基于虚拟筛选和分子动力学模拟发现新型ULK1抑制剂

Unc-51样自噬激活激酶1（unc-51-like autophagy activating kinase 1,ULK1）作为自噬启动的重要调控因子,是肿瘤治疗的关键靶点之一。首先,以已知ULK1抑制剂为基础构建药效团模型,通过药效团模型筛选、分子对接以及分子力学广义波恩表面积（Molecular Mechanics/Generalized Born Surface Area,MM/GBSA）结合自由能计算等方法,对含有52万多个类药性小分子的数据库进行虚拟筛选,得到具有较高理论亲和力的化合物。随后,50ns的分子动力学模拟验证了蛋白质-配体复合物结合的稳定性,最后10ns的平均结合自由能的计算研究进一步验证了配体的结合能力。结果表明,6个化合物（F5258-0159、F3407-0428、F0529-1100、F0696-3531、F3222-5280、F6525-5596）具有骨架新颖、分子对接分数和结合自由能数值优异及与ULK1的结合状态稳定等特点,可以作为新型潜在的ULK1抑制剂用于肿瘤治疗的研究,也为新型ULK1抑制剂的设计和研发提供新的研究思路。     

Neutron scattering from polymer blends under pressure

A procedure is used to analyze small-angle neutron-scattering (SANS) data from a pressurized polymer blend mixture (deuterated polystyrene/polyvinylmethylether). The Lattice-Fluid (LF) equation-of-state model is used along with a compressible random phase approximation (RPA) in order to obtain free volume fractions and intermonomer interaction potentials. Solving the two sets of equations (LF and RPA) self-consistently within the fitting procedure to the SANS data provides an improvement over the familiar incompressible RPA model. In this approach, the free volume fraction is the main varying parameter. Intermonomer interaction potentials were found to depend on pressure (weakly) and temperature (linearly). © 1995 John Wiley & Sons, Inc.     

An NMR method for the determination of protein-binding interfaces using dioxygen-induced spin-lattice relaxation enhancement

Using oxygen as a paramagnetic probe, researchers can routinely study topologies and protein-binding interfaces by NMR. The paramagnetic contribution to the amide (1)H spin-lattice relaxation rates (R(1)(P)) have been studied for uniformly (2)H,(15)N-labeled FB protein, a 60-residue three-helix bundle, constituting the B domain of protein A. Through TROSY versions of inversion-recovery experiments, R(1)(P) could be determined. R(1)(P) was then measured in the presence of a stoichiometric equivalent of an unlabeled Fc fragment of immunoglobulin (Ig) G, and the ratio of R(1)(P) of the FB-Fc complex to that of free FB [i.e., R(1)(P)(complex)/R(1)(P)(free)] was determined for each observable residue. Regions of helix I and helix II, which were previously known to interact with Fc, were readily identified as belonging to the binding interface by their characteristically reduced values of R(1)(P)(complex)/R(1)(P)(free). The method of comparing oxygen-induced spin-lattice relaxation rates of free protein and protein-protein complexes, to detect binding interfaces, offers greater sensitivity than chemical shift perturbation, while it is not necessary to heavily deuterate the labeled protein, as is the case in cross saturation experiments.     

Quantifying protein adsorption and function at nanostructured materials: enzymatic activity of glucose oxidase at GLAD structured electrodes

Nanostructured materials strongly modulate the behavior of adsorbed proteins; however, the characterization of such interactions is challenging. Here we present a novel method combining protein adsorption studies at nanostructured quartz crystal microbalance sensor surfaces (QCM-D) with optical (surface plasmon resonance SPR) and electrochemical methods (cyclic voltammetry CV) allowing quantification of both bound protein amount and activity. The redox enzyme glucose oxidase is studied as a model system to explore alterations in protein functional behavior caused by adsorption onto flat and nanostructured surfaces. This enzyme and such materials interactions are relevant for biosensor applications. Novel nanostructured gold electrode surfaces with controlled curvature were fabricated using colloidal lithography and glancing angle deposition (GLAD). The adsorption of enzyme to nanostructured interfaces was found to be significantly larger compared to flat interfaces even after normalization for the increased surface area, and no substantial desorption was observed within 24 h. A decreased enzymatic activity was observed over the same period of time, which indicates a slow conformational change of the adsorbed enzyme induced by the materials interface. Additionally, we make use of inherent localized surface plasmon resonances in these nanostructured materials to directly quantify the protein binding. We hereby demonstrate a QCM-D-based methodology to quantify protein binding at complex nanostructured materials. Our approach allows label free quantification of protein binding at nanostructured interfaces.     

