Molecular Dynamics Simulation of Chemokine Receptors in Lipid Bilayer: A Case Study on C–C Chemokine Receptor Type 2

Chemokine receptors belong to the membrane proteins that are included in many physiological phenomena. However, the mechanism of their action is unknown at the atomistic level in different aspects. In this study, a computational pipeline is exploited to investigate the molecular basis of how the structure of C–C chemokine receptor type 2, a prototypical chemokine receptor, is affected by lipid bilayer and an antagonist (INCB3344). This study includes homology modeling, molecular dynamics simulation in lipid bilayer, and docking. A detailed mechanism of INCB3344 has been described. Tyr 49, Trp 98, Tyr 120, His 121, and Glu 291 are proved to play important roles in binding. Integrating results obtained in this study and experimental data help us to suggest a two‐step ligand‐binding mechanism. The N‐terminus of protein first sticks out from the extracellular domain suitable for the contact with the antagonist. Binding of ligand to this segment leads to the geometrical changes to facilitate the ligand interactions with extracellular loop 2 of C–C chemokine receptor type 2. Finally, the interactions occurring between extracellular loop 2 and ligand induce conformational changes in C–C chemokine receptor type 2 structure. These changes bring the ligand closer to the binding pocket, allowing the interaction between INCB3344 and the residues of active site.     

Diffusion of lonizable Solutes Across Planar Lipid Bilayer Membranes: Boundary-Layer pH Gradients and the Effect of Buffers

The diffusion of weak acids or bases across planar lipid bilayer membranes results in aqueous boundary layer pH gradients. If not properly taken into account, such pH gradients will lead to errors in estimated membrane permeability coefficients, P _m. The role of the permeant concentration, the buffer capacity, and the physicochemical properties of both permeant and buffer on the magnitude and impact of such pH gradients have been explored. A theoretical model has been developed to describe the diffusion of both permeant and buffer species. Significant pH gradients develop depending on solution pH and the pK _a's, concentrations, and P _m values of both permeant and buffer. The relative error in experimentally determined P _m values was calculated as the ratio, r, between apparent P _m values (obtained from flux measurements using an equation which neglected boundary layer pH gradients) and its true value. Simulated r values ranged from 1 (0% error) to <0.01 (>100% error) for weak acids, decreasing with decreasing buffer capacity and increasing solute flux. The buffer capacity required for an r > 0.95 was calculated versus pH for permeants varying in pK _a and P _m. Membrane-permeable buffers significantly reduce boundary layer pH gradients through a feedback effect due to buffer cotransport. Apparent P _m values of p-hydroxymethyl benzoic acid across lecithin bilayer membranes at 25°C were obtained as a function of permeant concentration in various buffers [glycolic, 2-(N-morpholino)ethane-sulfonic, and formic acids]. Predictions agreed closely with experimental fluxes.     

Chemotherapy Drugs Form Ion Pores in Membranes Due to Physical Interactions with Lipids

We demonstrate the effects on membrane of the tubulin‐binding chemotherapy drugs: thiocolchicoside and taxol. Electrophysiology recordings across lipid membranes in aqueous phases containing drugs were used to investigate the drug effects on membrane conductance. Molecular dynamics simulation of the chemotherapy drug–lipid complexes was used to elucidate the mechanism at an atomistic level. Both drugs are observed to induce stable ion‐flowing pores across membranes. Discrete pore current–time plots exhibit triangular conductance events in contrast to rectangular ones found for ion channels. Molecular dynamics simulations indicate that drugs and lipids experience electrostatic and van der Waals interactions for short periods of time when found within each other’s proximity. The energies from these two interactions are found to be similar to the energies derived theoretically using the screened Coulomb and the van der Waals interactions between peptides and lipids due to mainly their charge properties while forming peptide‐induced ion channels in lipid bilayers. Experimental and in silico studies together suggest that the chemotherapy drugs induce ion pores inside lipid membranes due to drug–lipid physical interactions. The findings reveal cytotoxic effects of drugs on the cell membrane, which may aid in novel drug development for treatment of cancer and other diseases.     

Characterization of Lipid Membrane Dynamics by Simulation: 3. Probing Molecular Transport Across the Phospholipid Bilayer

Purpose. The goal of this study is to elucidate the role of the motions of the hydrocarbon chains of a phospholipid bilayer in penetrant diffusion. Penetrant size, as well as its position in the hydrocarbon core of the lipid bilayer, has also been explored regarding impact on the diffusion rate in a phospholipid bilayer. Methods. Molecular dynamics, MD, simulations were carried out on a model dimyristoyl phosphatidylcholine (DMPC) membrane bilayer with and without methanol and propanol as penetrants. The MD trajectories were analyzed in terms of estimating time and space properties. Results. These simulations show that torsion angle kink shifts in the hydrocarbon chains of phospholipids are natural occurrences in a bilayer assembly. The diffusion coefficients of methanol and propanol in a DMPC lipid bilayer, as calculated from the MD simulations, agree with experimental measurements. Both methanol and propanol show different diffusion rates in different regions of the hydrocarbon chain matrix of the lipid bilayer. Solute size has more impact on diffusion rate in the bilayer regions with high torsion angle order parameters, as compared to the regions with low torsion angle order parameters. Conclusions. The simulated transport behavior suggests that a kink shift diffusion mechanism is more likely to occur in regions with high torsion angle order parameters, and a free volume transport mechanism is more likely operative in the region with low torsion angle order parameters, mainly the center core of the bilayer. A three zone diffusion model is proposed for transport of a penetrant across a bilayer.     

