研究目的
To identify potent caspase-8 inhibitors from a library of fluorescent natural products using an AIEgen-based light-up probe to avoid interference from inborn fluorescence.
研究成果
The study successfully developed a caspase-8 specific AIEgen-based light-up probe that avoids fluorescence interference from natural products. It identified gossypol as a potent inhibitor, with (-)-gossypol being slightly more effective than (+)-gossypol. This approach is efficient for hit discovery in drug targeting.
研究不足
The probe showed non-specific binding with caspase-3, which could affect specificity in certain contexts. The screening was limited to a specific library of 86 compounds, and further optimization may be needed for broader applications.
1：Experimental Design and Method Selection:
Designed and synthesized a caspase-8 targeting light-up probe (C8-P1) based on an AIEgen (TPETH) with a large Stokes shift of 200 nm to avoid fluorescence interference from natural products. The probe was synthesized via thiol-ene reaction between maleimide-modified TPETH and peptide sequence Ac-DIETDC-NH
2：Sample Selection and Data Sources:
Screened a library of 86 fluorescent natural products with π electron conjugated systems, including alkaloids, polyenes, and flavonoids. Used caspase-8 enzyme and HeLa cells for in vitro and in situ assays.
3：List of Experimental Equipment and Materials:
Equipment includes UV lamp for fluorescence photos, HPLC with IE chiral column for enantiomer separation, confocal microscope for cellular imaging, and instruments for mass spectra and NMR verification. Materials include DMSO, HEPES buffer, H2O2, and various enzymes like pepsin, BSA, trypsin, lysozyme, caspases.
4：Experimental Procedures and Operational Workflow:
Synthesized and characterized C8-P
5：Evaluated its fluorescence response to caspase-8 and selectivity against other enzymes. Screened the natural product library by incubating compounds with caspase-8 and C8-P1, measuring fluorescence inhibition. Separated gossypol enantiomers using HPLC and tested their inhibition in vitro and in HeLa cells treated with H2OData Analysis Methods:
Measured fluorescence intensity changes, calculated IC50 values for inhibition, and used statistical methods for linear relationships and comparisons.
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