Cheng Qian,Hengqing Fu,Kevin A. Kovalchik,Huihui Li,David Da Yong Chen
A free solution method was developed for evaluating the specific binding affinity and stoichiometry of small molecules with oligo DNA subsequent to cation-induced G-quadruplex formation. A nonlinear curve fitting equation capable of extracting specific binding constants in the presence of nonspecific binding without the need for reference compounds was proposed and tested. Electrospray ionization mass spectrometry was first used to rapidly screen the small molecule candidates; then, the stoichiometry and affinity constants of the native state binding pair in solution were obtained with capillary electrophoresis frontal analysis (CE-FA). The B cell lymphoma 2 (Bcl-2) oncogene is directly responsible for the expression of Bcl-2 protein, which plays a significant role in cell apoptosis. The binding of a G-quadruplex formed in the promoter region of the Bcl-2 oncogene with a small molecule could stabilize the quadruplex structure and potentially regulate the transcription of Bcl-2. Four natural product drug candidates were tested for their ability to bind the Bcl-2 promoter G-quadruplex. Using this reference-free method based on CE-FA data, jatrorrhizine and palmatine were found to bind specifically to the Bcl-2 promoter G-quadruplex with stoichiometries of 4:1 and 3:1, respectively.
Dongsheng Ji,Jiahui Zhang,Jihai Liang,Zhi-Shu Huang,Bing Shu,Ding Li
Alzheimer's disease (AD) is a severe neurodegenerative disorder characterized by abnormal metabolism of β-amyloid (Aβ) precursor proteins and neuronal apoptosis, ultimately leading to cognitive dysfunction. The pathogenesis of AD is complex, and current single-target therapies are not effective in preventing the rapid progression of AD, which highlights the urgent need for developing multi-target drugs. In this study, a series of compounds were synthesized through a multi-targeting ligand strategy. After extensive screening and evaluation, we found a lead compound B14 , which showed excellent dual targeting ability for effectively alleviating neuronal apoptosis and oxidative stress damage of AD. In our molecular and cellular level experiments, B14 could target and stabilize the i-motif structure formed on the BCL-2 promoter to upregulate BCL-2 expression, which could also bind to Aβ and inhibit its deposition. In the Aβ 1-42 -induced cell model, B14 could maintain mitochondrial function and number, regulate intracellular reactive oxygen species (ROS) and Ca 2+ metabolism disorders, and effectively reduce Aβ 1-42 -induced apoptosis. Further studies showed that B14 also exhibited good ability to cross the blood-brain barrier (BBB), which significantly improved learning memory and cognitive deficits, reduced brain Aβ plaques, alleviated inflammation and restored oxidative stress markers in APP/PS1 mice. Our findings provide an innovative strategy of dual targeting BCL-2 promoter i-motif for transcriptional regulation and Aβ aggregation synergistically for mitigating AD pathologies. B14 represents a promising multi-target lead compound with a good potential for further development for AD treatment.
Jingwen Li,Yifei Li,Kai Zhang,Xiaomei Liu,Yuguo Tang,Kun Han
We developed a novel fluorescence detection method utilizing graphene quantum dots (GQDs) and manganese dioxide (MnO2) nanocomposites to enhance the sensitivity and specificity of in vitro monitoring of Bcl-2 gene expression, a key factor in tumor survival and treatment resistance. This method leverages the interplay between reduced glutathione (GSH) and hairpin DNA structures (H1 and H2) to simulate a cellular environment, enabling the redox-mediated disintegration of MnO2 nanosheets and the release of GQDs, restoring fluorescence. The presence of the Bcl-2 gene triggers a catalytic hairpin assembly (CHA) reaction, amplifying the Cy3 fluorescence signal and achieving a detection limit of 20.7 pM. The system demonstrated high specificity and stability, validated through control experiments with various DNA sequences. Optimization of key parameters, including MnO2 nanosheet concentration, GSH levels, and the ratio of hairpins H1 and H2, was critical for maximizing assay performance. The MnO2 nanosheets quench GQDs fluorescence and act as carriers for DNA probes, which are released upon reduction by GSH, mimicking intracellular redox conditions. The CHA reaction further amplifies the signal, allowing sensitive detection of low target gene levels. This biosensing platform integrates the unique properties of GQDs and MnO2 nanosheets, offering a real-time, sensitive, and selective method for monitoring Bcl-2 gene expression. This approach holds significant promise for early cancer diagnosis and personalized treatment strategies.
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