Study On The Inhibition Of Several Elastase Inhibitors

Elastase inhibitors can inhibit the over-decomposition of elastase and reduce the activity of elastase in living organisms,and treat emphysema,pancreatitis,cyst,tumor,virus infection,inflammation and injury caused by overexpression of elastase.Using the enzyme target activity screening model,molecular docking models and multispectral methods have been used to study the inhibitory mechanisms of several clinically and syntheic applied,ingredients derived from natural plants,and human derived elastic protease inhibitor.The results were as followed:The inhibitory effect of Sivelestat sodium（ONO-5046）on elastase was studied using the screening model,enzyme reaction kinetics and the Lineweaver-Burk double reciprocal method.The results showed that the IC₅₀=9.98μmol/L,the inhibition constant K_i=13.50×10^-6mol/L,the type of inhibition was a competitive inhibition,which showed that ONO-5046 with inhibitory effects on elastase was strong.The interaction of ONO-5046 and elastase was investigated using multispectral and molecular docking method.The combination of ONO-5046 and elastase was strong.ONO-5046 can bind with elastase to form a 1:1 complex.The thermodynamic parameters were calculated to beΔH<0,ΔS<0.Thermodynamic analysis suggested that hydrogen bonds and van der Waal’s forces were the main forces between ONO-5046 and elastase.The formation of the ONO-5046 complex can influence some conformation changes of elastase,the polarity and hydrophilicity of the environment around the amino acid residues in elastase increases,hydrophobicity decreases,the proportion ofα-helix andβ-fold structure decreased,the proportion of irregular crimp increased.The molecular docking simulation technique displayed that Sivelestat sodium and elastase had many kinds of interaction such as hydrogen bond,van der Waals force,electrostatic effect and salt bridge effect,which can form stable complex.There are multiple acyl groups,benzene rings,long conjugated structures,hydrophobic groups and anions,salt bridge with ARG in the molecular framework,the binding of elastase with ONO-5046 is beneficial.The active monomers of Lignan could inhibit the activity of elastase.With the nortracheloside and tracheloside from the lignans as the subject,The inhibition constant and inhibition type of tracheloside to elastase were determined by the screening model.The inhibition types of the inhibitor was competitive inhibition,K_{i（tracheloside）}=2.40×10^-3mol/L,The inhibitory activity of nortracheloside was weaker than that of tracheloside at the same concentration,which indicated that the inhibitory ability of tracheloside was stronger.Based on the previous work,the inhibition of elastase by six dibenzylbutyrolactone lignans,from strong to weak as followed:arctigenin,trachelogenin,mataeresinol,nortrachelogenin,tracheloside and nortrachelosid.In order to clarify the inhibitory effect of dibenzylbutyrolactone on elastase and structure-activity relationship,molecular docking technique was used to simulate the interaction between active monomers and elastase,The inhibitory activity of elastase on the 4-hydroxy of benzene was favorable,and that on the5-membered ring 8’,4-hydroxy of benzene ring was substituted by glycoside bond were unfavorable to the elastase inhibitory activity.Ulinastatin（UTI）is a protease inhibitor isolated from adult male urine,which can inhibit the activities of elastase,trypsin,granulocyte elastase and chymotrypsin.The screening model has been established to screen activities by using elastase as the target enzyme,the IC₅₀=22.50μmol/L,the inhibition constant K_i=19.80×10^-6mol/L,the type of inhibition was a competitive inhibition,which showed that UTI with inhibitory effects on elastase was strong.The quenching type of ulinastatin and elastase was static quenching,and the main binding force was hydrogen bond and van der Waals force.The results of synchronous fluorescence,ultraviolet-visible spectrometry,circular dichroism spectrometry and infrared spectrometry methods showed that ulinastatin changed the micro-environment of elastase and influenced its secondary structure.The K_iand IC₅₀of three kinds of elastic protease inhibitor showed that Sivelestat sodium had the strongest inhibitory ability,ulinastatin was next,and the active of Trachelospermum jasminoides was the weakest.From the structural analysis:the molecular framework of sivelestat sodium contains sulfonyl group,amide group,ester group,carboxyl group and benzene rings,which can form hydrogen bonds with the amino acid residue of elastase,trimethyl groups on the end carbon,which was hydrophobic interaction between the molecules,the oxygen anion in carbonyl group and the charged Arg in elastase exist salt bridge interaction.There were many kinds of forces between them,the binding ability was stronger,and the inhibitory activity was the best.Compared with Sivelestat sodium,though the Trachelospermum jasminoides has a structure of benzene ring,phenol hydroxy group,methoxy group and cyclolactone,the conjugation structure was short,the number of action sites was small,and the action force was weak,so its inhibitory activity is relatively weak.The conclusions above provide a reference to the design,synthesis,modification and modification of the new elastic protease inhibitor.