Synthesis and biological evaluation of novel peptides based on antimicrobial peptides as potential agents with antitumor and multidrug resistance‐reversing activities

Tumor chemotherapy, which plays an important role in the clinical treatment of metastatic cancer, is limited by low selectivity and drug resistance in clinical application. In our study, we selected antimicrobial peptide BP100 as a lead peptide, designed, and synthesized a series of novel antineoplastic peptides through solid‐phase synthesis. Among them, B4 and B8 showed excellent anticancer activity. As revealed by further investigations, these peptides could disrupt the cell membrane, trigger the cytochrome C release into cytoplasm, and ultimately lead to apoptosis. In addition, they also showed multidrug resistance‐reversing effects by performing effective antitumor activity against multidrug‐resistant cells. As a result, these peptides may possibly be regarded as a promising candidate for cancer treatment.     

两亲α-螺旋抗菌肽的分子设计研究现状与进展

抗菌肽以其广谱、快速、特异性作用及抗菌、抗病毒、抗肿瘤等活性,成为具有重要潜在价值的新型药物.最大限度地发挥其活性和降低毒性,是抗菌肽新药开发的首要问题.用分子设计手段改造抗菌肽已成为解决这一问题的关键.以两亲α-螺旋抗菌肽为对象,对分子设计过程中遵守阳离子性和两亲性的原则及影响活性的物理化学参数,可以采取序列修饰或全新设计的方法,以达到定向改造的目的.对分子设计在医药上的应用和发展前景进行了综述和展望.     

Effects of temporins on molecular dynamics and membrane permeabilization in lipid vesicles

Abstract: Temporins are a novel family of small (10–13 residues) cationic antimicrobial peptides recently isolated from the skin of the European red frog Rana temporaria. Although recently acquired evidence shows that temporins have the potential to kill bacteria by permeabilizing the cytoplasmic membrane, the molecular mechanisms of membrane selectivity and permeabilization are largely unknown. In this study, it was found that temporins cause the release of fluorescent markers entrapped in phosphatidylcholine liposomes in a manner that depends significantly on the size of the solute. Temporins were also shown to lack a detergent‐like effect on lipid vesicles, indicating that marker leakage caused by these peptides is not due to total membrane disruption but to perturbation of bilayer organization on a local scale. Binding of temporins to liposomes did lead to a small increase in lipid hydrocarbon chain mobility, as revealed by EPR spectroscopy of nitroxide‐labeled fatty acids incorporated in the bilayer. Reference experiments were conducted using the bee venom peptide melittin, whose properties and behavior in natural and model membrane systems are well known. Our findings for temporins are discussed in relation to the models proposed to date to account for the action of antimicrobial peptides on membranes.     

Activity Prediction and Structural Insights of Extracellular Signal‐Regulated Kinase 2 Inhibitors with Molecular Dynamics Simulations

A computational application to predict, probe and interpret the activities of a series of congeneric compounds inhibiting extracellular signal‐regulated kinase 2 protein kinase is presented. The study shows that molecular dynamics coupled with molecular mechanics Poisson–Boltzmann solvent accessible surface area free energy estimation is a suitable tool for investigating the experimental binding activities of ligands to protein kinases. Computed and experimental binding activities were found to be significantly correlated. Moreover, the interpretation of the X‐ray co‐crystal structure in conjunction with computational results shows that the hinge region of the protein insure the principal binding site via multiple hydrogen bonding interactions, whereas fine‐modulation of biological activities along the series is accomplished through the combination of weak and strong interactions that compete with water. These are located in the substituent moieties of the ligands interfacing with the DFG motif, the sugar region and the hydrophobic pocket of extracellular signal‐regulated kinase 2. The study suggests that a wider interaction framework that is well beyond the hinge region is required to predict and rationalize at molecular level the experimental biological activities of congeneric compound series.     

