4.2. Surface plasmon resonance (SPR)

SPR is a kind of biosensor technology used to study the interaction between various biomolecules. As a new biochemical analysis and detection tool, SPR is widely used in drug screening with the characteristics of label free, high sensitivity and low consumption [81]. In the classical SPR screening method, the target protein is coupled to the chip surface as the receptor, and then the analyte molecular solution flows through the chip surface. The compounds in the analyte solution that can bind with the receptor are used as the ligands. The binding between the ligand and the receptor leads to the increase of the surface mass of the sensor and the change of the refractive index. Through the computer control system, it is transformed into the sensing signal graph of time and resonance signal. By analyzing the sensing signal graph, the binding affinity and kinetic parameters of ligand and protein can be obtained, the affinity of compounds can be evaluated, and the compounds with good affinity can be selected [82]. The process of SPR is shown in Fig. 3 . The detection principle of SPR is based on the change of the refractive index of the chip surface, and it does not need to label the small molecules to be tested. Therefore, it can eliminate the influence of modified labeling on the activity of compounds, and the detection results can more truly reflect the affinity between compounds and proteins [83,84].

Schematic diagram of SPR. The target protein ACE2 or S protein are coupled to the chip surface as the receptor, and then, the analyte molecular solution flows through the chip surface. The compounds in the analyte solution that can bind with the. The binding between the ligand and the receptor leads to the increase in the surface mass of the sensor. The computer control system is transformed into the sensing signal graph of time and the resonance signal. By analyzing the sensing signal graph, the binding affinity and kinetic parameters of the ligand and protein can be obtained. The affinity of the compounds was evaluated and the compounds with good affinity were screened.

SPR technology can be used to rapidly screen lead compounds by detecting the K_D value of the interaction between the target protein and small molecules. Zhu [85]captured the ACE2-His protein as ligand, dissolved S-RBD-mFc in buffer solution as analyte to establish an SPR screening model of S-RBD and ACE2. When the compounds flowed through the chip surface fixed with ACE2 or S-RBD, the K_D value was determined to screen the compounds that could bind to ACE2 or S-RBD. Among them, demethylzeylasteral (19) can bind to S-RBD-mFc in mobile buffer solution with the lowest dissociation rate constant and the longest residence time. Demethylzeylaster (18) was placed in the analytical solution containing RBD and continuously flowed over the chip surface of ACE2 to observe its signal response. The results showed that demethylzeylaster (18) could also block the binding of S-RBD to ACE2.

SPR also can be used as in vitro molecular authentication model combined with VS. For complex system screening of active compounds, some non-specific components can be eliminated through VS. Then SPR can monitor the whole process of the selected compounds binding to the target protein in real time to further verify the binding ability of the compounds obtained from VS. Ye [86]used network pharmacology, molecular docking and SPR technology to explore the potential compounds and interaction mechanism of Toujie Quwen granules in the treatment of COVID-19. Firstly, the H–C-T network was constructed by network pharmacology, and the key compounds were screened to dock with ACE2. The selected quercetin (1), astragaloside IV(24), rutin (4) were tested by SPR. S Protein and ACE2 are fixed in CM5 chip by covalent bond, and the screened compound solution flows through the chip respectively to test the binding energy with S Protein and ACE2. The results showed that quercetin (1) and astragaloside IV (24) bound to SARS-CoV-2 S protein and inactivated it, while rutin (4) bound to ACE2.

Compared with the traditional inhibitor screening based on enzyme activity, SPR technology does not depend on the enzyme reaction process and is more efficient. It does not need to label the small molecules to be detected, and it can monitor the whole process of the binding of compounds with protein targets in real time, so as to analyze the binding of compounds with protein and generate sensing images to evaluate the affinity and kinetic information of candidate compounds. In addition, SPR has strong specificity, and it does not require high matrix of samples. It can also detect colored solutions such as traditional Chinese medicine extracts, which further improves the screening efficiency.