Probing molecular mechanism of inhibitor bindings to bromodomain-containing protein 4 based on molecular dynamics simulations and principal component analysis

ABSTRACT

It is well known that bromodomain-containing protein 4 (BRD4) has been thought as a promising target utilized for treating various human diseases, such as inflammatory disorders, malignant tumours, acute myelogenous leukaemia (AML), bone diseases, etc. For this study, molecular dynamics (MD) simulations, binding free energy calculations, and principal component analysis (PCA) were integrated together to uncover binding modes of inhibitors 8P9, 8PU, and 8PX to BRD4(1). The results obtained from binding free energy calculations show that van der Waals interactions act as the main regulator in bindings of inhibitors to BRD4(1). The information stemming from PCA reveals that inhibitor associations extremely affect conformational changes, internal dynamics, and movement patterns of BRD4(1). Residue-based free energy decomposition method was wielded to unveil contributions of independent residues to inhibitor bindings and the data signify that hydrogen bonding interactions and hydrophobic interactions are decisive factors affecting bindings of inhibitors to BRD4(1). Meanwhile, eight residues Trp81, Pro82, Val87, Leu92, Leu94, Cys136, Asn140, and Ile146 are recognized as the common hot interaction spots of three inhibitors with BRD4(1). The results from this work are expected to provide a meaningfully theoretical guidance for design and development of effective inhibitors inhibiting of the activity of BRD4.     

基于药效团的BRD4靶向小分子抑制剂虚拟筛选

BRD4靶点和多种肿瘤密切相关,是具有良好成药性的热门靶点。本文选取活性较好且结构差异较大的BRD4小分子抑制剂作为训练集分子,基于配体小分子共同特征(HipHop)方法使用Discovery Studio 3.0分子模拟软件构建了药效团。药效团通过测试集验证、ROC曲线验证(SE(sensitivity)=0.93765、SP(specificity)=0.89500、(AUC)=0.956),结果表明构建得到的药效团具有较强的可靠性和较高的可信度。药效团模型含有1个芳环中心、1个疏水基团、2个氢键受体四个药效特征元素。此药效团被用于ZINC数据库进行虚拟筛选,共筛选了861203个分子,命中率为0.782%。再对筛选得到的分子经过分子对接、ADMET成药性预测、构象分析并讨论分子-蛋白相互作用模式,最终得到了21个有潜力的BRD4小分子抑制剂。     

Degradation of the BAF Complex Factor BRD9 by Heterobifunctional Ligands

The bromodomain-containing protein BRD9, a subunit of the human BAF (SWI/SNF) nucleosome remodeling complex, has emerged as an attractive therapeutic target in cancer. Despite the development of chemical probes targeting the BRD9 bromodomain, there is a limited understanding of BRD9 function beyond acetyl-lysine recognition. We have therefore created the first BRD9-directed chemical degraders, through iterative design and testing of heterobifunctional ligands that bridge the BRD9 bromodomain and the cereblon E3 ubiquitin ligase complex. Degraders of BRD9 exhibit markedly enhanced potency compared to parental ligands (10- to 100-fold). Parallel study of degraders with divergent BRD9-binding chemotypes in models of acute myeloid leukemia resolves bromodomain polypharmacology in this emerging drug class. Together, these findings reveal the tractability of non-BET bromodomain containing proteins to chemical degradation, and highlight lead compound dBRD9 as a tool for the study of BRD9.     

Molecular Dynamic Simulations of Bromodomain and Extra-Terminal Protein 4 Bonded to Potent Inhibitors

Bromodomain and extra-terminal domain (BET) subfamily is the most studied subfamily of bromodomain-containing proteins (BCPs) family which can modulate acetylation signal transduction and produce diverse physiological functions. Thus, the BET family can be treated as an alternative strategy for targeting androgen-receptor (AR)-driven cancers. In order to explore the effect of inhibitors binding to BRD4 (the most studied member of BET family), four 150 ns molecular dynamic simulations were performed (free BRD4, Cpd4-BRD4, Cpd9-BRD4 and Cpd19-BRD4). Docking studies showed that Cpd9 and Cpd19 were located at the active pocket, as well as Cpd4. Molecular dynamics (MD) simulations indicated that only Cpd19 binding to BRD4 can induce residue Trp81-Ala89 partly become α-helix during MD simulations. MM-GBSA calculations suggested that Cpd19 had the best binding effect with BRD4 followed by Cpd4 and Cpd9. Computational alanine scanning results indicated that mutations in Phe83 made the greatest effects in Cpd9-BRD4 and Cpd19-BRD4 complexes, showing that Phe83 may play crucial roles in Cpd9 and Cpd19 binding to BRD4. Our results can provide some useful clues for further BCPs family search.     

