Theoretical Studies Of Glucosidase Catalytic Mechanisms And Interactions Of Several Novel Drug Molecules

As the main energy sources of a variety of organisms, the oligosaccharides may be converted from the most abundant biopolymers-cellulose on earth, or be obtained by the monosaccharide polymerization. The relizations of the two transformations are depended on the the catalytic effects of the glycosidases. The enzymes to hydrolyze the cellulose are called as glycohydrolase, and the monosaccharide polymerizations are catalyzed by glycosynthases. At present, various kinds of crystals for the glycohydrolases and glycosynthases have been crystallized and the catalytic residues in the active sites have been identifized by one point mutations. Though people have paied much attention to the catalytic reactions of glycosidases in theory, many reaction mechanisms of glycosidase are still not very clear. In this research, we used the combined quantum mechanics and the molecular mechanics (QM/MM) methods to study several kinds of glycosidases catalytic mechanisms.Biomacromolecules, such as vitamin D receptor, progesterone receptor, human EP3receptor and so on, are served as the targets for drugs design and synthesis, which are mainly used to treat human major diseases, such as osteoporosis, Paget's disease of bone, mastalgia, peripheral arterial disease and so on. The investigations of interactions between drugs and biomacromolecules are thought to be important in treatment of major diseases and the foundation for drugs design and synthesis. In order to explore the binding modes and inhibition mechanisms of several inhibitors that interact with vitamin D receptor, progesterone receptor, human EP3receptor from molecular prespective, we use the theoretical chemistry methods (molecular simulations, QM/MM and3D-QSAR) to study the binding modes, conformational changes and3D-QSAR based drug designs. Many valuable results have been got, which are described as follows:(1) The catalytic mechanism studies of the rice BGlu1β-glucosidase.The glycosylation and deglycosylation mechanisms of the rice BGlu1β-glucosidase. The rice BGlu1(namely Os3bglu7), which evolves from the microbial enzymes, is more closely related to plant enzymes and more efficiently to hydrolyze cello-oligosaccharides. In order to explore the catalysis mechanism of this enzyme, the QM/MM method was used to study the glycosylation mechanism of rice BGlu1 P-glucosidase in complex with laminaribiose. The calculation results reveal that the glycosylation step experiences a concerted process from the reactant to the glycosyl-enzyme complex with an activation barrier of15.7kcal/mol, in which an oxocarbenium cation-like transition state (TS) is formed. At the TS, the terminal saccharide residue planarizes toward the half-chair conformation, and the glycosidic bond cleavage is promoted by the attacks of proton donor (E176) on glycosidic oxygen and nucleophilic residue (E386) on the anomeric carbon of laminaribiose. Both the nucleophilic glutamate (E386) and acid/base catalyst (E176) establish shorter hydrogen bridges with the C2-hydroxyl groups of sugar ring, which play an important role in the catalytic reaction of rice BGlul β-glucosidase. This study determines the pathway of the catalytic mechanism and achieves a better description of the glycosylation process.It is proposed that the catalysis of BGlul follows a double-displacement mechanism involving a glycosylation and a deglycosylation steps. The deglycosylation step of the substrate laminaribiose catalyzed by rice BGlul β-glucosidase. by using QM/MM approach. The calculation results reveal that the nucleophilic water (Watl) attacks to the anomeric C1, and the deglycosylation step experiences a barrier of21.4kcal/mol from the glycosyl-enzyme intermediate to the hydrolysis product, in which an oxocarbenium cation-like TS is formed. At the TS, the covalent glycosyl-enzyme bond is almost broken (distance of2.45A), and the new covalent bond between the attacking oxygen of the water molecule and C1is basically established (length of2.14A). In addition, a short hydrogen bridge is observed between the nucleophilic E386and the C2-OH of sugar ring (distance of1.94A) at the TS, which facilitates the ring changing from a chair form to half-chair form, and stabilizes the oxocarbenium cation-like TS.So far, we have studied the whole mechanism of the substrate laminaribiose catalyzed by rice BGlul β-glucosidase. We hope our studies could give a better understanding of the hydrolysis mechanism of β-glucosidase in future.? The synthesis mechanism of the rice BGlul β-glucosidase E386mutants. The mutant of a β-glycosidase could act as the glycosynthase