Studies On Inhibitor Design And Molecular Mechanism Of Metabolic Enzymes(PHGDH And PGAM1)and Thymine DNA Glycosylase

It is of important significance to studying the dynamic behaviors of biological macromolecules and 3-dimensional structures of small organic molecules,as well as the principles of molecular recognition between biologically relevant molecules,during the mechanism based drug discovery pipeline.Conformational search of organic molecules and/or macromolecules can be applied to exploring the knowledge of macromolecular structure-to-function relationships and driving the design of small molecule modulators to regulate the functions of biomolecules.In the first chapter of this thesis,the principle of molecular conformational search,the source of initial coordinates,the methodological improvements and the conformational convergence are discussed in term of organic small molecules and biological macromolecules.The conformations of small molecules captured experimentally by X-ray crystallography are mainly deposited in the Cambridge Structural Database(CSD)and Protein Data Bank(PDB)Database.New NMR methodology can also accurately quantify the accessible 3D-space of small molecules in physiologically-relevant solvents.Another important source of structural information for small organic ligands is computational method,which can quickly identify physically relevant and diverse conformers so as to explore their conformational space reasonably.Conformational sampling of small organic molecules algorithms described in this thesis includes exhaustive systematic search,assembly of constitutive fragments,distance geometry constraints,genetic algorithm,monte carlo and molecular dynamics simulations.The biological molecule coordinates are mainly derived from the PDB database,which contains the structures from X-ray crystallography,NMR and Cryo-electron microscopy.The simulation techniques for conformational sampling and optimization of biomolecules are mainly forcefield-based molecular modeling including monte carlo and molecular dynamics simulations and enhanced sampling methods.Here several methods and strategies for enhanced sampling are described in detail,including replica exchange molecular dynamics(REMD),umbrella sampling,metadynamics,local elevation methods and steered molecular dynamics.The conformational search methods were widely used to solve problems in other chapters of this doctoral thesis.Recent advances in the understandings of cancer metabolism have highlighted the vital role of the serine synthetic pathway.Phosphoglycerate dehydrogenase(PHGDH)catalyzes the first step of serine synthetic pathway and has been notably shown as the putative role in cancer development because of its high expression level in a number of human tumors.In 2016,a great number of compounds have been reported to show inhibition activity against PHGDH.A virtual screening strategy that combines pharmacophore modeling,molecular docking and cheminformatics techniques was performed to find novel scaffolds with high PHGDH inhibition activity.We bought 126 compounds for bioactivity test and finally found 4 compounds which can potentially inhibit the activity of PHGDH.Compound 140 is the best one with the value of IC50 19μM.Binding mode analysis by molecular docking approach revealed that compound 140,compound 154 and compound 92 have similar binding conformations with NAD+,and compound 140 is amenable for further structural optimization.Phosphoglycerate mutase 1(PGAM1)was reported to be commonly overexpressed in many human cancers.Progress in understanding of the relationship between molecular mechanisms of PGAM1 and cancer development will provide the rationale to support the development of cancer therapy.The C-terminal portion of available crystal structures of PGAM1 and its homologous proteins is partially disordered due to the weak definite electron density.We expected that the C-terminal region of PGAM1 is conformationally variable and may play a vital role in substrate entrance and product release during the catalytic cycle.The C-terminal region is predicted to be highly flexible by PONDR-FIT server(Disprot).Monte carlo and molecular dynamics simulations revealed that the C-terminal region is inherently dynamic.Large-scale conformational changes were observed in the C-terminal segment,which leads to the structural transition of PGAM1 from the closed state to the open state.The C-terminal segment influenced the binding of 2,3-BPG.The electrostatic potential of open and closed states were verified and showed the positive charged catalytic pocket of PGAM1.These findings describe dynamic features of C-terminal region in detail and will help to design a novel and efficient inhibitor of PGAM1.A swing mechanism of C-terminal region was proposed to facilitate further studies of catalytic mechanism and physiological function on its homologues.Epigenetics is one of the most rapidly burgeoning fields in the life sciences.The role of DNA methylation was showed on human disease and development.Two crystal structures of DNA decamer comprising different cytosine modifications(one is 5-formylcytosine and the other is 5-carboxycytosine)were determined to a resolution of 1.0 ? using X-ray crystallography by professor Liang Zhang.We found that one DNA structure containing 5fC is A-DNA,whereas the other one with 5caC is B-DNA.We wanted to investigate if 5fC and 5caC have distinct impacts on the structure of the DNA double helix,as well as the mechanisms by which TDG recognizes the oxidized modifications.Molecular dynamics simulations and analyses of DNA with modified cytosines,TDG and their complex were conducted to address our puzzles.As a result,both structures in the presence of 5fC or 5caC show the features of B-DNA during simulations.The most obvious effect is the opening of the minor groove at 5fC/5caC sites.The effect induced by 5caC is significantly larger than that of 5fC.Furthermore,two distinct conformational states of TDG were observed due to the movements of loop 198-204 via molecular dynamics simulation of the apo TDG.In further simulations of the complex of TDG and specific modified base DNA,we found that lysine 201 could form a stable hydrogen bond with carboxyl group of 5caC and the hydrogen bond was not appeared between the oxygen atom of 5fC and lysine 201.DNA structure can be distorted by the arginine 275 in our simulations.These results provide good explanation of specific differences of recognition mechanism of TDG toward 5fC and 5caC on the atomic level.The conclusions of our theoretical simulations support the in-depth studies for substrate recognition and catalysis of TDG.