关键词: 毛细管电泳前沿分析, 电喷雾质谱, 相互作用, G-四链体, 天然产物

Abstract:

The relationship between a drug and its target directly affects its pharmacology and efficacy. Drug-target binding ability and binding stoichiometry are essential characterization data in pharmaceutical research. The c-myb proto-oncogene encodes a crucial transcription factor that is involved in proliferation, differentiation, and maturation during hematopoiesis. Recent studies have found that the human oncogene c-myb is overexpressed in cancer tissues such as colorectal cancer. C-myb has become a potential therapeutic target for colorectal cancer, leukemia, and other cancers. A guanine (G)-rich DNA sequence located in the promoter region of c-myb can be spontaneously folded to form an intra-molecular G-quadruplex (G4) with cationic induction. The specific recognition of small molecules can stabilize this G4 folding, thus regulating the transcription and expression of c-myb. In this study, pressure assisted capillary electrophoresis frontier analysis (PACE-FA) combined with electrospray ionization mass spectrometry (ESI-MS) was used to investigate the interactions between the human c-myb promoter G4 and natural product molecules. In PACE-FA, an external pressure (no more than 13.8 kPa) in the same direction of the migration of the analyte was used in capillary electrophoresis frontier analysis (CE-FA), which greatly sped up the analysis while maintaining the accuracy of the results. Meanwhile, the combination of PACE-FA and ESI-MS could rapidly determine the affinity and stoichiometric relationship between binding molecules and targets. First, the intramolecular parallel-stranded G4 formation of the c-myb promoter sequence in the presence of cations (K⁺, NH₄⁺) was investigated by circular dichroism (CD) and ESI-MS. Then, ESI-MS was used to rapidly screen the natural products for candidate molecules that bound the target G4 DNA. The binding interactions were measured by mixing the c-myb DNA with each natural product separately in a 1:4 molar ratio, and then directly infusing these mixtures into the ESI-MS system. From the ESI-MS spectra, IR_a values calculated from the relative intensities of DNA and its complex ions were used to probe the binding affinities of the natural products. This parameter denotes the relative binding affinity for a small molecule with the G4 DNA. Three natural products were identified through the screen, and their binding affinities to G4 DNA were ranked as follows:pseudolaric acid B > scopolamine butylbromide > nuciferine. Considering that both specific and non-specific binding existed in the solution phase, a free solution method using PACE-FA was developed to further test the binding ability of these products to the c-myb promoter G4 DNA. In the PACE-FA experiments, the pre-equilibrated mixture of the c-myb G4 DNA and the selected ligand was injected into the capillary prior to separation. Upon applying voltage and an external pressure (6.9 kPa) to the capillary, different species of analytes in the sample migrated at their own rates due to their different sizes and charges. The results showed that scopolamine butylbromide could bind specifically to target G4 DNA with 1:1 stoichiometry and a binding constant of 1.18×10⁵ L/mol. Nuciferine's binding to G4 DNA showed a linear increasing trend due to nonspecific binding; thus nuciferine is a nonspecific binder. Although pseudolaric acid B showed high affinity for the c-myb G4 DNA and 1:1 and 1:2 G4-bound complex ions were observed in ESI-MS measurements, the PACE-FA results indicated that pseudolaric acid B did not bind to target G4 DNA in free solution. Therefore, scopolamine butylbromide could be the best candidate to regulate the transcription of the c-myb oncogene, and is expected to be a precursor for anticancer drugs. In this work, PACE-FA has allowed for significant improvements to the conventional CE-FA technique. This combination of PACE-FA and ESI-MS not only reduced the time needed for binding analysis, but also improved the accuracy and specificity of the affinity analysis compared to conventional binding approaches. Furthermore, this combination could be used to screen other targeted drug candidates and to evaluate their interaction mechanisms.

Key words: capillary electrophoresis frontal analysis (CE-FA), electrospray ionization mass spectrometry (ESI-MS), interaction, G-quadruplex, natural product