Structural Determination of Three Different Series of Compounds as Hsp90 Inhibitors Using 3D-QSAR Modeling, Molecular Docking and Molecular Dynamics Methods

Hsp90 is involved in correcting, folding, maturation and activation of a diverse array of client proteins; it has also been implicated in the treatment of cancer in recent years. In this work, comparative molecular field analysis (CoMFA), comparative molecular similarity indices analysis (CoMSIA), molecular docking and molecular dynamics were performed on three different series of Hsp90 inhibitors to build 3D-QSAR models, which were based on the ligand-based or receptor-based methods. The optimum 3D-QSAR models exhibited reasonable statistical characteristics with averaging internal q(2) > 0.60 and external r(2) (pred) > 0.66 for Benzamide tetrahydro-4H-carbazol-4-one analogs (BT), AT13387 derivatives (AT) and Dihydroxylphenyl amides (DA). The results revealed that steric effects contributed the most to the BT model, whereas H-bonding was more important to AT, and electrostatic, hydrophobic, H-bond donor almost contributed equally to the DA model. The docking analysis showed that Asp93, Tyr139 and Thr184 in Hsp90 are important for the three series of inhibitors. Molecular dynamics simulation (MD) further indicated that the conformation derived from docking is basically consistent with the average structure extracted from MD simulation. These results not only lead to a better understanding of interactions between these inhibitors and Hsp90 receptor but also provide useful information for the design of new inhibitors with a specific activity.     

NMR chemical shift perturbation study of the N-terminal domain of Hsp90 upon binding of ADP,AMP-PNP,geldanamycin, and radicicol

Hsp90 is one of the most abundant chaperone proteins in the cytosol. In an ATP-dependent manner it plays an essential role in the folding and activation of a range of client proteins involved in signal transduction and cell cycle regulation. We used NMR shift perturbation experiments to obtain information on the structural implications of the binding of AMP-PNP (adenylyl-imidodiphosphate-a non-hydrolysable ATP analogue), ADP and the inhibitors radicicol and geldanamycin. Analysis of (1)H,(15)N correlation spectra showed a specific pattern of chemical shift perturbations at N210 (ATP binding domain of Hsp90, residues 1-210) upon ligand binding. This can be interpreted qualitatively either as a consequence of direct ligand interactions or of ligand-induced conformational changes within the protein. All ligands show specific interactions in the binding site, which is known from the crystal structure of the N-terminal domain of Hsp90. For AMP-PNP and ADP, additional shift perturbations of residues outside the binding pocket were observed and can be regarded as a result of conformational rearrangement upon binding. According to the crystal structures, these regions are the first alpha-helix and the "ATP-lid" ranging from amino acids 85 to 110. The N-terminal domain is therefore not a passive nucleotide-binding site, as suggested by X-ray crystallography, but responds to the binding of ATP in a dynamic way with specific structural changes required for the progression of the ATPase cycle.     

Exploring Large Domain Motions in Proteins Using Atomistic Molecular Dynamics with Enhanced Conformational Sampling

Conformational transitions in multidomain proteins are essential for biological functions. The Apo conformations are typically open and flexible, while the Holo states form more compact conformations stabilized by protein-ligand interactions. Unfortunately, the atomically detailed mechanisms for such open-closed conformational changes are difficult to be accessed experimentally as well as computationally. To simulate the transitions using atomistic molecular dynamics (MD) simulations, efficient conformational sampling algorithms are required. In this work, we propose a new approach based on generalized replica-exchange with solute tempering (gREST) for exploring the open-closed conformational changes in multidomain proteins. Wherein, selected surface charged residues in a target protein are defined as the solute region in gREST simulation and the solute temperatures are different in replicas and exchanged between them to enhance the domain motions. This approach is called gREST selected surface charged residues (gREST_SSCR) and is applied to the Apo and Holo states of ribose binding protein (RBP) in solution. The conformational spaces sampled with gREST_SSCR are much wider than those with the conventional MD, sampling open-closed conformational changes while maintaining RBP domains’ stability. The free-energy landscapes of RBP in the Apo and Holo states are drawn along with twist and hinge angles of the two moving domains. The inter-domain salt-bridges that are not observed in the experimental structures are also important in the intermediate states during the conformational changes.     

