Theoretical Study Of Peptide Deformylase And Inhibitor And Study Of Resonance Of The Heavy-light-heavy Reaction System

Two parts are included in this thesis:one part is about the interactions and character of peptide deformylase(PDF)and inhibitor by docking and molecular dynamics simulation(MD),and the other is about the partial potential energy surface (PPES)and formation mechanism of the scattering resonance states of three and polyatomic systems by quantum chemistry methods,and they are described as follows.Part 1With the wide-spread use of antibacterial drug,the increase of multi-drug resistant bacterial has been one of the rigorous challenges and created an urgent demand for new antibiotic with novel mechanisms of action and new structures.The absence of deformylase activity in mammal cells,coupled with the observation that the gene encoding PDF(def)is present in all sequenced pathogenic bacterial genomes,has made PDF an attractive target for antibacterial chemotherapy.PDF and its inhibitor became a hot frontier research topic in the late 90s of 20th Century,for its clear biochemistry process,crystal structure and active site.High homology in gene of PDF makes the PDF inhibitor a class of potential broad-spectrum antibacterial agent.Bb-83698 from British Biotech& Genesoft and VRC-4887 from Vicuron have been under clinic trial and showed attractive effects for Gram-positive and gram-negative bacteria.So the discovery,development and application of PDF inhibitors have became a new active field of antibiotic research.Based on the crystal structures of PDF,we investigate the binding modes between PDF and inhibitors,structure-activity relationship by docking and molecular dynamics simulation.In addition,for improvement of theoretical design and enhancement of deficiency for docking methods,we review the binding energy of target molecule with MD simulation.Followings are the abstracts of our studies.Chapter 1 is the preface of the dissertation.In the part 1,we introduce the history development of PDF.We concentrate on the aspects of the function of PDF in bacteria,the structure and type of PDF,type and structure-activity relationship(SAR) of inhibitor,advance of clinical drug and computer-aided drug design.In the part 2, we introduce the history background,the classification,the current experimental and the theoretical research,and the tendency of the development of the scattering resonance states.In chapter 2,we investigate the binding modes and SAR between different type of hydroxamic acids derivatives and PDF by docking methods.Four types of inhibitors, including proline-3-alkylsuccinyl and N-alkyl urea hydroxamic acids derivatives are investigated.Firstly these inhibitors are docked into PDF,then eighteen inhibitors are selected to build the linear equation between scoring function and IC₅₀,finally the inhibitory activities of rest five inhibitors are predicted.By comparison of their binding modes,we can analyze their structure-activity relationships.In chapter 3,we investigate the binding modes between different types of inhibitors and PDF,and SAR by docking methods.Four types of inhibitors,including hydroxamic acids derivatives and N-formylhydroxylamine derivatives,are investigated.The results show that there is a good linear relationship between AutoDock results and the experimental values.By comparison of their binding modes, some new binding pockets are found,which will be helpful for the design of new inhibitor.In chapter 4,automated docking and molecular dynamics simulation are applied to investigate the binding of macrocyclic inhibitors to E.coli PDF.These macrocyclic peptidomimetic inhibitors show the potent inhibitory activities against Gram-positive and Gram-negative pathogens.We mainly analyze the effects of cycle size on the binding modes and binding free energy,and the predicted binding free energy approaches its experimental value.In chapter 5,automated docking and molecular dynamics simulation are applied to investigate the binding of BB-83698 to the B.stea PDF.BB-83698 is a first potent PDF inhibitor to enter clinical trials,and B.Stea PDF is a representative typeâ…¡PDF, so it will be significant to explore the their binding modes.Firstly BB-83698 is docked into the active site of PDF,and three binding modes are obtained by scoring function.Then molecular dynamics simulation and binding free energy calculation are performed to judge most favorable for inhibitor binding,and the dynamics structure are also compared with that of crystal complex.In chapter 6,the binding modes of the peptide thiol inhibitors and E.coli PDF are investigated by docking and molecular dynamics simulation.The stereoisomer shows potent antibacterial activities against Gram-positive bacteria and moderate activity against Gram-negative bacteria,in which the L-ligands are more biologically active than D-ligands.We analyze the effects of binding modes and free binding energy on inhibitory activity,AutoDock results are in good agreement with the experimental values,which demonstrates a good linear relationship.In chapter 7,automated docking and targeted molecular dynamics simulation are applied to investigate the conformational changes of substrate pocket of LiPDF.In the actinonin-bound enzyme complex,the substrate pocket adopts a semi-open conformation.While the opening is not large enough for inhibitor entering,for the CD-loop restricts its access to the substrate pocke.To understand how the actinonin bind with substrate pocket,we perform a MD run separately on ligand-free and actinonin-bound LiPDF.In chapter 8,automated docking and molecular dynamics simulation are applied to investigate the binding of substrate,product to E.coli PDF.The binding of substrate and the release of product are involved in the catalysis process of PDF,so it is important to understand the interaction mechanism between substrate,product and E. coli PDF,which is in favor of the design of new PDF inhibitor.We compare the stabilized conformations of free,substrate-bound and product-bound enzyme,analyze the conformational changes of residues around avtive site when substrate and product bind with enzyme,and also compare the conformation of product-bound complex with that of crystal complex.The innovative points in the first part are summarized as follows:1.The different types of hydroxamic acids derivatives show different inhibitory activities.