Determination Of The Protonation State Of The ASP Dyad And The Relevance To The Activity Of The Inhibitors

β-secretase(memapsin 2,BACE-1)is a critical enzyme in the pathogenesis of Alzheimer’s disease(AD),and it can cleave the amyloid precursor protein(APP)to theβamyloid peptide(Aβ).Subsequent oligomerization and aggregation of Aβlead to neuritic plaque which is the important pathological features of AD patients.Thus,inhibition of BACE-1 has been implicated as a promising therapeutic target to suspend even cure the AD.There is an aspartic dyad(asp dyad),contains two aspartic acid residues,lies in the catalytic region of BACE-1,is a key factor in the catalytic hydrolysis for the aspartic proteases.It has been proposed that such catalytic hydrolysis is initiated by asp dyad with a nucleophilic water molecule through an acid-base catalysis mechanism.Therefore,designing compounds bound to the asp dyad can be an efficient strategy to discover the inhibitors of BACE-1.Plenty of studies point that the aspartic dyad can adopt multiple protonation state while different inhibitor binding to the enzyme.At the same time,the various protonation states of asp dyad are of great important to the binding of inhibitors.A recent study revealed that the change of the asp dyad’s protonation state makes a bounded inhibitor to be inactive.Hence,it helps in developing the high efficiency inhibitors of BACE-1 if we can quickly determine the asp dyad’s protonation states with various inhibitors.However,the asp dyad is a quite complicated system as two asp residues are titrable.As a result,the net charge of the asp dyad has three possibilities,i.e.-2,-1and 0,and there a multi protonation states when the net charge is-1 or 0.Thus,the determination of protonation state of asp dyad is very important and challenging.To promote the development of drug design and discovery,we will mainly explore and develop the approaches in determination of the protonation of asp dyad,and then study the relevance between the asp dyad’s protonation states and the activity of the inhibitors.In Chapter One,we briefly introduced the background of the project,involving the structure of the BACE-1 protease,the mechanism of BACE-1 in AD,the significance and the research progress of the determination of the protonation of the asp dyad and the computational methods used in this paper.In Chapter Two,we compared the reliability and efficiency of the conventional MD simulation and TI method in the determination of the asp dyad’s protonation state with two protonation state models when their net charge is-1.There are researches had successfully used conventional MD approach to determine the preferred protonation state of asp dyad.However,in our study,it is found that the force fields and the controlling parameters can significantly influence the success of the prediction using the conventional MD simulations.In theory,the free energy changes between possible protonation states can be used to determine the protonation state and thermodynamic integration could be a proper method to do this.We illustrated that protonation state prediction by thermodynamic integration(TI)method is insensitive to versions of force fields or to values of controlling parameter cut off.For all the force fields and cut off values evaluated in the present study,the predictions of asp dyad’s protonation state by TI calculations were consistent with each other.Contrary to the intuition that conventional MD is more efficient.In Chapter Three,we developed a fast and reliable thermodynamic approach for determining protonation state of asp dyad and test it in four possible protonation states with the asp dyad’s net charge is-1.It is hard to be determined by calculating free energy changes between possible protonation states,because the free energy changes due to protein conformational flexibility are usually much larger than that originated from the different locations of protons.Sophisticated methods like free energy perturbation(FEP),thermodynamic integration(TI)are therefore usually used for this purpose.However,they are computationally expensive.In present study,we have developed a simple thermodynamic approach to effectively eliminate the free energy changes arising from proteins conformational flexibility and to only estimate the free energy changes originated from the locations of protons,providing a fast and reliable method for determining the protonation state of asp dyad.The test of this approach on totally 14 asp dyad’s systems,shows that the predictions from this approach are all consistent with experiments or with the computationally expensive TI calculations.In Chapter Four,to extend the prediction scope to all three valence states of the asp dyad,including totally nine kinds protonated states,we construct the thermodynamic cycle between the model molecular system and the calculated system,combining the new method described in previous chapter.There are two advantages in our new method:Firstly,the establishment of thermodynamic cycle,avoiding the calculation of the absolute solvation energy of proton,which eliminates the introduction of extra errors;Secondly,the appropriate selection of the model system,making the theoretical calculation error of the calculated molecular system and model molecular system is equal,which offsets the system errors caused by theoretical calculation.To verify the effectiveness of the method,we selected a BACE-1 system as the model molecular system whose pK_a of two aspartic residues are known,and predicted the protonation state of three BACE-1 system with their asp dyad’s protonation states are figured out.In Chapter Five,in order to better screen the inhibitors of BACE-1 systems,we determined the protonation state of a series BACE-1 systems with different inhibitors,and explored the relationship between its structure and activity.The results showed that the protonation state of the asp dyad is very important for the study of inhibitors,and the fitted polynomial between the activity data and the distances in the catalytic active center could provide a fantastic strategy for the design of the AD’s new drugs.