Rational design of a sensitivity-enhanced tracer for discovering efficient APC-Asef inhibitors
Jie Zhong1,2,3, Yuegui Guo4, Shaoyong Lu2
1State Key Laboratory of Medical Genomics, National Research Center for Translational Medicine at Shanghai, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Abstract:
The adenomatous polyposis coli (APC)-Rho guanine nucleotide exchange factor 4 (Asef) protein-protein interaction (PPI) is essential for colorectal cancer metastasis, making it a promising drug target. Herein, we obtain a sensitivity-enhanced tracer (tracer 7) with a high binding affinity (Kd = 0.078 μM) and wide signal dynamic range (span = 251 mp). By using tracer 7 in fluorescence-polarization assays for APC-Asef inhibitor screening, we discover a best-in-class inhibitor, MAI-516, with an IC50 of 0.041 ± 0.004 μM and a conjugated transcriptional transactivating sequence for generating cell-permeable MAIT-516. MAIT-516 inhibits CRC cell migration by specifically hindering the APC-Asef PPI. Furthermore, MAIT-516 exhibits no cytotoxic effects on normal intestinal epithelial cell and colorectal cancer cell growth. Overall, we develop a sensitivity-enhanced tracer for fluorescence polarization assays, which is used for the precise quantification of high-activity APC-Asef inhibitors, thereby providing insight into PPI drug development.
Insights
Researchers developed a sensitive tracer and identified a novel inhibitor, MAIT-516, targeting the APC-Asef protein-protein interaction crucial for colorectal cancer metastasis. This discovery offers a new avenue for developing effective colorectal cancer therapies.
Area of Science:
- Biochemistry
- Molecular Biology
- Oncology
Background:
- The adenomatous polyposis coli (APC)-Rho guanine nucleotide exchange factor 4 (Asef) protein-protein interaction (PPI) is critical for colorectal cancer (CRC) metastasis.
- Targeting this APC-Asef PPI presents a promising therapeutic strategy for CRC.
Purpose of the Study:
- To develop a sensitive tracer for fluorescence polarization assays to screen for APC-Asef inhibitors.
- To identify and characterize novel inhibitors of the APC-Asef PPI for CRC treatment.
Main Methods:
- Development of a sensitivity-enhanced tracer (tracer 7) with high binding affinity.
- Utilizing fluorescence polarization assays for high-throughput screening of APC-Asef inhibitors.
- Characterization of inhibitor efficacy (IC50) and cell permeability.
Main Results:
- Tracer 7 demonstrated high binding affinity (Kd = 0.078 μM) and a wide signal dynamic range.
- A novel inhibitor, MAI-516, was identified with potent activity (IC50 = 0.041 ± 0.004 μM).
- The cell-permeable derivative, MAIT-516, effectively inhibited CRC cell migration by targeting the APC-Asef PPI without cytotoxicity.
Conclusions:
- A novel, sensitive fluorescence polarization assay was established for APC-Asef PPI inhibitor screening.
- MAIT-516 represents a promising, non-cytotoxic therapeutic candidate for colorectal cancer.
- This work provides valuable insights into PPI-targeted drug development for cancer therapy.
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