新型二氟甲基磷酸类酪氨酸蛋白磷酸酯酶1B抑制剂的分子动力学模拟和结合自由能计算

通过分子对接建立了一系列含二氟甲基磷酸基团(DFMP)或二氟甲基硫酸基团(DFMS)的抑制剂与酪氨酸蛋白磷酸酯酶1B(PTP1B)的相互作用模式, 并通过1 ns的分子动力学模拟和molecular mechanics/generalized Born surface area (MM/GBSA)方法计算了其结合自由能. 计算获得的结合自由能排序和抑制剂与靶酶间结合能力排序一致; 通过基于主方程的自由能计算方法, 获得了抑制剂与靶酶残基间相互作用的信息, 这些信息显示DFMP/DFMS基团的负电荷中心与PTP1B的221位精氨酸正电荷中心之间的静电相互作用强弱决定了此类抑制剂的活性, 进一步的分析还显示位于DFMP/DFMS基团中的氟原子或其他具有适当原子半径的氢键供体原子会增进此类抑制剂与PTP1B活性位点的结合能力.     

Microscopic modes and free energies of 3-phosphoinositide-dependent kinase-1 (PDK1) binding with celecoxib and other inhibitors

Celecoxib, also known as Celebrex (approved by FDA in 1998) and remembered as the fastest-selling drug in history, was used as a cyclooxygenase-2 (COX-2) selective inhibitor having both anti-inflammatory and anticancer activities. Most recent studies have revealed that the apoptotic activity of celecoxib (and its derivatives) is actually independent of the COX-2 inhibitory activity and that celecoxib also inhibits the kinase activity of 3-phosphoinositide-dependent protein kinase-1 (PDK1), suggesting that the well-known anticancer activity of celecoxib is not due to the inhibition of COX-2, but possibly is due to the inhibition of PDK1. It is highly desirable to develop new celecoxib derivatives as PDK1-specifc inhibitors to avoid the side effects of COX-2 inhibitors. To understand how PDK1 binds with celecoxib and its derivatives, we have performed extensive molecular docking and combined molecular dynamics (MD) simulations and molecular mechanics/Poisson-Boltzmann surface area (MM-PBSA) binding free energy calculations on eight representative PDK1 inhibitors, leading to the finding of a new, more favorable binding mode which is remarkably different from the previously proposed binding mode. Based on the determined most stable binding structures, the calculated binding free energies are all in good agreement with the corresponding experimental data, and the biological activity data available for celecoxib and its derivatives can be better interpreted. The obtained new insights, concerning both the binding mode and computational protocol, will be valuable not only for future rational design of novel, more potent PDK1-specific inhibitors as promising anticancer therapeutics, but also for rational design of drugs targeting other proteins.     

Tracking oxytetracyline mobility across environmental interfaces by second harmonic generation

This work examines the binding behavior of the antibiotic oxytetracycline (OTC) to mineral oxide/water interfaces in the presence and absence of organic functional groups using the interface-specific technique second harmonic generation (SHG). Studies show that OTC binding to fused quartz, methyl ester, carboxylic acid, and alkyl interfaces is fully reversible and highly dependent on solution pH, with appreciable adsorption occurring only at pH 8. Relative surface coverage at pH 8 is highest for the polar organic-functionalized surfaces, and surface saturation occurs for the methyl ester-functionalized fused quartz/water interface at 2 x 10(-5) M. Adsorption isotherm measurements indicate that the binding process is controlled by hydrogen bonding and hydrophobic interactions, with free energies of adsorption on the order of -40 kJ/mol for all interfaces studied. The results indicate that OTC transport in the environment will depend heavily on soil pH and composition and have implications for the development of bacterial antibiotic resistance.     