â‘ The proline group approaches Cys90 and makes van der Waals interacts with pyrrole group of Pro94.â‘¡The N-alkyl urea group makes the P3' phenyl group to form the van der Waals and hydrophobic interactions with Ile44,Ile86å’ŒLeu125, and phenolic hydroxyl can form the new hydrogen bond with Arg97.â‘¢If electron-withdrawing group such as OH or F is linked atαposition,F1 and F2 can form the new hydrogen bond with Gly45.â‘£isoxazole side chain inserts into the Sl'pocket,which results in that the inhibitor can not form the new hydrogen bond with Gly89,and also no corresponding P3' side chain is used to interact with enzyme. So the introduction of electron-withdrawing group atαposition,N-alkyl urea group at P1' and P2' positions,or proline group at P2'position will in favor of the inhibitor binding to enzyme.In addition,the P3' side chain can be modified to make it form the hydrogen bond and hydrophobic interactions,which will improve the inhibitor's binding affinity greatly.2.When the different types of inhibitors docked into E.coli PDF,there are four pockets(A,B,C and D)near the active site,which can accommodate the side chain of inhibitor.The residues in pocket A are Ile128,Cys129,Gln131 and His132.B is shallow and has less residues including Ile44,Leu72,Cys129.Pocket C is composed of Gly43,Ile44,Gly45å’ŒLeu46.D has the most residues such as Leu46,Cys90, Leu91,Ser92,Pro94,Glu95,Gln96 and Leu141.In the binding modes of 3,8 and PDF,new binding pocket are found.The residues on one side of the pocket include Val16,Pro19,Ile57,Val59,Glu64,Arg113,Cys129,Met134,Val138,Lys140,Tyr145. Those on the other side are Glu76,Leu77,Lys80,Gld83,Arg109,Ala110,Phe118 and Aso123.Considering the interactions between pockets and inhibitor will be helpful in designing the inhibitor with high binding affinity.3.We discuss the effects of cycle size on inhibitory activity in detail.The results show that,a common character is that macrocycle inserts into the S1'pocket and interacts with hydrophobic residues.The 13-and 14-membered maerocycles of 1 and 2 lie away from S1' pocket residues,and their interactions are very weak.6 to 8 have the similar binding mode,their macrocycles can't be completely accommodated by the S1'pocket,and the necessary hydrogen bonds,van der Waals interactions and hydrophobic interactions can't be formed with the active site.While compounds 3 to5 fit nicely into active site.On one hand,compounds their macrocycles of 3 to 5 make more close contacts with the hydrophobic residues.On the other hand,the more hydrogen bonds between inhibitor and Ile44,Gly45 and Arg97 make the complex more stable.4.Exploring the binding modes between BB-83698 and B.stea PDF will be in favor of the design of new inhibitor.The results show that large conformational changes take place for Mode 2,leading the ligand to deviate from the crystal positions of the complex greatly,while most negative relative binding free energy indicates it may be a new binding mode.The binding mode and binding free energy of Mode 3 show this mode is not favorable for inhibitor's binding to PDF.The average structure of the Mode 1 complex suggests that the fundamental hydrogen bond interactions between inhibitor and Ile59 and Gly109 stabilize the binding of ligand to protein.In addition,the close interactions between piperazine ring and Pro57 and Ile59 and between the benzo-[1,3]-dioxole group and Pro57,Ile59 and Leu146 seem to be very important for the binding of inhibitor to B.Stea PDF.The introduction of hydrophilic group onto the benzo-[1,3]-dioxole ring will probably greatly increase the binding affinity of ligand.5.The binding modes of thiol stereoisomers are investigated.The results show that the L-ligands have more hydrogen bonds,van der Waals interactions and hydrophobic interactions with E.coli PDF than those of D-ligands.At the P2' position, L-ligands can makes van der Waals interactions with Leu91 and hydrogen bonds with Cys90,Ile93,Glu95 and Gln96.P3' side chain of L-ligand interacts with not only hydrophobic residues Ile44,Va162,Ile86,Leu125 and Ile128 but also with hydrophilic residues Glu42 and Glu87.The above results indicate that the P2' and P3' side chain of L-ligand can be modified into the multifunctional grouPs in favor of interaction with above mentioned residues.Although the peptide thiol inhibitor has weaker inhibitory activity than hydroxamic acids derivatives and N-formylhydroxylamine derivatives,the stereochemistry is also fit for them.6.This chapter analyzes the conformational changes of LiPDF by docking and targeted MD methods.The results show that Tyr71 plays an important role of mediating the movements of CD-loop.The average structure obtained from the MD simulation approaches its crystal structure.In the free LiPDF,the hydrogen bond between Tyr71 and Arg108 results in the closure of the substrate pocket.In the presence of actinonin,Tyr71 firstly hydrogen-bonds with actinonin,then it goes across the substrate pocket and hydrogen-bonds with Arg108.The re-opening of the substrate pocket occurs with the breaking of the hydrogen bond between Tyr71 and Arg108 and leaving of CD-loop from