Water molecules inside protein structure affect binding of monosaccharides with HIV‐1 antibody 2G12

Water molecules inside biomolecules constitute integral parts of their structure and participate in the functions of the proteins. Some of the X‐ray crystallographic data are insufficient for analyzing a series of ligand–protein complexes in the same condition. We theoretically investigated antibody binding abilities of saccharide ligands and the effects of the inner water molecules of ligand–antibody complexes. Classical molecular dynamics and quantum chemical simulations using a model with possible water molecules inside the protein were performed with saccharide ligands and Human Immunodeficiency Virus 1 neutralizing antibody 2G12 complexes to estimate how inner water molecules of the protein affect the dynamics of the complexes as well as the ligand–antibody interaction. Our results indicate the fact that d ‐fructose's strong affinity to the antibody was partly due to the good retentiveness of solvent water molecules of the ligand and its stability of the ligand's conformation and relative position in the active site. © 2016 Wiley Periodicals, Inc.     

含磷嘧啶类CDK9抑制剂的分子对接、3D-QSAR和分子动力学模拟

选取64个具有潜力的含磷嘧啶类细胞周期依赖性蛋白激酶(CDK9)小分子抑制剂,采用分子对接方法研究了该类小分子与CDK9的结合作用,结果表明,分子构象、氢键形成、疏水性和氨基酸残基Cys106在此类抑制剂与CDK9的结合过程中具有重要作用.在配体叠合的基础上,运用比较分子力场分析(Co MFA)、比较分子相似性指数分析(Co MSIA)和Topomer Co MFA(T-COMFA)研究了分子结构与抑制活性的关系,发现由训练集立体场、静电场和疏水场组合的Co MSIA模型为最优模型,其内部交叉验证相关系数(Q2=0.557)、非交叉验证相关系数(R2=0.959)和外部预测相关系数(r2=0.863)具有统计学意义,该模型的三维等值线图直观显示了化合物的活性与其三维结构的关系.根据这些结果设计了10个具有新结构的含磷嘧啶类化合物,分子对接和分子动力学模拟结果表明,新化合物和CDK9的结合模式与原化合物64相同,自由能分析从理论上证明了新化合物64d的CDK9抑制活性优于化合物64,并且显示含磷基团与残基Asp109的静电场能在化合物与CDK9作用过程中有重要作用.     

Enhanced sampling molecular dynamics simulations correctly predict the diverse activities of a series of stiff-stilbene G-quadruplex DNA ligands

Ligands with the capability to bind G-quadruplexes (G4s) specifically, and to control G4 structure and behaviour, offer great potential in the development of novel therapies, technologies and functional materials. Most known ligands bind to a pre-formed topology, but G4s are highly dynamic and a small number of ligands have been discovered that influence these folding equilibria. Such ligands may be useful as probes to understand the dynamic nature of G4 in vivo, or to exploit the polymorphism of G4 in the development of molecular devices. To date, these fascinating molecules have been discovered serendipitously. There is a need for tools to predict such effects to drive ligand design and development, and for molecular-level understanding of ligand binding mechanisms and associated topological perturbation of G4 structures. Here we study the G4 binding mechanisms of a family of stiff-stilbene G4 ligands to human telomeric DNA using molecular dynamics (MD) and enhanced sampling (metadynamics) MD simulations. The simulations predict a variety of binding mechanisms and effects on G4 structure for the different ligands in the series. In parallel, we characterize the binding of the ligands to the G4 target experimentally using NMR and CD spectroscopy. The results show good agreement between the simulated and experimentally observed binding modes, binding affinities and ligand-induced perturbation of the G4 structure. The simulations correctly predict ligands that perturb G4 topology. Metadynamics simulations are shown to be a powerful tool to aid development of molecules to influence G4 structure, both in interpreting experiments and to help in the design of these chemotypes.