Conformational Transitions and Convergence of Absolute Binding Free Energy Calculations

The Binding Energy Distribution Analysis Method (BEDAM) is employed to compute the standard binding free energies of a series of ligands to a FK506 binding protein (FKBP12) with implicit solvation. Binding free energy estimates are in reasonably good agreement with experimental affinities. The conformations of the complexes identified by the simulations are in good agreement with crystallographic data, which was not used to restrain ligand orientations. The BEDAM method is based on λ -hopping Hamiltonian parallel Replica Exchange (HREM) molecular dynamics conformational sampling, the OPLS-AA/AGBNP2 effective potential, and multi-state free energy estimators (MBAR). Achieving converged and accurate results depends on all of these elements of the calculation. Convergence of the binding free energy is tied to the level of convergence of binding energy distributions at critical intermediate states where bound and unbound states are at equilibrium, and where the rate of binding/unbinding conformational transitions is maximal. This finding mirrors similar observations in the context of order/disorder transitions as for example in protein folding. Insights concerning the physical mechanism of ligand binding and unbinding are obtained. Convergence for the largest FK506 ligand is achieved only after imposing strict conformational restraints, which however require accurate prior structural knowledge of the structure of the complex. The analytical AGBNP2 model is found to underestimate the magnitude of the hydrophobic driving force towards binding in these systems characterized by loosely packed protein-ligand binding interfaces. Rescoring of the binding energies using a numerical surface area model corrects this deficiency. This study illustrates the complex interplay between energy models, exploration of conformational space, and free energy estimators needed to obtain robust estimates from binding free energy calculations.     

Charge optimization of the interface between protein kinases and their ligands

Examining the potential for electrostatic complementarity between a ligand and a receptor is a useful technique for rational drug design, and can demonstrate how a system prioritizes interactions when allowed to optimize its charge distribution. In this computational study, we implemented the previously developed, continuum solvent-based charge optimization theory with a simple, quadratic programming algorithm and the UHBD Poisson-Boltzmann solver. This method allows one to compute the best set of point charges for a ligand or ligand region based on the ligand and receptor shape, and the receptor partial charges, by optimizing the binding free energy obtained from a continuum-solvent model. We applied charge optimization to a fragment of the heat-stable protein kinase inhibitor (PKI) of protein kinase A (PKA), to three flavopiridol inhibitors of CDK2, and to cyclin A which interacts with CDK2 to regulate the cell cycle. We found that a combination of global (involving every charge) and local (involving only charges in a local region) optimization can give useful hints for designing better inhibitors. Although some parts of an inhibitor may already contribute significantly to binding, we found that they could still be the most important targets for modifications to obtain stronger binders. In studying the binding of flavopiridol inhibitors to CDK2, comparable binding affinity could be obtained regardless of whether the net charges of the inhibitors were constrained to -2, -1, 0, 1, or 2 during the optimization. This provides flexibility in inhibitor design when a certain net charge of the inhibitor is desired in addition to strong binding affinity. For the study of the PKA-PKI and CDK2-cyclin A interfaces, we identified residues whose charge distributions are already close to optimal and those whose charge distributions could be refined to further improve binding.     

蛋白质相互作用: 界面分析, 结合自由能计算与相互作用设计

蛋白质相互作用在生命活动中起着重要作用. 研究蛋白质间相互作用的本质有助于了解生命活动中这些基本单元的作用. 本文主要综述了近期蛋白质相互作用研究的进展, 包括蛋白质相互作用界面的基本性质, 蛋白质结合自由能的计算方法, 不同相互作用在蛋白质结合/解离中的角色和差异, 以及上述知识在蛋白质相互作用设计中的应用. 蛋白质相互作用界面的特性, 例如界面大小、保守性以及结构的动态性质, 使得具有生物功能的蛋白质相互作用界面区别于非特异性的晶体堆积界面. 生物功能界面的一个重要结构特征是界面上存在着关键残基以及相对独立的相互作用模块. 利用多种方法, 如MM-PBSA、统计平均势以及不同的相互作用自由能模型, 可以在不同的精度上计算蛋白质相互作用自由能. 利用蛋白质相互作用界面的特点, 从不同的角度进行蛋白质相互作用对的设计与改造, 近年来已经有了不少成功的例子, 但还存在着很大的挑战. 我们认为在今后的蛋白质相互作用设计中, 考虑各种因素对蛋白质结合与解离的动力学过程的影响将有助于提高人类控制蛋白质相互作用的能力.     