Arg108.The hydrophilicity of Tyr71 suggests us that new inhibitor can be modified at P3' according to its different purposes.7.The dynamics simulation of substrate,product and PDF complexes shows that the larger flexibility of CD-loop is helpful for the binding of substrate and release of product.To make the substrate enter into the active site,the CD-loop interacts with Glu42 and Glu64,which forces the residues around the binding pocket to move towards CD-loop,and the pocket becomes larger,at the same time,the loose residues Ile86,Leu125 and Ile128 approach substrate.After the deformyling of substrate, Leu91 moves to P2' side chain,and Ile86,Leu125 and Ile128 form the hydrophobic interactions with the side chain of Methionine.The two oxygen atoms of Glu133 move together with iron,which is necessary for the catalysis of Glu133.When the product leaves the active pocket,CD-loop and the residues around the active pocket begin to leave from product,and the hydrogen bonds,van der Waals interactions and hydrophobic interactions between product and enzyme become weak gradually and disappear finally.These three different simulations will be helpful for further understanding the catalytic mechanism of PDF.Part 2The research on the scattering resonance state is always the important frontier work in the chemical dynamics research area.The scattering resonance state is the quasi-bound state or transient steady state formed in chemical reactions,which controls some key properties of the reaction system,i.e.,the ratio of the products,the distribution of the energy and the space of the products.In most recent years,the rapid developments of the experimental research on the scattering resonance states accelerate the theoretical research work consumedly.The most remarkable achievements are the high-resolution threshold photodetachment spectroscopy results of the D M Neumark et al.and the crossed molecular beams experiments performed by Kopin Liu,which prove the opinion about the existence of scattering resonance state.But there are still lots of unsolved problems of the theoretical research on the formation mechanism of scattering resonance states.The main contents are introduced as follows.In chapter 9 we introduce the current theoretical methods by which the scattering resonance states are investigated.The theoretical research mainly involves the construction of the potential energy surface and the calculation of the resonance states with the quantum reactive scattering theory.Then the theoretical basis and the construction method of the partial potential energy surface(PPES)are introduced in detail.At last we explain how to investigate the scattering resonance states in the chemical reactions with the PPES.In chapter 10,by the PPESs of these systems constructed here,we discuss some important characters(like resonance lifetime,etc.)of the scattering resonance states in H+NO,X+CH₄(X=H,F,Cl,Br)and OH+H₂Oreaction systems.And then we compare the results with the data reported in the literature,and give the reasonable explanation for the experimental results about molecular beam of these systems.The innovative points in these results are summarized as follows:1.The first point is the construction of the PPES.The PPES is not equal to the traditional simple reduced-dimensional potential energy surface(RD-PES).The PPESs constructed here are the productions of the coupling of the vibrational freedom and the minimum energy reactive pathway,and the potential energy of the system only includes the reactive coordinate s.So the complicated problems of the PESs are reduced reasonably.The dynamics potential well in PPES gives us reasonable explanation of the formation mechanism of the scattering resonance states,and some interrelated data such as resonance energy are obtained by the calculations on the PPESs.Therefore,the PPES should be a useful tool for the research on the scattering resonance states.2.The scattering resonance states of the H+NO→N+OH or O+NH are investigated.The asymmetrical wells are emerged in the transition state region for every reaction pathway,when coupling of both the translation along the reaction coordinate and vibration(adopting vibrational adiabatic approximation)perpendicular to reaction coordinate is adopted.We see that just the dynamic Eying Lake traps the reaction system and forms quasi-bound state,so this resonance is called Feshbach resonance.Using the PPES,we estimate the lifetime of the complex is 0.85 ps,which approaches Han K.L.and Schatz's computational results.3.Systemic investigation was done for the scattering resonance states of the HLH systems,X+CH₄→HX+CH₃(X=H,F,Cl,Br),and the rules relevant to these systems were summarized.All these reactions belong to asymmetrical state-to-state reactions with the potential curves which are asymmetric along the reactive coordinate.So the curves in the PPESs of these reactions also have considerable similarity.On the other hand,because of the difference of the mass of the heavy atoms,there is remarkable difference in depth and width of the potential well on the potential curves between these reactions.And the lifetime of the scattering resonance state increases with the increase of the mass of the heavy atoms correspondingly.Among these reactions,the two reactions Cl+CH₄→HCl+CH₃ and Br+CH₄→HBr+CH₃ are the ones with long lifetime resonance states.4.The investigation is done for the scattering resonance states of OH+H₂O→H₂O+OH LHL reaction system.All reactions belong to symmetrical state-to-state reactions with the potential curves which are symmetric along the reactive coordinate. The potential well can explain the formation mechanism of the scattering resonance state.Finally,the lifetime of the scattering resonance state is estimated.