to efficiently synthesize oligosaccharides with high yields. In this study, the mechanisms of oligosaccharide synthesis promoted by rice BGlul E386mutants (E386G, E386S and E386A) have been studied by using QM/MM approach. This mechanism is a single step, in which the abstract of the proton by the acid/base E176, the formation of the glycosidic bond and the departure of the leaving group are concerted. The energy barriers are sensitive to sterically hindered interactions of the mutated residues. The energy barriers of E386Q E386S and E386A are calculated to be22.4,25.7and28.2kcal/mol, where the sequence is consistent with the experimental results. Besides, the role of the incoming crystal water Watl in the active site is also explored. Our results gives a good explanation of why the activity of Gly is better than Ser, and Ser than Ala, which cannot be explained from the crystal structure alone.(2) The hydrolysis mechanism of Bacteroides thetaiotaomicron a-glucosidase BtGH97a. Bacteroides thetaiotaomicron (B. thetaiotaomicron), a Gram-negative anaerobe, is a bacterial symbiont. It is a dominant member of the intestinal microbiota of human gut. BtGH97a is one member of glycoside hydrolases (GHs) families GH97. This kind of enzyme plays a major role in the breakdown of polysaccharides ingested in the diet into a form that could be absorbed and utilized by the host. In this paper, the hydrolysis mechanism of pNP-Glc catalyzed by BtGH97a was firstly studied by using QM/MM approach. Two possible reaction pathways were considered. In the first pathway, a water molecule deprotonated by a nucleophilic base (here E439or E508) attacks firstly on the anomeric carbon of pNP-Glc, then a proton from an acid residue (E532) attacks on the glycosidic oxygen to finish the hydrolysis reaction (named as nucleophilic attack-first pathway). In the second pathway, the proton from E532attacks firstly on the glycosidic oxygen, then the water deprotonated by the nucleophilic base attacks on the anomeric carbon of pNP-Glc (named as proton attack-first pathway). Our calculation results indicate that the nucleophilic attack-first pathway is favourable in energy, in which the nucleophilic attack process is the rate-determining step with an energy barrier of15.4kcal/mol in the case of residue E508as nucleophilic base. In this rate-determining step, the deprotonation of water and the attack on the anomeric carbon are concerted. In the proton attack-first pathway, the proton attack on the glycosidic oxygen is the rate-determining step, and the energy barrier is24.1kcal/mol. We conclude that the hydrolysis mechanism would follow nucleophilic attack-first pathway. Our present work has answered some meaningful questions, such as which residue is the most favorable nucleophilic base to assist the hydrolysis reaction, and which pathway is the most possible.(3) The Structural Rearrangement of vitamin D receptor and3D-QSAR study of its inhibitors. The structural rearrangement of vitamin D receptor ligand binding domain. Vitamin D receptor (VDR) is a member of nuclear hormone receptor super family. The activity of VDR is induced by the natural hormone and its analogues. The structural rearrangement of the ligand binding domain (LBD) of human Vitamin D receptor (hVDR) complex ed with1α,25-dihydroxyvitamin D3(natural ligand) and its analogues (denoted as b and c) was studied by molecular dynamics (MD) simulations. MD simulations revealed that these ligands could induce different structural changes of LBD, in which la,25-dihydroxyvitamin D3only led to a minute change, suggesting that LBD adopted its canonical active conformation upon binding the natural ligand, while b and c could provoke a clear structural rearrangement of the LBD. In complex of hVDR-LBD/b, it is found that helix6(H6) and subsequent loop6-7shift outwards and the last turn of H11shifts away from H12, which generate a new cavity at the bottom of the binding pocket in order to accommodate the extra butyl group on the side chain of ligand b. As for hVDR-LBD/c, the steric exclusion of the second side chain of ligand c makes the N-terminal of H7move outsides and C-terminal of H11close to H12, expanding the bottom of the pocket. These calculation results agree well with the experimental observations. The results provide the train of thought for the understanding of the interactions between the inhibitors and VDR and further for the design of new VDR inhibitors with reducing the calcemic side effects.3D-QSAR study of a series of vitamin D3-26,23-lactone analogs.3D-QSAR approach is one of the most useful tool for drug design. It can design potential molecules with high predictive activities. The ligand-based3D-QSAR