Enhanced sampling molecular dynamics simulations and solution-phase experiments come together to demonstrate the diverse effects of G4-interactive small molecules.     

Net charge changes in the calculation of relative ligand‐binding free energies via classical atomistic molecular dynamics simulation

The calculation of binding free energies of charged species to a target molecule is a frequently encountered problem in molecular dynamics studies of (bio‐)chemical thermodynamics. Many important endogenous receptor‐binding molecules, enzyme substrates, or drug molecules have a nonzero net charge. Absolute binding free energies, as well as binding free energies relative to another molecule with a different net charge will be affected by artifacts due to the used effective electrostatic interaction function and associated parameters (e.g., size of the computational box). In the present study, charging contributions to binding free energies of small oligoatomic ions to a series of model host cavities functionalized with different chemical groups are calculated with classical atomistic molecular dynamics simulation. Electrostatic interactions are treated using a lattice‐summation scheme or a cutoff‐truncation scheme with Barker–Watts reaction‐field correction, and the simulations are conducted in boxes of different edge lengths. It is illustrated that the charging free energies of the guest molecules in water and in the host strongly depend on the applied methodology and that neglect of correction terms for the artifacts introduced by the finite size of the simulated system and the use of an effective electrostatic interaction function considerably impairs the thermodynamic interpretation of guest‐host interactions. Application of correction terms for the various artifacts yields consistent results for the charging contribution to binding free energies and is thus a prerequisite for the valid interpretation or prediction of experimental data via molecular dynamics simulation. Analysis and correction of electrostatic artifacts according to the scheme proposed in the present study should therefore be considered an integral part of careful free‐energy calculation studies if changes in the net charge are involved. © 2013 The Authors Journal of Computational Chemistry Published by Wiley Periodicals, Inc.     

Effect of monovalent ion binding on molecular dynamics of the S100-family calcium-binding protein calbindin D9k

Calbindin D_9k is a member of the S100 subfamily of EF-hand calcium binding proteins, and has served as an important model system for biophysical studies. The fast timescale dynamics of the calcium-free (apo) state is characterized using molecular dynamics simulations. Order parameters for the backbone NH bond vectors are determined from the simulations and compared with experimentally derived values, with a focus on the dynamics of calcium-binding site I. There is a significant discrepancy between simulated and experimental order parameters for site I residues in the case of no ion bound in site I. However, it was found in the simulations that a Na⁺ ion can bind in site I, and the resulting order parameters determined from the simulations are in excellent agreement with experiment. Comparisons are made to X-ray structures of other S100 family members in which Na⁺ ions were observed or suggested to be bound in site I. © 2019 Wiley Periodicals, Inc.     

Live‐Cell‐Templated Dynamic Combinatorial Chemistry

Dynamic covalent chemistry combines in a single step the screening and synthesis of ligands for biomolecular recognition. In order to do that, a chemical entity is used as template within a dynamic combinatorial library of interconverting species, so that the stronger binders are amplified due to the efficient interaction with the target. Here we employed whole A549 living cells as template in a dynamic mixture of imines, for which amplification reflects the efficient and selective interaction with the corresponding extracellular matrix. The amplified polyamine showed strong interaction with the A549 extracellular matrix in on‐cell NMR experiments, while combination of NMR, SPR, and molecular dynamics simulations in model systems provided insights on the molecular recognition event. Notably, our work pioneers the use of whole living cells in dynamic combinatorial chemistry, which paves the way towards the discovery of new bioactive molecules in a more biorelevant environment.     

Live-Cell-Templated Dynamic Combinatorial Chemistry

Dynamic covalent chemistry combines in a single step the screening and synthesis of ligands for biomolecular recognition. In order to do that, a chemical entity is used as template within a dynamic combinatorial library of interconverting species, so that the stronger binders are amplified due to the efficient interaction with the target. Here we employed whole A549 living cells as template in a dynamic mixture of imines, for which amplification reflects the efficient and selective interaction with the corresponding extracellular matrix. The amplified polyamine showed strong interaction with the A549 extracellular matrix in on-cell NMR experiments, while combination of NMR, SPR, and molecular dynamics simulations in model systems provided insights on the molecular recognition event. Notably, our work pioneers the use of whole living cells in dynamic combinatorial chemistry, which paves the way towards the discovery of new bioactive molecules in a more biorelevant environment.     

