Study on the interaction mechanism of trypsin/pepsin-doxycycline hyclate-quercetin ternary system

The interaction mechanism of trypsin/pepsin-doxycycline hyclate-quercetin ternary system was investigated by the molecular docking, molecular dynamics simulation and multiple spectroscopic methods. The results show that when doxycycline hyclate and quercetin are bound with trypsin/pepsin in sequence to form the ternary system, the different sequence of doxycycline hyclate/quercetin binding has different effects on the binding affinity of the two drugs and trypsin/pepsin. However, the binding site of the two drugs on trypsin/pepsin is basically the same as that of the corresponding binary system, except for pepsin-doxycycline hyclate (first)-quercetin ternary system. For the trypsin-quercetin (first)-doxycycline hyclate ternary system, the activity of trypsin is inhibited due to the presence of quercetin. The doxycycline hyclate that binding first even affects the binding site of quercetin and the inhibition of pepsin activity. When doxycycline hyclate and quercetin bind with trypsin/pepsin at the same time, the binding site of doxycycline hyclate and quercetin on trypsin/pepsin is completely different from their corresponding binary system. The values of binding constant of 10⁴ L mol⁻¹ indicate that the binding affinity are all moderate. In ternary system, the free concentration of doxycycline hyclate and quercetin in trypsin/pepsin is higher than that in binary system. Hydrophobic interactions, electrostatic forces and hydrogen bonds are the main non-covalent forces. The extent of unfolding of trypsin/pepsin peptide backbone in ternary system is greater than that in binary system. The effects of binary and ternary systems on the secondary structure of trypsin/pepsin are not significantly different, and both results in the reduction of trypsin/pepsin β-sheet.