for82inhibitors of25-dehydro-la-hydroxyvitamin D3-26,23-lactone analogs has been studied by using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) models. The established CoMFA model in training set gives the cross-validated q2value of0.516and non-cross-validated r2ncv value of0.667, while the CoMSIA model represents a q2of0.517and r2ncv of0.632. In general, the predictive ability of CoMFA model is superior to CoMSIA model, in which the r2pred of CoMFA is0.639and that of CoMSIA is0.619. Based on the CoMFA contour maps, some key structural characters of vitamin D3analogs responsible for inhibitory activity are identified, and some new C2a-modified24-alkylvitamin D3lactone analogs with high predicted values are designed. The ligand functional group mutations by FEP simulation and docking studies reveal the rationality of the molecular design. In this study, we design some newly designed inhibitors not only possess higher predicted pIC50values but also with better binding model. We expect to give some targeted lead compounds for the drug design and discovery research.(4)3D-QSAR study of progesterone receptor (PR) inhibitors. The receptor-based CoMFA and CoMSIA were performed on a series of54PR inhibitors. The established CoMFA model in training set gives statistically significant results with the cross-validated q2of0.534and non-cross-validated r2ncv of0.947. The best CoMSIA model was derived by the combination use of steric and hydrophobic fields with a q2of0.615and r2ncv of0.954. A test set of18compounds were utilized to validate the predictive abilities of the two models. The values of predicted correlation coefficient r2pred are0.681and0.677for CoMFA and CoMSIA, respectively. Based on the CoMFA maps, the key structural characters of PR inhibitors are identified. Two binding models of oxindoles and benzimidazol-2-ones are given by the QM/MM calculations. This may provide useful theoretical information for drug design of PR inhibitors.(5)3D-QSAR study of human EP3receptor antagonists. Prostanoids generated in vivo act with EP3receptor, and play a key role in vascular homeostasis, such as platelet function regulation. In this study, the ligand-based CoMFA and CoMSIA methods were performed on a series of57selective human EP3receptor antagonists. The best CoMFA and CoMSIA models in training set derive statistically significant results with the cross-validated q2values of0.505and0.492, non-cross-validated r2ncv values of0.937and0.921, respectively. A test set of20compounds was utilized to validate the predictive abilities of the two models. The values of predicted correlation coefficient r2pred are0.549and0.540for CoMFA and CoMSIA, respectively. Based on the CoMFA maps, the key structural characters of human EP3inhibitors are identified. This may provide useful information for drug design.The primary innovations are as follows:? We systematically investigated the remaining hydrolysis mechanisms of the remaining glucosidase (rice BGlul β-glucosidase) as well as oligosaccharide synthesis mechanism catalyzed by the corresponding mutants by the combined QM/MM methods. Comparing to the traditional quantum chemistry methods, our research methods consider fully the interactions of the active center residues and the corresponding protonated states of the catalytic residues, which could provide much accurate data of the structures and energy barriers closer to the experimental fact. This could give better explanations of the catalysis essence of rice BGlul glucosidase, which could not be explained from the crystal structure alone (such as the order of the catalyzed oligosaccharide synthesis mechanisms of E386G, E386S and E386A mutant).We systematically investigated the inverting hydrolysis mechanism of Bacteroides thetaiotaomicron a-glucosidase BtGH97a. Two possible reaction pathways were designed. The transition states and energy barriers for each pathway were investigated. The work resolved which residue was the most favorable nucleophilic base to assist the hydrolysis reaction, and which pathway was the most possible.For a series of new drug molecules (vitamin D3inhibitors, progesterone receptor inhibitors and human EP3receptor inhibitors), the binding modes and highly predictive structure-activity relationship modes were constructed by the molecular docking and3D-QSAR methods, and some new inhibitors of vitamin D3inhibitors, progesterone receptor inhibitors and human EP3receptor inhibitors with higher predictive activities were designed. According the newly designed molecules, we introduce the combined QM/MM method to predict their binding modes, which may provide theoretical guidances for drug design and synthesis in the future.