Single run measurements of drug-protein binding by high-performance frontal analysis capillary electrophoresis and mass spectrometry

A novel drug-protein binding measurement method based on high-performance frontal analysis and capillary electrophoresis (HPFA/CE) is presented. A single run measurement approach is proposed to circumvent utilization of a calibration curve that is often performed with HPFA. A sensitive mass spectrometer is applied as a detector enabling the measurement of in vitro protein binding at lower drug concentrations. Unbound free fraction and binding constants can be determined by a single run measurement by consecutive injections of an internal drug standard, a buffer plug and a drug-protein mixture. Effects of injection volumes on peak height and plateau profile were investigated in two different separation systems, non-volatile buffer and volatile buffer, with UV and mass spectrometry detection, respectively. A simplified one-to-one binding model is employed to evaluate the proposed method by using both single and multiple drug concentrations to measure the unbound free fraction and calculate the binding constants of some selected compounds. The method is suitable for rapid and direct screening of the binding of a drug to a specific protein or drug-plasma protein binding.     

Toward the discovery of inhibitors of babesipain-1, a Babesia bigemina cysteine protease: in vitro evaluation, homology modeling and molecular docking studies

Babesia bigemina is a protozoan parasite that causes babesiosis, a disease with a world-wide distribution in mammals, principally affecting cattle and man. The unveiling of the genome of B. bigemina is a project in active progress that has already revealed a number of new targets with potential interest for the design of anti-babesiosis drugs. In this context, babesipain-1 has been identified as a proteolytically active enzyme whose three-dimensional structure has not been resolved yet, but which is known to be inhibited by cysteine proteases inhibitors such as E64, ALLN, leupeptin, and vinyl sulfones. In this work, we introduce (1) a homology model of babesipain-1; (2) a comparison between babesipain-1 and falcipain-2, a cysteine protease of the malaria parasite Plasmodium falciparum; (3) in vitro data for babesipain-1 inhibition by HEDICINs and HECINs, previously reported as modest inhibitors of falcipain-2; and (4) the docked binding conformations of HEDICINs and HECINs in the model of babesipain-1. HEDICINs presented similar preferred binding conformations for both babesipain-1 and falcipain-2. However, in vitro bioassay shows that HEDICINs and HECINs are better inhibitors of babesipain-1 than of falcipain-2, which could be explained by observed differences between the active pockets of these proteins in silico. Results presented herein provide a valuable contribution to future computer-aided molecular design of new babesipain-1 inhibitors.     

van der Waals interaction between internal aqueous droplets and the external aqueous phase in double emulsions

A mathematical model for analyzing the van der Waals interaction between the internal aqueous droplets (W(1)) and the external aqueous phase (W(2)) of double emulsions has been established. The effects of Hamaker constants of the materials forming the system, especially those of the two different adsorbed surfactant layers with uniform density (A(1) and A(2)), on the van der Waals interaction were investigated. The overall van der Waals interaction across the oil film is a combined result of four individual parts, that is, W(1)-W(2), A(1)-A(2), W(1)-A(1), and A(2)-W(2) van der Waals interaction, and it may be either attractive or repulsive depending on many factors. It was found that the overall van der Waals interaction is dominated by the W(1)-W(2) interaction at large separation distances between the W(1)/O and O/W(2) interfaces, while it is mostly determined by the A(1)-A(2) interaction when the two interfaces are extremely close. Specifically, in the cases when the value of the Hamaker constant of the oil phase is intermediate between those of W(1) and W(2) and there is a thick oil film separating the two interfaces, a weak repulsive overall van der Waals interaction will prevail. If the Hamaker constant of the oil phase is intermediate between those of A(1) and A(2) and the two interfaces are very close, the overall van der Waals interaction will be dominated by the strong repulsive A(1)-A(2) interaction. The repulsive van der Waals interaction at such cases helps stabilize the double emulsions.     

