In Silico Study Of Protein-ligand Interactions

With the completion of the human genome project, the post-genomic era, mainlyfocusing on functional genomics and proteomics, has come. Proteins are importantcomponents of organisms playing indispensable role in diverse life processes. The study ofrelationship between structure and function of protein is the pioneering and hot issue incurrent life science. The recognition and interaction between proteins and ligands are the mainways in which proteins exert their biological functions in various life activities, such as generegulation, signal transduction and immune response. Therefore, the study of the interactionmechanism between protein and ligand is of great significance for understanding theintracellular activities of organisms, and also providing necessary theoretical basis for theresearch and development of new drugs.Since determining structure of protein complex with experimental approach remainschallenging. Recently, with the continuous progress in computers’ processing ability and therapid development and extensive application of theoretical simulation, the molecularmodeling methods, such as molecular dynamics (MD) simulation, molecular docking and freeenergy computation, have become important tools for exploring the interaction process ofprotein with its ligand. Molecular modeling not only enables us to understand and reveal theessence of life phenomena in levels of molecule, subunit or even atom, but also providesstrong theoretical support to experimental results. With the improvement of molecularmodeling theory and technology advances, molecular modeling methods are extensively beingused in the research of protein structure-function relationship, protein-ligand mutualrecognition, as well as rational drug design.The codling moth, Cydia pomonella (L.), an important economic pests occurring almostworldwide, has developed field resistance to various pesticides due to the overuse ofpesticides. Uncovering the resistance mechanism of this pest could make valuablecontribution to its management. The diamondback moth (DBM), Plutella xylostella, adestructive pest damaging cruciferae crops, has developed field resistance to almost allavailable pesticides. Cantharidin has been reported being toxic to DBM implying its potentialuse for DBM control. However, to date, it is nearly impossible to obtain large-scale amount ofcantharidin. The action site of cantharidin on DBM is unclear. The study of cantharidin targetsite in DBM could facilitate the molecular-oriented design of cantharidin-like compounds. Protein phosphatase5(PP5) plays key role in the dephosphorylation processes ofmultiple signalpathway-related proteins, such as p53, apoptosis signal-regulating kinase1，and mitogen-activated protein kinase, etc. Its overexpression has been proved associatedwiththe proliferation of many tumor cells. PP5is regarded as a promising target for anticancertherapies. Cantharidin is a potent inhibitor of PP5, but its high cytotoxicity limits its clinicalapplication. The study of key interaction between cantharidin and PP5will benefit andpromote the design of higher specific and less cytotoxic cantharidin-like antitumor drug.The main content of this dissertation includes the following two parts:1. Molecular simulation of interactions between protein and ligand applications in thefield of pest control in agriculture(1) A key amino acid associated with acephate detoxification by Cydiapomonellacarboxylesterases based on molecular dynamics with alanine scanning andsite-directed mutagenesisInsecticide-detoxifying carboxylesterases(CEs) gene CpCE-1was cloned from C.pomonella. Molecular dynamics(MD) simulation and computational alanine scanning (ASM)indicate that Asn232in CpCE-1constitutes an approximate binding hotspot with a bindingfree energy difference (ΔΔGbind) value of3.66kcal/mol. The catalytic efficiency (kcat/Km)of N232A declined dramatically, and the half inhibitory concentrations (IC50) value increasedby more than230folds. Metabolism assay in vitro reveals that the acephate could bemetabolized by wild CpCE-1, whereas N232A mutation is unable to metabolize the acephate,suggesting that the hot-spot Asn232is a crucial residue toacephate metabolism. Mutationsdetection suggests that low frequency of Asn232replacement occured in Europe field strains.Our MD, CAS, site-directed mutagenesis and metabolism studies introduce a new amino acidresidue Asn232involved in the metabolism of the acephate with CpCE-1, andthemethodology is reliable in insecticide resistance mechanism research and prediction of keyamino acids in a protein which associated with specific physiological and biochemicalfunctions.(2) Study of cantharidin and its analogs on DBM serine/threonine protein phosphatases(PSPs).The3D models of five DBM PSPs members, PP1, PP2A, PP4, PP5, and PP6wereconstructed. These five models shared high similarity especially in their active domains.Molecular docking revealed cantharidin and its analogs anchor into their active sites. As theirhigh identity on active domains, we only selected the3D model of PP5for the following study.The rank of binding free energy of cantharidin and its analogs on DBM PP5was consistentwith their toxicity (LC50) on DBM larvae. We further tested the inhibitory activity (IC50) of cantharidin and its analogs against the recombinant PP5protein, which was also inaccordance with thebinding free energy and the LC50values. The high correlation of bindingfree energy, LC50, and IC50, indirectly proved that the predicted binding model obtained bymolecular docking is close to the natural one. Based on this binding model, we explored theinhibitory mechanism of cantharidin and its analogs on DBM PP5from the aspect of the keyinteraction between protein and ligand. We found that PP5key residues involving in bindinginteraction are sensitive to the skeletons of cantharidin and its analogs, the slight structuralmodifications of thesechemicals could lead to the changes of their inhibitory activity. Wepresume that the inhibitory ability of cantharidin and its analogs on DBM PSPs determinetheir toxicity to DBM larvae. Our finding could significantly contribute to the design andscreen of cantharidin-like pesticides with novel action model.2. Protein-ligand interactions in the target of human diseasesInsights into the key interactions between human protein phosphatase5andcantharidin at the atomic level using molecular dynamics simulations and site-directedmutagenesis.Protein phosphatase5(PP5) is a promising novel target for anticancer therapies.High-resolution crystal structures of PP5c soaked with two inhibitors, cantharidin andnorcantharidin, have been reported. However, the key interactions between the cantharidinand PP5c at the atomic level remain undefined. In this study, the computational approaches ofquantum mechanics (QM), molecular dynamics (MD), binding free energy, free energydecomposition and alanine scanning mutagenesis (ASM), combined with site-directedmutagenesis, were employed to uncover the key interactions between PP5c and threeinhibitors: cantharidin, norcantharidin and endothall. The binding free energies predicted byMM-PBSAare in close qualitative agreement with the in vitro IC50values. The hydrogen bond(H-bond) and hydrophobic interactions between the three inhibitors and adjacent residues areanalyzed and discussed. H-bonds of His129and Arg225greatly contribute to the binding ofcantharidin and norcantharidin. Involvement of the H-bonds betweenArg225and Val254makes endothall binding more stable. The ASM results obtained are consistent with those ofsite-directed mutagenesis experiments qualitatively, suggesting that His129and Arg225arehotspots which play a critical role inPP5c’s binding with three inhibitors. Moreover, His129is indispensable for substrate catalysis. This work provides insight into the mechanism ofcantharidin and its analogs binding to PP5c at the atomic level and will facilitate modificationof cantharidin-like chemicals to rationally develop more specific and less cytotoxic drugs.