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Published on: August 5, 2022
A multiplexed time-resolved fluorescence resonance energy transfer ultrahigh-throughput screening assay for targeting
Wukun Ouyang1, Qianjin Li1, Qiankun Niu1
1Department of Pharmacology and Chemical Biology, Emory University School of Medicine, Atlanta, GA 30322, USA.
Abstract:
The transforming growth factor-beta (TGFβ) signaling pathway plays crucial roles in the establishment of an immunosuppressive tumor microenvironment, making anti-TGFβ agents a significant area of interest in cancer immunotherapy. However, the clinical translation of current anti-TGFβ agents that target upstream cytokines and receptors remains challenging. Therefore, the development of small-molecule inhibitors specifically targeting SMAD4, the downstream master regulator of the TGFβ pathway, would offer an alternative approach with significant therapeutic potential for anti-TGFβ signaling. In this study, we present the development of a cell lysate-based multiplexed time-resolved fluorescence resonance energy transfer (TR-FRET) assay in an ultrahigh-throughput screening (uHTS) 1536-well plate format. This assay enables simultaneous monitoring of the protein‒protein interaction between SMAD4 and SMAD3, as well as the protein‒DNA interaction between SMADs and their consensus DNA-binding motif. The multiplexed TR-FRET assay exhibits high sensitivity, allowing the dynamic analysis of the SMAD4-SMAD3-DNA complex at single-amino acid resolution. Moreover, the multiplexed uHTS assay demonstrates robustness for screening small-molecule inhibitors. Through a pilot screening of an FDA-approved bioactive compound library, we identified gambogic acid and gambogenic acid as potential hit compounds. These proof-of-concept findings underscore the utility of our optimized multiplexed TR-FRET platform for large-scale screening to discover small-molecule inhibitors that target the SMAD4-SMAD3-DNA complex as novel anti-TGFβ signaling agents.
Insights
Researchers developed a novel assay to screen for drugs targeting the SMAD4 protein, a key regulator in the transforming growth factor-beta (TGFβ) pathway. This new method identified potential small-molecule inhibitors for cancer immunotherapy.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- The transforming growth factor-beta (TGFβ) signaling pathway is critical for creating an immunosuppressive tumor microenvironment, making it a key target in cancer immunotherapy.
- Current therapeutic strategies targeting upstream components of the TGFβ pathway have faced clinical translation challenges.
- Developing inhibitors for SMAD4, the downstream regulator of TGFβ signaling, presents a promising alternative therapeutic approach.
Purpose of the Study:
- To develop a robust assay for screening small-molecule inhibitors targeting the SMAD4 protein.
- To identify novel compounds that can modulate the SMAD4-SMAD3-DNA complex for anti-TGFβ signaling.
Main Methods:
- Development of a cell lysate-based multiplexed time-resolved fluorescence resonance energy transfer (TR-FRET) assay.
- Implementation of the assay in an ultrahigh-throughput screening (uHTS) 1536-well plate format.
- Simultaneous monitoring of SMAD4-SMAD3 protein-protein and SMADs-DNA protein-DNA interactions.
Main Results:
- The multiplexed TR-FRET assay demonstrated high sensitivity and dynamic analysis capabilities for the SMAD4-SMAD3-DNA complex.
- The assay proved robust for screening small-molecule inhibitors in a uHTS format.
- Pilot screening of an FDA-approved compound library identified gambogic acid and gambogenic acid as potential hit compounds.
Conclusions:
- The developed multiplexed TR-FRET assay is a valuable platform for large-scale screening of novel anti-TGFβ signaling agents.
- Targeting the SMAD4-SMAD3-DNA complex with small molecules offers a promising strategy for cancer immunotherapy.
- The identified hit compounds warrant further investigation as potential therapeutic agents.
