Study On The Binding Mechanism And Drug Loading Properties Of Sorafenib And G-Quadruplex

C-MYC G-quadruplex（G4）is a unique DNA mainly distributed in the promoter region,which can affect the transcriptional regulation process of organisms and has a high correlation with cancer.However,c-MYC protein lacks a clear target,so it has important biological significance to regulate proto-oncogene expression through small molecule stable G-quadruplex indirectly.Sorafenib（Sor）is a single drug approved by the European Union for treating patients with advanced hepatocellular carcinoma and renal cell carcinoma.However,it was found in clinical use that it showed dose-dependent anti-tumor activity,and its clinical application was limited due to poor bioavailability and various biological side effects.Drug loading platform is the main factor affecting drug efficacy.Reasonable selection and design of carriers can regulate the release rate of small molecules,so it is essential to find a suitable carrier to carry sorafenib.Based on the above reasons,this subject took sorafenib as the drug model.It used various analytical techniques to explore the drug loading potential of G-quadruplex,focusing on the binding mode,binding ability,and binding mechanism between them and evaluating the drug loading performance of G-quadruplex.This study helps discover the potential mechanism of sorafenib in vivo,providing primary data for the design and screening of G-quadruplex stabilizer and providing a reference basis for the application of G-quadruplex in the direction of the natural carrier.The main research contents and results are as follows:（1）Using molecular docking and molecular dynamics simulation,a visual combination model was built,and the dynamic stability of the model was discussed.The simulation results showed that the groove binding behavior of sorafenib to G-quadruplex was mainly driven by hydrogen bonds and hydrophobic forces.Through the dynamic simulation of 200ns,it was found that the binding energy of the system fluctuated around-100 KJ/mol,and with the increase of the number of hydrogen bonds,indicating that the binding was spontaneous,and the formation of new hydrogen bonds might be beneficial to improve the binding stability.The predicted results can provide theoretical guidance for subsequent experiments.（2）The binding mechanism of sorafenib with G-quadruplex was explored through a variety of biophysical experiments,such as ultraviolet spectroscopy,fluorescence titration,viscosity method,circular dichroism,and so on.The circular dichroism spectrum was used to analyze the conformation changes of the G-quadruplex.The results showed that the G-quadruplex had negative and positive peaks at 245 nm and 265 nm,respectively;The addition of sorafenib caused a slight change in circular dichroic signal,indicating that DNA can form parallel structure after annealing in this solution,and sorafenib will not change its original structure type.The viscosity experiment is used to determine the accurate binding mode.The test results showed that the concentration of small molecules didn’t affect the measurement results of the relative viscosity of the solution,indicating that the binding mode of the system should be groove binding.The binding constant measured by ultraviolet spectroscopy was（1.2±0.2）×10⁵ M^-1,indicating they had medium strength binding ability.Fluorescence resonance energy transfer experiment was used to evaluate the structural stability of the G-quadruplex.The analysis results showed that adding small molecules increased the chain-breaking temperature,indicating that sorafenib has the potential to stabilize the G-quadruplex structure.Molecular fluorescence spectrum and time-resolved fluorescence spectrum were used to determine the fluorescence quenching mechanism,and found that the bimolecular rate quenching constant（K_q）was more significant than 2×10¹⁰M^-1s^-1.The monitored fluorescence lifetime is almost not affected by the G-quadruplex.This result jointly proved that they formed a ground-state complex with a binding ratio of1:1,and the quenching mechanism of this system belonged to static quenching.To sum up,this study uses the G-quadruplex to build a natural carrier model for the delivery of sorafenib,mainly from two aspects of the interaction mechanism and drug loading performance.The results show that the groove binding mode dominated by hydrogen bond and hydrophobic force between sorafenib and G-quadruplex is the basis for drug delivery of this carrier.The evaluation results of drug loading capacity can also explain the details of the study of the mechanism of action.These findings help to understand their binding characteristics and provide basic reference information for the future development of natural drug delivery platforms.