Interactions between chlorinated dioxins and a positively charged molecular probe: New molecular interaction potential

Interaction with the ligand binding domain of receptors for natural chemicals present one potential mechanism for the biological effects of environmental chemicals. Evidence suggests that the electrostatic interaction between the ligand and the receptor is an important component for binding to some of the relevant receptors. The presence of charged residues near the binding site suggests that the charge distribution of the free ligand may be different from the charge distribution of the ligand as it approaches the binding domain of the protein. In this study a new type of potential is computed for a series of dibenzo-p-dioxin (dioxin) ligands. This quantum mechanically computed potential results from interaction between the ligand and a trimethyl ammonium probe at a set of grid points. This interaction potential is compared with the molecular electrostatic potential computed from the wave function of the isolated ligands. Three types of local minima are found: (1) above the oxygen; (2) above the conjugated ring; and (3) above the chlorine(s). The molecular electrostatic potential emphasizes the minima associated with the chlorine atoms and, in that potential, the minima associated with the oxygen atoms disappear with chlorination. In the new potential, the minima over the oxygen atoms are maintained even in tetrachlorodioxin. As chlorination is increased the differences between the two potentials increases. The new potential shows the influence of the π-cation interaction, which is largest when there is little substitution on the ring. The presence of the probe induces a dipole component of 1 debye perpendicular to the plane of the ligand. Local minima in the interaction potential are then used as starting structures for the determination of the most stable ligand–probe complexes. The most stable structures are obtained from the minima associated with the oxygen atoms. These structures are stabilized by a hydrogen bond formation between the probe and the oxygen and the molecule is bent by 30° about the O(SINGLE BOND)O axis. For this series of molecules, the new potential retains some of the features that determine the hydrogen bond whereas the molecular electrostatic potential does not. © 1998 John Wiley & Sons, Inc. J Comput Chem 19: 673–684, 1998     

On the magnitude of the chelate effect for the recognition of proteins by pharmacophores scaffolded by self-assembling oligonucleotides

The simultaneous interaction of the binding moieties of a bidentate ligand on adjacent epitopes of a target protein represents an attractive avenue for the discovery of specific, high-affinity binders. We used short DNA fragments in heteroduplex format to scaffold pairs of binding molecules with defined spatial arrangements. Iminobiotin derivates were coupled either via bifunctional linkers or by using various oligonucleotides, thus allowing monovalent or bivalent binding to streptavidin. We determined the binding affinities of the synthesized constructs in solution. We also investigated the efficiency of recovery of superior bidentate ligands in affinity capture experiments, by using both radioactive counts and DNA microarrays as readouts. This analysis confirmed the suitability of the DNA heteroduplex as a scaffold for the identification of synergistic pairs of binding moieties, capable of a high-affinity interaction with protein targets by virtue of the chelate effect.     

Structure-based discovery of novel inhibitors of Mycobacterium tuberculosis CYP121 from Indonesian natural products

Tuberculosis (TB) continues to be a serious global health threat with the emergence of multidrug-resistant tuberculosis (MDR-TB) and extremely drug-resistant tuberculosis (XDR-TB). There is an urgent need to discover new drugs to deal with the advent of drug-resistant TB variants. This study aims to find new M. tuberculosis CYP121 inhibitors by the screening of Indonesian natural products using the principle of structure-based drug design and discovery. In this work, eight natural compounds isolated from Rhoeo spathacea and Pluchea indica were selected based on their antimycobacterial activity. Derivatives compound were virtually designed from these natural molecules to improve the interaction of ligands with CYP121. Virtual screening of ligands was carried out using AutoDock Vina followed by 50 ns molecular dynamics simulation using YASARA to study the inhibition mechanism of the ligands. Two ligands, i.e., kaempferol (KAE) and its benzyl derivative (KAE3), are identified as the best CYP121 inhibitors based on their binding affinities and adherence to the Lipinski’s rule. Results of molecular dynamics simulation indicate that KAE and KAE3 possess a unique inhibitory mechanism against CYP121 that is different from GGJ (control ligand). The control ligand alters the overall dynamics of the receptor, which is indicated by changes in residue flexibility away from CYP121 binding site. Meanwhile, the dynamic changes caused by the binding of KAE and KAE3 are isolated around the binding site of CYP121. These ligands can be developed for further potential biological activities.     

