Guanfacine Transdermal Patch with Various Acrylic Pressure-Sensitive Adhesives.
AAPS PharmSciTech
January 2025
Xiangya School of Pharmaceutical Science, Central South University, Changsha, 410006, Hunan, China.
Acrylic pressure-sensitive adhesives (PSAs) are widely applied in transdermal drug delivery systems (TDDS). However, the molecular mechanisms underlying the effect of functional groups of PSAs on drug release and transdermal permeation properties remain insufficiently clear. In this study, we investigated the effect of acrylic PSAs' functional groups on the in vitro release and transdermal permeation properties of a model drug guanfacine (GFC).
View Article and Find Full Text PDFModular Photoswitchable Molecular Glues for Chemo-optogenetic Control of Protein Function in Living Cells.
Angew Chem Int Ed Engl
January 2025
Umeå Universitet: Umea Universitet, Department of Chemistry, Department of Chemistry, 90187, Umeå, SWEDEN.
Optogenetic systems using photosensitive proteins and chemically induced dimerization/proximity (CID/CIP) approaches enabled by chemical dimerizers (also termed molecular glues), are powerful tools to elucidate the dynamics of biological systems and to dissect complex biological regulatory networks. Here, we report a versatile chemo-optogenetic system using modular, photoswitchable molecular glues (sMGs) that can undergo repeated cycles of optical control to switch protein function on and off. We use molecular dynamics (MD) simulations to rationally design the sMGs and further expand their scope by incorporating different photoswitches, resulting in sMGs with customizable properties.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!