Understanding microscopic binding of human microsomal prostaglandin E synthase-1 with substrates and inhibitors by molecular modeling and dynamics simulation

Microsomal prostaglandin E synthase-1 (mPGES-1) is a promising target for development of next-generation anti-inflammatory drugs. It is crucial for rational design of the next-generation anti-inflammatory drugs to know the three-dimensional (3D) structure of mPGES-1 trimer and to understand how mPGES-1 binds with substrates and inhibitors. In the current work, a 3D structural model of human mPGES-1 trimer has been developed, for the first time, by performing combined homology modeling, molecular docking, and molecular dynamics simulation. The 3D structural model enables us to understand how mPGES-1 binds with its substrates/inhibitors, and the key amino acid residues for the mPGES-1 binding with ligands have been identified. The detailed 3D structures and calculated binding free energies for mPGES-1's binding with substrates and inhibitors are all consistent with available experimental data, suggesting that the 3D model of the mPGES-1 trimer and the enzyme-ligand binding modes are reasonable. The new structural insights obtained from this study should be valuable for rational design of next-generation anti-inflammatory drugs.     

Polar discontinuities and 1D interfaces in monolayered materials

Interfaces are the birthplace of a multitude of fascinating discoveries in fundamental science, and have enabled modern electronic devices, from transistors, to lasers, capacitors or solar cells. These interfaces between bulk materials are always bi-dimensional (2D) ‘surfaces’. However the advent of graphene and other 2D crystals opened up a world of possibilities, as in this case the interfaces become one-dimensional (1D) lines. Although the properties of 1D nanoribbons have been extensively discussed in the last few years, 1D interfaces within infinite 2D systems had remained mostly unexplored until very recently. These include grain boundaries in polycrystalline samples, or interfaces in hybrid 2D sheets composed by segregated domains of different materials (as for example graphene/BN hybrids, or chemically different transition metal dichalcogenides). As for their 2D counterparts, some of these 1D interfaces exhibit polar characteristics, and can give rise to fascinating new physical properties. Here, recent experimental discoveries and theoretical predictions on the polar discontinuities that arise at these 1D interfaces will be reviewed, and the perspectives of this new research topic, discussed.     

Computational alanine scanning of the 1:1 human growth hormone-receptor complex

The MM-PBSA (Molecular Mechanics-Poisson-Boltzmann surface area) method was applied to the human Growth Hormone (hGH) complexed with its receptor to assess both the validity and the limitations of the computational alanine scanning approach. A 400-ps dynamical trajectory of the fully solvated complex was simulated at 300 K in a 101 A x 81 A x 107 A water box using periodic boundary conditions. Long-range electrostatic interactions were treated with the particle mesh Ewald (PME) summation method. Equally spaced snapshots along the trajectory were chosen to compute the binding free energy using a continuum solvation model to calculate the electrostatic desolvation free energy and a solvent-accessible surface area approach to treat the nonpolar solvation free energy. Computational alanine scanning was performed on the same set of snapshots by mutating the residues in the structural epitope of the hormone and the receptor to alanine and recomputing the deltaGbinding. To further investigate a particular structure, a 200-ps dynamical trajectory of an R43A hormone-receptor complex was simulated. By postprocessing a single trajectory of the wild-type complex, the average unsigned error of our calculated deltadeltaGbinding is approximately1 kcal/mol for the alanine mutations of hydrophobic residues and polar/charged residues without buried salt bridges. When residues involved in buried salt bridges are mutated to alanine, it is demonstrated that a separate trajectory of the alanine mutant complex can lead to reasonable agreement with experimental results. Our approach can be extended to rapid screening of a variety of possible modifications to binding sites.     

Photosensitized paraquat-induced structural alterations and free radical mediated fragmentation of serum albumin