Effect of Polyphosphorylation on Behavior of Protein Disordered Regions

Proteins interact with many charged biological macromolecules (polyelectrolytes), including inorganic polyphosphates. Recently a new protein post-translational modification, polyphosphorylation, or a covalent binding of polyphosphate chain to lysine, was demonstrated in human and yeast. Herein, we performed the first molecular modeling study of a possible effect of polyphosphorylation on behavior of the modified protein using replica exchange molecular dynamics simulations in atomistic force field with explicit water. Human endoplasmin (GRP-94), a member of heat shock protein 90 family, was selected as a model protein. Intrinsically disordered region in N-terminal domain serving as a charged linker between domains and containing a polyacidic serine and lysine-rich motif, was selected as a potent polyphosphorylation site according to literature data. Polyphosphorylation, depending on exact modification site, has been shown to influence on the disordered loop flexibility and induce its further expanding, as well as induce changes in interaction with ordered part of the molecule. As a result, polyphosphorylation in N-terminal domain might affect interaction of HSP90 with client proteins since these chaperones play a key role in protein folding.     

静电作用力算法对分子动力学模拟结果的影响分析

蛋白质分子和界面之间的作用在药物输送以及生物分离等领域至关重要。利用分子动力学模拟考察蛋白质分子在界面附近的行为是最近10年研究的热点。在早期的工作中,Wang等发现同电荷离子交换介质可用于辅助蛋白质复性,但其机理不甚明确。在利用分子动力学模拟研究其分子机理时发现,不同静电作用力参数对模拟结果有直接的影响。因此,通过全原子分子动力学模拟考察不同静电参数条件对模拟结果的影响,展示此过程的构象和能量变化,分析了造成结果差异的原因。研究结果揭示了不同静电参数对模拟结果的影响,为进一步研究蛋白质在界面表面的行为奠定了一定的理论基础。     

Conformation-dependent dynamics of macromolecular ions in the gas phase under an electrostatic field: A molecular dynamics simulation

Highly charged macromolecular ions exhibit various conformations in gas. Intramolecular charge-to-charge interaction induces a transformation from a globular structure into a stretched one. The change in the molecular conformation brings a complex dynamic behavior of ions under an electrostatic field. In the present study, we visualized the movement of a monovalent and multiply charged straight chain macromolecules, polyethylene glycol (PEG), by a molecular dynamics (MD) simulation. The simulation showed that a singly charged PEG ion (899?<?M_W < 4,643) takes a globular conformation. The electrical mobilities of these ions determined from the migration distance under an electric field were compared with the experimental data and those determined by the classical Mason–Schamp theory under an assumption of spherical shape. As a result, we obtained a good agreement between the MD, theoretical, and experimental data for the monovalent ions. We also found that the MD simulation successfully predicts the electrical mobility of the multiply charged stretched PEG ions, but the classical theory fails. We were able to visualize the periodic bending and stretching motion by the MD simulation. This unique motion results from the localization of charges on the PEG molecule and may have a significant effect on the dynamic behavior of macromolecule ions in gas.

Copyright © 2019 American Association for Aerosol Research     

Computational Studies of Allosteric Regulation in the Hsp90 Molecular Chaperone: From Functional Dynamics and Protein Structure Networks to Allosteric Communications and Targeted Anti-Cancer Modulators

Computational studies of allosteric interactions have witnessed a recent renaissance fueled by growing interest in the modeling of complex molecular assemblies and biological networks. Allosteric interactions of the molecular chaperone Hsp90 with a diverse array of cochaperones and client proteins allow for molecular communication in signal transduction networks. In this review, recent developments in the understanding of allosteric interactions in the context of structural, functional, and computational studies of the Hsp90 chaperone are discussed. A comprehensive analysis of structural and network-based models of protein allostery is provided. Computational and experimental approaches and advances in the understanding of Hsp90 interactions and regulatory mechanisms are reviewed to provide a systematic and critical view of the current progress and most challenging questions in the field. The current status and future prospects for translational research, bridging the basic science of chaperones with the discovery of anti-cancer therapies, are also highlighted.     

