Study On The Interaction Of Several Anticoccidial Drugs With The Biomacromolecules By The Spectroscopic And Molecular Docking Technique

Exploring the mechanism between anticoccidial drugs and serum albumins and DNA in organisms at the molecular level will provide a basis for clarifying the transport and metabolism of veterinary drugs remaining in food in vivo,meamwhile,it will also provide guidance for the design of new veterinary drug molecules and molecular modifications.The interaction of several common anticoccidial drugs between serum albumins and DNA were studied by multispectral methods and molecular docking technique.The main contents of the study were as follows:The interaction between amprolium hydrochloride/dinitolmide and bovine serum albumin?BSA?was studied by fluorescence spectroscopy and molecular docking technique.The results showed that the quenching processes of BSA fluorescence by the two drugs were static quenchings.The binding constants Ka(amprolium hydrochloride-BSA:1.056×10⁴ L mol^-1;dinitolmide-BSA:0.948×10⁴ L mol^-1)and binding sites?n?at different temperatures were calculated.The values of?H,?S and?G indicated that electrostatic force plays an important role in the binding process of amprolium hydrochloride/dinitolmide to BSA.Based on F?rster nonradiative energy transfer theory?FRET?,the binding distance of the two drugs to BSA was calculated as 3.27 nm?3.91 nm.Metal ions such as K⁺,Ca²⁺,Cu²⁺,Zn²⁺,Fe³⁺and Mg²⁺have no significant effect on the binding of amprolium hydrochloride/dinitolmide to BSA.Molecular docking technique and site competition experiments proved that the two drugs bind to the sub-domain IIA site of BSA,the competition experiments further confirmed the correctness of the conclusion.The effect of the addition of the two drugs on the conformation of BSA was investigated by infrared spectroscopy,circular dichroism spectroscopy and synchronous fluorescence spectroscopy.Under the condition of pH=7.4,the interaction of pyrimethamine/trimethoprim with BSA and human serum albumin?HSA?was studied by spectroscopy and molecular docking technique.The results of fluorescence quenching spectroscopy and time-resolved fluorescence spectroscopy showed that the quenching processes of BSA/HSA fluorescence of the two drugs were static quenchings.The Ka and n at 291K,301 K and 311 K were calculated.Site competition and molecular docking technique confirmed that pyrimethamine and trimethoprim bind to the sub-domain IIA site of BSA/HSA,respectively.UV-vis absorption spectroscopy confirmed that the interaction between the two drugs and BSA/HSA did occur.From the thermodynamic data,it could see that the main force of pyrimethamine/trimethoprim binding to BSA/HSA was electrostatic force.FRET was used to calculate the binding distance between the two drugs and BSA/HSA?pyrimethamine-BSA/HSA:1.86 nm/2.24 nm;trimethoprim-BSA/HSA:2.02 nm/3.59 nm?.Metal ions had little effects on the binding of the drug-protein system.The results of infrared spectroscopy and circular dichroism spectroscopy showed that the addition of the two drugs had an effect on the secondary structure of proteins.From the results of synchronous fluorescence spectroscopy,it can be seen that pyrimethamine/trimethoprim changed the microenvironment near the tryptophan?Trp?residues.The interaction of nitrofurazone with BSA/HSA was explored using spectroscopy and molecular docking technique.The results of fluorescence quenching spectroscopy and time-resolved fluorescence spectroscopy showed that the quenching processes of BSA/HSA fluorescence by nitrofurazone were static quenchings.The calculated Ka of nitrofurazone and BSA/HSA at different temperatures were 6.306×10⁴ L mol^-11 and 1.669×10⁴ L mol^-1,respectively,and the values of n were about 1.UV-Vis absorption spectroscopy proved that there was indeed an interaction between the two drugs and BSA/HSA.The results of circular dichroism spectroscopy and infrared spectroscopy showed that nitrofurazone altered the secondary structure of the protein.Synchronous fluorescence spectroscopy showed that the addition of the drugs changed the microenvironment near the Trp residue,resulted in a decrease in the polarity and an increase in hydrophobicity.From the results of molecular docking technique and site competition experiments,nitrofurazone bound to the sub-domain IIA site of the protein.The binding force of nitrofurazone-BSA/HSA was calculated as electrostatic force.The binding distance was 3.42 and 2.99 nm,respectively,which indicated the energy transfer in the static quenching process was non-radiative.The interaction between furazolidone/nitrofurazone and DNA was investigated by multi-spectroscopy,cyclic voltammetry and molecular docking techniques.From the results of time-resolved fluorescence spectroscopy,the fluorescence lifetimes of DNA-AO and furazolidone/nitrofurazone-DNA-AO were almost unchanged,which indicated that the ground state complex formed by the binding of drugs to DNA was non-fluorescent.The results of the circular dichroism and infrared spectroscopy showed that the addition of the two drugs changed the secondary structure of DNA.Viscosity experiments showed that with the increase of furazolidone concentration,the relative viscosity of DNA increased,and the change of nitrofurazone concentration had little effect on the relative viscosity of DNA.In addition,the results of melting temperature?T_m?,ionic strength,site competition experiments and molecular docking technique all demonstrated the intercalation binding mode for furazolidone to DNA and groove binding mode for nitrofurazone to DNA.The binding constants?K_a?obtained as 3.66×10⁴ L mol^-11 and 3.95×10⁴ L mol^-1 for furazolidone-DNA and nitrofurazone-DNA from UV-vis absorption spectroscopy,respectively.