Protein-ligand binding free energy estimation using molecular mechanics and continuum electrostatics. Application to HIV-1 protease inhibitors

A method is proposed for the estimation of absolute binding free energy of interaction between proteins and ligands. Conformational sampling of the protein-ligand complex is performed by molecular dynamics (MD) in vacuo and the solvent effect is calculated a posteriori by solving the Poisson or the Poisson-Boltzmann equation for selected frames of the trajectory. The binding free energy is written as a linear combination of the buried surface upon complexation, SASbur, the electrostatic interaction energy between the ligand and the protein, Eelec, and the difference of the solvation free energies of the complex and the isolated ligand and protein, deltaGsolv. The method uses the buried surface upon complexation to account for the non-polar contribution to the binding free energy because it is less sensitive to the details of the structure than the van der Waals interaction energy. The parameters of the method are developed for a training set of 16 HIV-1 protease-inhibitor complexes of known 3D structure. A correlation coefficient of 0.91 was obtained with an unsigned mean error of 0.8 kcal/mol. When applied to a set of 25 HIV-1 protease-inhibitor complexes of unknown 3D structures, the method provides a satisfactory correlation between the calculated binding free energy and the experimental pIC5o without reparametrization.     

An integrated in silico screening strategy for identifying promising disruptors of p53-MDM2 interaction

The p53 protein, also called guardian of the genome, plays a critical role in the cell cycle regulation and apoptosis. This protein is frequently inactivated in several types of human cancer by abnormally high levels of its negative regulator, mouse double minute 2 (MDM2). As a result, restoration of p53 function by inhibiting p53-MDM2 protein–protein interaction has been pursued as a compelling strategy for cancer therapy. To date, a limited number of small-molecules have been reported as effective p53−MDM2 inhibitors. X-ray structures of MDM2 in complex with some ligands are available in Protein Data Bank and herein, these data have been exploited to efficiently identify new p53-MDM2 interaction antagonists through a hierarchical virtual screening strategy. For this purpose, the first step was aimed at compiling a focused library of 686,630 structurally suitable compounds, from PubChem database, similar to two known effective inhibitors, Nutlin-3a and DP222669. These compounds were subjected to the subsequent structure-based approaches (quantum polarized ligand docking and molecular dynamics simulation) to select potential compounds with highest binding affinity for MDM2 protein. Additionally, ligand binding energy, ADMET properties and PAINS analysis were also considered as filtering criteria for selecting the most promising drug-like molecules. On the basis of these analyses, three top-ranked hit molecules, CID_118439641, CID_60452010 and CID_3106907, were found to have acceptable pharmacokinetics properties along with superior in silico inhibitory ability towards the p53-MDM2 interaction compared to known inhibitors. Molecular docking and molecular dynamics results well confirmed the interactions of the final selected compounds with critical residues within p53 binding site on the MDM2 hydrophobic clefts with satisfactory thermodynamics stability. Consequently, the new final scaffolds identified by the presented computational approach could offer a set of guidelines for designing promising anti-cancer agents targeting p53-MDM2 interaction.     

Role and Perspective of Molecular Simulation-Based Investigation of RNA–Ligand Interaction: From Small Molecules and Peptides to Photoswitchable RNA Binding

Aberrant RNA–protein complexes are formed in a variety of diseases. Identifying the ligands that interfere with their formation is a valuable therapeutic strategy. Molecular simulation, validated against experimental data, has recently emerged as a powerful tool to predict both the pose and energetics of such ligands. Thus, the use of molecular simulation may provide insight into aberrant molecular interactions in diseases and, from a drug design perspective, may allow for the employment of less wet lab resources than traditional in vitro compound screening approaches. With regard to basic research questions, molecular simulation can support the understanding of the exact molecular interaction and binding mode. Here, we focus on examples targeting RNA–protein complexes in neurodegenerative diseases and viral infections. These examples illustrate that the strategy is rather general and could be applied to different pharmacologically relevant approaches. We close this study by outlining one of these approaches, namely the light-controllable association of small molecules with RNA, as an emerging approach in RNA-targeting therapy.