A Clickable Photosystem I,Ferredoxin, and Ferredoxin NADP+ Reductase Fusion System for Light-Driven NADPH Regeneration**

Photosynthetic organisms like plants, algae, and cyanobacteria use light for the regeneration of dihydronicotinamide dinucleotide phosphate (NADPH). The process starts with the light-driven oxidation of water by photosystem II (PSII) and the released electrons are transferred via the cytochrome b₆f complex towards photosystem I (PSI). This membrane protein complex is responsible for the light-driven reduction of the soluble electron mediator ferredoxin (Fd), which passes the electrons to ferredoxin NADP⁺ reductase (FNR). Finally, NADPH is regenerated by FNR at the end of the electron transfer chain. In this study, we established a clickable fusion system for in vitro NADPH regeneration with PSI−Fd and PSI−Fd−FNR, respectively. For this, we fused immunity protein 7 (Im7) to the C-terminus of the PSI−PsaE subunit in the cyanobacterium Synechocystis sp. PCC 6803. Furthermore, colicin DNase E7 (E7) fusion chimeras of Fd and FNR with varying linker domains were expressed in Escherichia coli. Isolated Im7−PSI was coupled with the E7−Fd or E7−Fd−FNR fusion proteins through high-affinity binding of the E7/Im7 protein pair. The corresponding complexes were tested for NADPH regeneration capacity in comparison to the free protein systems demonstrating the general applicability of the strategy.     

Approaches to Introduce Helical Structure in Cysteine-Containing Peptides with a Bimane Group

An i−i+4 or i−i+3 bimane-containing linker was introduced into a peptide known to target Estrogen Receptor alpha (ERα), in order to stabilise an α-helical geometry. These macrocycles were studied by CD and NMR to reveal the i−i+4 constrained peptide adopts a 3₁₀-helical structure in solution, and an α-helical conformation on interaction with the ERα coactivator recruitment surface in silico. An acyclic bimane-modified peptide is also helical, when it includes a tryptophan or tyrosine residue; but is significantly less helical with a phenylalanine or alanine residue, which indicates such a bimane modification influences peptide structure in a sequence dependent manner. The fluorescence intensity of the bimane appears influenced by peptide conformation, where helical peptides displayed a fluorescence increase when TFE was added to phosphate buffer, compared to a decrease for less helical peptides. This study presents the bimane as a useful modification to influence peptide structure as an acyclic peptide modification, or as a side-chain constraint to give a macrocycle.     

Homology modeling of NR2B modulatory domain of NMDA receptor and analysis of ifenprodil binding

NMDA receptors are glutamate-gated ion channels (iGluRs) that are involved in several important physiological functions such as neuronal development, synaptic plasticity, learning, and memory. Among iGluRs, NMDA receptors have been perhaps the most actively investigated for their role in chronic neurodegeneration such as Alzheimer's, Parkinson's, and Huntington's diseases. Recent studies have shown that the NTD of subunit NR2B modulates ion channel gating through the binding of allosteric modulators such as the prototypical compound ifenprodil. In the present paper, the construction of a three-dimensional model for the NR2B modulatory domain is described and docking calculations allow, for the first time, definition of the ifenprodil binding pose at an atomic level and fully explain all the available structure-activity relationships. Moreover, in an attempt to add further insight into the ifenprodil mechanism of action, as it is not completely clear if it binds and stabilizes an open or a closed conformation of the NR2B modulatory domain, a matter, which is fundamental for the rational design of NMDA antagonists, MD simulations followed by an MM-PBSA analysis were performed. These calculations reveal that the closed conformation of the R1-R2 domain, rather than the open, constitutes the high affinity binding site for ifenprodil and that a profound stabilization of the closed conformation upon ifenprodil binding occurs. Thus, for a rational design and/or for virtual screening experiments, the closed conformation of the R1-R2 domain should be taken into account and our 3D model can provide valuable hints for the design of NR2B-selective antagonists.     

