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A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules
Published on: March 10, 2017
Cell-based high-throughput screening using a target-NanoLuc fusion construct to identify molecular glue degraders of
Muyu Xu1, Jinying Qiu1, Lin Tan1
1Centre of Translational Research, Shenzhen Bay Laboratory, Guangming District, Shenzhen Guangdong 518132 China wshen@cpl.ac.cn jeffrey.hill@beigene.com xumy@szbl.ac.cn.
Abstract:
Oncoprotein c-Myc (Myc) plays a critical role in regulating cellular gene expression. Although Myc dysregulation is found in more than 70% of cancers and can facilitate tumor initiation and progression, it is still considered to be an "undruggable" oncotarget years after its first discovery. Recent advances in the field of targeted protein degradation provide alternative Myc-targeting strategies. Here, we develop the first Myc-NanoLuc fusion plasmid transfected cell-based high-throughput screening assay to identify Myc-downregulating small molecules. We verified the effectiveness of our assay by demonstrating that previously known Myc-downregulating compounds (G9 and SY-1365) were successfully identified from a library of bioactive compounds with established biological function. Next, we screened another 108 800 compounds from the diverse ChemDiv library collection, and 14 novel Myc-downregulating compounds were identified after cherry-pick triplicate confirmation, counter-screening, dose-response and western blotting experiments. A cellular thermal shift assay further demonstrated that five out of the 14 Myc-downregulating compounds bound to endogenous Myc protein in crude 293T whole-cell lysate. Subsequently, compound C1 was shown to selectively degrade Myc protein at a DC50 value of around 5 μM. Further characterization showed that C1 killed cancer cells with high Myc expression at a lower dose than it killed cancer cells with low Myc expression. Moreover, C1 selectively reduced the expression of various Myc-target genes. Intriguingly, co-immunoprecipitation showed that C1 functionally acted like a molecular glue to aggregate Myc proteins and block Myc/Max interaction. The self-aggregation of Myc and the dissociation of the Myc/Max dimer by C1 promoted Myc degradation. Using a target-NanoLuc fusion strategy in our novel cell-based high-throughput screening system, we identified a molecular glue-like small molecule degrader of Myc.
Insights
Researchers developed a novel high-throughput screening assay to identify small molecules that reduce oncoprotein c-Myc (Myc). This approach identified a new molecular glue-like compound that degrades Myc, offering a promising strategy against cancers with Myc dysregulation.
Area of Science:
- Oncology
- Molecular Biology
- Drug Discovery
Background:
- Oncoprotein c-Myc (Myc) is crucial for gene expression and frequently dysregulated in over 70% of cancers.
- Myc's role in tumorigenesis makes it a key target, but its "undruggable" status has limited therapeutic strategies.
- Targeted protein degradation offers novel approaches to target proteins like Myc.
Purpose of the Study:
- To develop a high-throughput screening (HTS) assay for identifying Myc-downregulating small molecules.
- To discover novel compounds that can target and degrade the Myc oncoprotein.
- To characterize the mechanism of action of identified Myc degraders.
Main Methods:
- Developed a Myc-NanoLuc fusion plasmid transfected cell-based HTS assay.
- Screened over 108,800 compounds from the ChemDiv library.
- Utilized triplicate confirmation, counter-screening, dose-response, western blotting, cellular thermal shift assay, and co-immunoprecipitation for validation and mechanistic studies.
Main Results:
- Successfully identified known Myc-downregulating compounds (G9, SY-1365) using the assay.
- Discovered 14 novel Myc-downregulating compounds, with five showing direct binding to endogenous Myc.
- Identified compound C1, a molecular glue-like degrader, selectively reducing Myc protein levels and killing Myc-high cancer cells.
Conclusions:
- The novel HTS assay is effective for identifying Myc-downregulating compounds.
- Compound C1 demonstrates selective Myc degradation via a molecular glue mechanism, promoting Myc aggregation and dissociation from Max.
- This study provides a promising small molecule degrader of Myc, offering a new therapeutic avenue for Myc-driven cancers.