Photosensitization of paraquat with photosynthetically active radiations (PAR) induced substantial production of both the hydroxyl radicals (*OH) and superoxide anions (O(2)(*-)) under in vitro conditions. Addition of transition metal ion, Cu (II) enhanced the paraquat-induced *OH radical production by 1.8-fold. Treatment of bovine serum albumin (BSA) with photosensitized paraquat resulted in a dose dependent fragmentation of protein. The quantitative analysis revealed the release of 73 microM acid soluble amino groups and 450 microM carbonyl groups from treated albumin at the highest albumin-paraquat molar ratio of 1:8 in presence of 200 microM Cu (II). The results with the free radical quenchers such as mannitol and superoxide dismutase (SOD) clearly reflected the involvement of *OH radicals in protein fragmentation process. The fluorescence data revealed significantly higher binding of paraquat with serum albumin. The binding constants (K(a)) and binding capacity (n) of albumin for paraquat were determined to be 3.4 x 10(5) l/mole and 12.9, respectively. Fluorescence emission spectra exhibited significant quenching of the intrinsic fluorescence of albumin upon addition of paraquat at increasing molar ratios. This is attributed to induced conformational changes in protein structure upon paraquat interaction at specific sites on albumin molecule. Most likely, the alkyl group transfers occur from N1 and/or N1' positions of paraquat to the electron rich sites at critical amino acid residues on treated protein. At higher paraquat concentrations, the albumin-paraquat interaction resulted in adduct formation with concurrent protein alkylation and free radical mediated fragmentation. Thus, on the basis of these results, the paraquat-protein interaction leading to alkylation, structural alterations and/or fragmentation of biological macromolecules has been suggested as an important factor for agrochemical-induced toxicity.     

Fluorescence Anisotropy Assays Reveal Affinities of C- and O-Glycosides for Concanavalin A(1)

The free energies of binding of various C- and O-glycosides to the lectin concanavalin A were measured using fluorescence anisotropy. Fluorescein derivatives of mannose and glucose were synthesized and were shown to bind to concanavalin A with free energies of -5.1 and -4.3 kcal mol(-)(1), respectively. Competition experiments were performed to determine the binding energies of different nonfluorescent carbohydrates, and the results were in excellent agreement with the binding energies determined by microcalorimetry. The minimum carbohydrate epitope that fills the lectin carbohydrate binding site, methyl 3,6-di-O-(alpha-mannopyranosyl)-alpha-mannopyranoside, competes directly for the site with the fluorescent ligands, indicating that the fluorescent ligands bind specifically. The binding affinities of C-glycosides to concanavalin A were compared with those of O-glycosides. The free energies of binding for corresponding C- and O-glycosides differed by less than 0.5 kcal mol(-)(1), indicating that recognition properties of C- and O-glycosides are very similar. It was found that for some ligands the use of a carbon linkage rather than an oxygen linkage caused the specificity of binding to decrease slightly.     

An estimation method of binding free energy in terms of ABEEMσπ/MM and continuum electrostatics fused into LIE method

A method is proposed for the estimation of absolute binding free energy of interaction between proteins and ligands. The linear interaction energy method is combined with atom‐bond electronegativity equalization method at σπ level Force field (fused into molecular mechanics) and generalized Born continuum model calculation of electrostatic solvation for the estimation of the absolute free energy of binding. The parameters of this method are calibrated by using a training set of 24 HIV‐1 protease–inhibitor complexes (PDB entry 1AAQ). A correlation coefficient of 0.93 was obtained with a root mean square deviation of 0.70 kcal mol^?1. This approach is further tested on seven inhibitor and protease complexes, and it provides small root mean square deviation between the calculated binding free energy and experimental binding free energy without reparametrization. By comparing the radii of gyration and the hydrogen bond distances between ligand and protein of three training model molecules, the consistent comparison result of binding free energy is obtained. It proves that this method of calculating the binding free energy with appropriate structural analysis can be applied to quickly assess new inhibitors of HIV‐1 proteases. To test whether the parameters of this method can apply to other drug targets, we have validated this method for the drug target cyclooxygenase‐2. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2011     

乙酰胆碱酯酶AChE与1,7-二氮杂咔唑抑制剂的作用机理的分子动力学模拟

通过分子对接、分子动力学（MD）模拟以及成键自由能分析方法,从原子水平上模拟研究了3种1,7-二氮杂咔唑衍生物（分别记为M1、M2和M3）与AChE的结合模式及相互作用机理,分析和讨论了研究体系的静电相互作用和范德华相互作用（vdW）。用MM-PBSA方法计算的3种抑制剂与AChE之间的结合自由能与抑制剂的实验生物活性数据（IC₅₀值）相对应。分析结果表明,残基S286与抑制剂之间形成的氢键作用有利于抑制剂与AChE之间的结合。范德华相互作用,尤其是抑制剂与关键残基W279和Y334的作用,对抑制剂与AChE之间的结合自由能有较大的贡献,在区分抑制剂M1（或M2）和M3的生物活性上发挥着重要的作用。