Inhibition of Hepatitis C Replication by Targeting the Molecular Chaperone Hsp90: Synthesis and Biological Evaluation of 4,5,6,7-Tetrahydrobenzo[1,2-d]thiazole Derivatives

Cellular chaperones that belong to the heat-shock protein 90 (Hsp90) family are a prerequisite for successful viral propagation for most viruses. The hepatitis C virus (HCV) uses Hsp90 for maturation, folding, and modification of viral proteins. Based on our previous discovery that marine alkaloid analogues with a 4,5,6,7-tetrahydrobenzo[1,2-d]thiazole-2-amine structure show inhibition of HCV replication and binding to Hsp90, a series of twelve novel compounds based on this scaffold was designed and synthesized. The aim was improved Hsp90 affinity and anti-HCV activity. Through structural optimization, improved binding to Hsp90 and specific HCV inhibition in genotype 1b and 2a replicon models was achieved for three compounds belonging to the newly synthesized series. Furthermore, these compounds efficiently inhibited replication of full-length HCV genotype 2a in a reporter virus RNA assay with IC₅₀ values ranging from 0.03 to 0.6 μm .     

Hyrtiosal, from the marine sponge Hyrtios erectus, inhibits HIV-1 integrase binding to viral DNA by a new inhibitor binding site

HIV-1 integrase (IN) is composed of three domains, the N-terminal domain (NTD, residues 1-50), the catalytic core domain (CCD, residues 51-212), and the C-terminal domain (CTD, residues 213-288). All the three domains are required for the two known integration reactions. CCD contains the catalytic triad and is believed to bind viral DNA specifically, and CTD binds viral DNA in a nonspecific manner. As no clear evidence has confirmed the involvement of NTD in DNA binding directly, NTD has not been seriously considered and less is known about its function in viral replication. In the current work, using a SPR technology-based assay, the HIV-1 viral DNA was determined to bind directly to NTD with a K(D) value of 8.8 microM, suggesting that the process of preintegrated complex formation for HIV-1 IN might involve the direct interaction of NTD with viral DNA in addition to binding of viral DNA to the catalytic core domain and C-terminal domain. Moreover, such viral DNA/IN binding could be inhibited by the marine product hyrtiosal from the marine sponge Hyrtios erectus with an IC(50) of 9.60+/-0.86 microM. Molecular dynamic analysis correlated with a site-directed mutagenesis approach further revealed that such hyrtiosal-induced viral DNA/IN binding inhibition was caused by the fact that hyrtiosal could bind HIV-1 NTD at Ser17, Trp19, and Lys34. As hyrtiosal was recently discovered by us as a protein tyrosine phosphatase 1B (PTP1B) inhibitor,1 this work might also supply multiple-target information for this marine product, and the verified HIV-NTD/HIV-1 IN interaction model could have further implications for new HIV-1 IN inhibitor design and evaluation.     

Frustrated Interfaces Facilitate Dynamic Interactions between Native Client Proteins and Holdase Chaperones

Molecular chaperones are crucial for cellular life to ensure that all proteins obtain their right fold and functionality. Many chaperones promiscuously bind a wide spectrum of client proteins, ranging from nascent to quasi-native and native proteins. Several recent studies have investigated, at atomic resolution, how chaperones interact with native proteins. Native proteins feature a wide variety of structural conformations, and therefore, a given chaperone cannot accomplish full surface complementarity to all of its client proteins. This limitation is circumvented by the recognition of frustrated regions on the client protein surface by the chaperone. In this interaction mode, the chaperone forms a multitude of transient local interactions with some segments of the client, whereas other parts are transiently not in favorable interactions. A permanent rearrangement of the client conformation on the chaperone occurs. Reconfiguration on the chaperone surface also gives the client a chance to fold into its correct, minimally frustrated conformation.     

