2.3. Estimation of binding affinities by ITC

In this study, ITC was performed by Nano ITC (TA Instruments, USA) to estimate the binding affinities between the compounds and the spike protein. Sample solutions containing the target protein (10 μM) and 8 natural product samples, including amentoflavone (200 μM), candidine (100 μM), cephalinone D (100 μM), dioscin (246 μM), epimedinc (200 μM), saikosaponin C (200 μM), torvoside K (200 μM), and celastrol (100 μM), were all dissolved in dimethyl sulfoxide (DMSO) and diluted with protein elution buffer. The detection of protein concentration was estimated by the Bradford method with BSA as the standard in this study. During the experiment, 50 μL of each compound was placed in a syringe and titrated into 300 μL of spike protein contained in the cell of the ITC instrument using 20 additions at 2.5 μL/time at 5 min intervals. The parameters for this instrument were set to a stirring rate of 300 rpm within the cell and a constant temperature of 25 °C. All solutions were degassed by a degassing station from TA Instruments (Lindon, UT, USA) and were filtered through a 0.22-μm membrane filter (F-Millipore™) before the titration experiments began. We titrated the DMSO diluted buffer into the same buffer to be the background signal. All samples are deducted background signal (The buffer into the buffer). To estimate the strength of the interaction between each compound and protein, the dissociation constant (K_d), reaction stoichiometry (n), c value (c), and the thermodynamic parameters, including entropy (ΔS), enthalpy (ΔH), and Gibbs free energy change (ΔG), were all measured by the NanoAnzalyze software (TA Instruments, USA). Thermodynamic parameters were calculated according to thermodynamics formulas^{22, 23, 24}:

(R: gas constant, T: absolute temperature, K_a: binding constant).

ITC data with c values between 1 and 1000 is an acceptable binding constants.²⁴ The c values were calculated according to the equation:

(n: stoichiometry of interaction, K_a: binding constant, [M]_T: total macromolecular concentration, K_d: dissociation constant).