Theoretical Studies On The Inhibitory Mechanisms Of Peptide Inhibitors And Aspirin-like Molecules

Enzymes are macromolecular biological catalysts and plays a fundamentally important role in controlling and performing most life processes.Abnormal enzyme activity leads to metabolic disorders,diseases and even death,but enzyme inhibitors can limit enzyme activity,thus improving symptoms.Experimental studies can provide crucial and indispensable information concerning the mechanism,thermodynamics,and kinetics for enzymatic reactions.However,experimental data often provide indirect evidence,can neither study the reaction mechanism at the molecular level,and nor directly determine the structure of the transition state.Computational simulations can yield atomistic or even electronic information regarding the effects of site-specific interactions on the reaction process,the reaction path,and the structure of the transition state.At present,computer-aided design has played an important role in drug design and the improvement of enzyme activity,which is helpful to understand the complex interactions in the enzyme catalysis and provide a theoretical basis for the design of novel drugs and enzymes.In this paper,we studied the protease hydrolysis mechanisms of the two kinds of peptide inhibitors and the binding mechanism of aspirin-like molecules in cyclooxygenase in detail and constructed the relationship between the characteristic structure of inhibitors and their inhibitory ability.The main achievements of this paper are summarized as follows:1.Theoretical studies of the inhibitory mechanism of peptide inhibitorsProteinases are potentially damaging in living systems due to their function of breaking down proteins and peptides,so it's important to keep their activities strictly under control.Due to its strong inhibitory activity,high selectivity and non-toxicity,peptide inhibitors are regarded as the focus of drug research,but they are easily hydrolyzed by homologous enzymes and lose their inhibitory activity.Some peptide inhibitors can resist proteolysis ranging from hours to even years,but the protease hydrolysis mechanism has not yet attained a consensus.Experimentally,the process of hydrolysis of peptide inhibitors cannot be observed at the molecular level,nor can the rate of each step distinguished.But the existing theoretical work mainly focuses on acylation reaction.In this paper,we employed Born Oppenheimer pseudo bond ab initio QM/MM MD simulation combined with umbrella sampling,and studied the whole protease hydrolysis mechanisms of sunflower trypsin inhibitor-1(SFTI-1)and chymotrypsin inhibitor 2(CI2)in detail.For SFTI-1,the acylation reaction is a rate-determining step,and it follows the standard mechanism.A combination of static non-bonded interactions and dynamic motions along the reaction coordinate could account for different hydrolysis rates between them.A comparison among SFTI-1 and three analogs with similar non-bonded interactions further discovered a positive correlation between the mobility of inhibitors and the hydrolysis rates.Apart from the cyclic backbone and disulfide bond,intramolecular hydrogen bonds also increase the rigidity of the backbone of inhibitors,and therefore hinder inhibitor motions to resist proteolysis.These new detailed mechanistic insights suggest the need to consider inhibitor motions in the rational design of peptide inhibitors.For CI2,the acylation reaction is very fast,and mainly a low-barrier hydrogen bond between His64 and Asp32 in the transition state together with lack of covalent backbone constraints makes the peptide bond of CI2 break more easily than other serine protease inhibitors.But the hydrolysis rate depends not only on the energy barrier of deacylation,but also on the concentration of deacylation reaction complex(EA2).The reversibility of EA2 back to the enzyme-inhibitor complex(EI)and retarded access of water reduce the concentration of EA2 to further hinder the hydrolysis of CI2.Instead of dissociation constant of inhibitors,we suggest employing the free energy at EA2 to predict the relative hydrolysis rates of mutants of CI2,which are testified by the experimental relative hydrolysis rates.Up to now,our simulations suggested that the inhibitory mechanism for serine protease inhibitors is clear:the proteolysis of peptides with backbone cycles are mainly retarded in acylation reaction,such as SFTI-1;while for those without backbone cycles,the reversibility from acyl-enzyme to El and retention of leaving group play a decisive role in hindering the hydrolysis of CI2.2.Theoretical study of the inhibitory mechanism of aspirin-like moleculesThe cyclooxygenase(COX)catalyzes the conversion arachidonic acid into prostaglandins.COX exits in mainly two isoform:COX-1 is a constitutive enzyme,while COX-2 is an induced enzyme,produces prostaglandins involved in mainly pathological processes such as inflammation,hyperalgesia and even cancer and would be an important pharmacological target.Aspirin(Acetylsalicylic acid,ASA)is currently the only covalent anti-inflammatory drug,but due to its own acidity and lack of COX2 selectivity,it will produce side effects such as peptic ulcer and dyspepsia.Improving aspirin and reducing its toxicity is a hot spot in drug design.A series of aspirin-like molecules were synthesized experimentally,substitution of the carboxyl moiety of aspirin with alkynylsulfide groups confers significantly COX-2 selectivity,among which APHS is the most potent.Experimental results showed that the potent inhibition for COX-2 of APHS could be achieved by high non-covalent binding even in the absence of acetylation.In this paper,we employed molecular docking,molecular dynamics simulations and binding free energy calculation,and studied the binding mechanism of APHS and its analogues inactivating COX-2.Our studies suggested three possible binding modes of APHS with COX-2,and discovered a newly preferred binding pattern with the lowest binding free energy.The preference to COX-2 for APHS mainly owes to the balance between entropy effect and unfavorable polar interactions(Polar=?Eele+??GPB).Comparative studies found that the increase of non-polar interaction and the decrease of entropy loss increases the binding of APHS in COX-2.The findings of our work provide reference information and theoretical basis for the design of new aspirin-like molecules to reduce side effects in gastrointestinal and blood.