Structural Basis for Design of New Purine-Based Inhibitors Targeting the Hydrophobic Binding Pocket of Hsp90

Inhibition of the molecular chaperone heat shock protein 90 (Hsp90) represents a promising approach for cancer treatment. BIIB021 is a highly potent Hsp90 inhibitor with remarkable anticancer activity; however, its clinical application is limited by lack of potency and response. In this study, we aimed to investigate the impact of replacing the hydrophobic moiety of BIIB021, 4-methoxy-3,5-dimethylpyridine, with various five-membered ring structures on the binding to Hsp90. A focused array of N⁷/N⁹-substituted purines, featuring aromatic and non-aromatic rings, was designed, considering the size of hydrophobic pocket B in Hsp90 to obtain insights into their binding modes within the ATP binding site of Hsp90 in terms of π–π stacking interactions in pocket B as well as outer α-helix 4 configurations. The target molecules were synthesized and evaluated for their Hsp90α inhibitory activity in cell-free assays. Among the tested compounds, the isoxazole derivatives 6b and 6c, and the sole six-membered derivative 14 showed favorable Hsp90α inhibitory activity, with IC₅₀ values of 1.76 µM, 0.203 µM, and 1.00 µM, respectively. Furthermore, compound 14 elicited promising anticancer activity against MCF-7, SK-BR-3, and HCT116 cell lines. The X-ray structures of compounds 4b, 6b, 6c, 8, and 14 bound to the N-terminal domain of Hsp90 were determined in order to understand the obtained results and to acquire additional structural insights, which might enable further optimization of BIIB021.     

Analysis of cation-π interactions to the structural stability of RNA binding proteins

Cation-π interactions play an important role to the stability of protein structures. In this work, we have analyzed the influence of cation-π interactions in RNA binding proteins. We observed cation-π interactions in 32 out of 51 RNA binding proteins and there is a strong correlation between the number of amino acid residues and number of cation-π interactions. The analysis on the influence of short (<±3 residues), medium (±3 or ±4 residues) and long range contacts (>±4 residues) showed that the cation-π interactions are mainly formed by long-range contacts. The cation-π interaction energy for Arg-Trp is found to be the strongest among all interacting pairs. Analysis on the preferred secondary structural conformation of the residues involved in cation-π interaction indicates that the cationic Lys and Arg prefer to be in α-helices and β-strands, respectively, whereas the aromatic residues prefer to be in strand and coil regions. Most of the cation-π interactions forming residues in RNA binding proteins are conserved among homologous sequences. Further, the cation-π interactions have distinct roles to the stability of RNA binding proteins in addition to other conventional non-covalent interactions. The results observed in the present study will be useful in understanding the contribution of cation-π interactions to the stability of RNA binding proteins.     

Homology Modelling of the GABA Transporter and Analysis of Tiagabine Binding

A homology model of the human GABA transporter (GAT‐1) based on the recently reported crystal structures of the bacterial leucine transporter from Aquifex aeolicus (LeuT) was developed. The stability of the resulting model embedded in a membrane environment was analyzed by extensive molecular dynamics (MD) simulations. Based on docking studies and subsequent MD simulations of three compounds, the endogenous ligand GABA and two potent inhibitors, (R)‐nipecotic acid and the anti‐epilepsy drug tiagabine, various binding modes were identified and are discussed. Whereas GABA and (R)‐nipecotic acid, which are both substrates, are stabilised with residues located deep inside the occluded state binding pocket (including residues Tyr 60 and Ser 396), tiagabine, which contains a large aliphatic side chain, is stabilised in a binding mode that extends from the substrate binding pocket (i.e., stabilised by Phe 294) to the extracellular vestibule, where the side chain is stabilised by aliphatic residues. The tiagabine binding mode, reaching from the substrate binding site to the extracellular vestibule, forces the side chain of Phe 294 to adopt a distinct conformation from that found in the occluded conformation of the transporter. Hence, in presence of tiagabine, GAT‐1 is constrained in an open‐to‐out conformation. Our results may be of particular interest for the design of new GAT‐1 inhibitors.     

Restructuring an interdomain linker in the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex of Escherichia coli

The lipoyl, subunit-binding and catalytic domains of the dihydrolipoamideacetyltransferase subunits (E2p) of the Escherichia coli pyruvatedehydrogenase complex are connected by linker sequences whichare characteristically rich in alanine and proline residues.By facilitating domain movement these linkers are thought topromote interactions between the three types of active sitethat participate in the catalytic cycle of the complex. To investigatefunctional constraints associated with linker composition andsequence, the natural linker of an E2p subunit containing onelipoyl domain was replaced by shorter sequences containing:mixtures of alanine plus proline residues; mainly alanine; mainlyproline; and mainly charged residues. Each artificial linkerpossessed a central histidine residue for assessing linker flexibilityby ¹H-NMR spectroscopy. The resultant complexes exhibited 181%(proline), 74–79% (alanine plus proline), 63% (alanine)and 7% (charged residues) of parental activity compared witha value of 75% expected for a complex with a comparably shortenedlinker. The ¹H-NMR spectra showed that the alanine plus prolinelinkers are flexible but the alanine linker and the prolinelinker are relatively inflexible. Substantial variations inlinker sequence and composition were tolerated without lossof function, and the enhanced activity conferred by the prolinelinker was attributed to the combined effects of length andrelative inflexibility.     

Study of specific interactions in inclusion complexes of amine-terminated PAMAM dendrimer/flavonoids by experimental and computational methods

Polyamidoamine (PAMAM) dendrimers have a multifunctional structure, able to encapsulate molecules for pharmacological applications. We evaluated the specific interaction that govern the encapsulating and affinity of one group of natural and synthetic flavonoids into the G₅-PAMAM dendrimers. The complexation and capture percent of one flavonoid series into G₅-PAMAM dendrimers, under neutral and acid pH conditions, were studied through UV–Vis spectroscopy. Additionally, only three of the flavonoids (two synthetic and one natural) were studied by high-performance liquid chromatography (HPLC) and molecular dynamic (MD) simulation, at neutral pH to calculate the affinity constants (K_d) and binding free energies (ΔG_b). From spectroscopic results, we observed that the encapsulation was much more rapid at low pH than at neutral pH, which was attributed to a greater number of cavities inside the dendrimer. The MD simulations suggested that the more compact molecular structure at neutral pH reduces the capture kinetics. Finally, the relative binding free energies calculated using MD simulations showed the same tendency as the experimental data for the three complexes. These affinities appear to be due to a complex balance of different contributions, which cannot be attributed to hydrogen bonds or charge–charge interactions alone. Nevertheless, we suggest that a protocol including UV–Vis, HPLC, and MD simulation can be a powerful predictive tool to determine the affinity of drug binding to nanocarriers.     

Effects of protein properties on adsorption and transport in polymer‐grafted ion exchangers: A multiscale modeling study

We use multiscale modeling to study how the molecular properties of a protein affect its adsorption and transport in ion exchange chromatography matrices with either open pores or charged polymers grafted into the pore structure. Coarse‐grained molecular dynamics (MD) simulations of lysozyme, bovine serum albumin, and immunoglobulin show that higher protein net charge leads to greater partitioning into the polymer‐grafted pore space but slower diffusion there due to favorable electrostatic interactions, while larger size decreases both pore space partitioning and diffusion due to steric effects of the polymers. Mass transfer simulations based on the MD results show that the polymer‐grafted systems can enhance the adsorption kinetics if pore space partitioning and diffusion are both sufficiently high. The simulations illustrate that to achieve fast adsorption kinetics, there is a tradeoff between favorable binding and rapid diffusion which largely depends on the charge and size of the protein. © 2017 American Institute of Chemical Engineers AIChE J, 63: 4564–4575, 2017