Multilamellar Liposomes and Solid-Supported Lipid Membranes (TRANSIL): Screening of Lipid-Water Partitioning Toward a High-Throughput Scale

Purpose. Lipid-water partitioning of 187 pharmaceuticals has been assessed with solid-supported lipid membranes (TRANSIL) in microwell plates and with multilamellar liposomes for a data comparison. The high-throughput potential of the new approach was evaluated. Methods. Drugs were incubated at pH 7.4 with egg yolk lecithin membranes either on a solid support (TRANSIL beads) or in the form of multilamellar liposomes. Phase separation of lipid and water phase was achieved by ultracentrifugation in case of liposomes or by a short filtration step in case of solid-supported lipid membranes. Results. Lipid-water partitioning data of both approaches correlate well without systematic deviations in the investigated lipophilicity range. The solid-supported lipid membrane approach provides high-precision data in an automated microwell-plate setup. The lipid composition of the solid-supported lipid membranes was varied to study the influence of membrane change on lipid-water partitioning. In addition, pH-dependent measurements have been performed with minimal experimental effort. Conclusions. Solid-supported lipid membranes represent a valuable tool to determine physiologically relevant lipid-water partitioning data of pharmaceuticals in an automated setup and is well suited for high-throughput data generation in lead optimization programs.     

Stabilized helical peptides: overview of the technologies and its impact on drug discovery

Introduction: Protein-protein interactions are predominant in the workings of all cells. Until now, there have been a few successes in targeting protein-protein interactions with small molecules. Peptides may overcome some of the challenges of small molecules in disrupting protein-protein interactions. However, peptides present a new set of challenges in drug discovery. Thus, the study of the stabilization of helical peptides has been extensive.

Areas covered: Several technological approaches to helical peptide stabilization have been studied. In this review, stapled peptides, foldamers, and hydrogen bond surrogates are discussed. Issues regarding design principles are also discussed. Furthermore, this review introduces select computational techniques used to aid peptide design and discusses clinical trials of peptides in a more advanced stage of development.

Expert opinion: Stabilized helical peptides hold great promise in a wide array of diseases. However, the field is still relatively new and new design principles are emerging. The possibilities of peptide modification are quite extensive and expanding, so the design of stabilized peptides requires great attention to detail in order to avoid a large number of failed lead peptides. The start of clinical trials with stapled peptides is a promising sign for the future.     

Structural models and binding site prediction of the C-terminal domain of human Hsp90: a new target for anticancer drugs

Heat shock protein 90 is a valuable target for anticancer drugs because of its role in the activation and stabilization of multiple oncogenic signalling proteins. While several compounds inhibit heat shock protein 90 by binding the N-terminal domain, recent studies have proved that the C-terminal domain is important for dimerization of the chaperone and contains an additional binding site for inhibitors. Heat shock protein 90 inhibition achieved with molecules binding to the C-terminal domain provides an additional and novel opportunity to design and develop drugs. Therefore, for the first time, we have investigated the structure and the dynamic behaviour of the C-terminal domain of human heat shock protein 90 with and without the small-middle domain, using homology modelling and molecular dynamics simulations. In addition, secondary structure predictions and peptide folding simulations proved useful to investigate a putative additional alpha-helix located between H18 and beta20 of the C-terminal domain. Finally, we used the structural information to infer the location of the binding site located in the C-terminal domain by using a number of computational tools. The predicted pocket is formed by two grooves located between helix H18, the loop downstream of H18 and the loop connecting helices H20 and H21 of each monomer of the C-terminal domain, with only two amino acids contributing from each middle domain.     

Computational insights into the different catalytic activities of CYP3A4 and CYP3A5 toward schisantherin E

The cytochromes CYP3A4 and CYP3A5 share 84% sequence identity, but they exhibit different catalytic activities toward some substrates. Schisantherin E (SE) was recently identified as a selective substrate of CYP3A5, which exhibited catalytic efficiency that was more than 23 times higher than CYP3A4. At present, however, the structural determinants responsible for the different catalytic activities of the two enzymes toward SE have not been fully understood. In this study, a combination of molecular docking, molecular dynamic simulations, and binding free energy calculation was performed on the CYP3A4/CYP3A5‐SE systems to investigate the issue. The results demonstrate that Ser119 in CYP3A4 and Glu374 in CYP3A5 formed direct hydrogen bonding with SE, respectively. Additionally, one water molecule located between the B‐C loop and the I helix mediated different hydrogen‐bonding networks between CYP3A4/3A5 and SE. The residue differences (Phe/Leu108 and Leu/Phe210) triggered the distinct conformational changes of the Phe‐cluster residues, especially Phe213 and Phe215, which formed stronger hydrophobic interactions with SE in CYP3A5. The calculated binding free energies were consistent with the experimental results.     

Integrating Pharmacophore into Membrane Molecular Dynamics Simulations to Improve Homology Modeling of G Protein‐coupled Receptors with Ligand Selectivity: A2A Adenosine Receptor as an Example

Homology modeling has been applied to fill in the gap in experimental G protein‐coupled receptors structure determination. However, achievement of G protein‐coupled receptors homology models with ligand selectivity remains challenging due to structural diversity of G protein‐coupled receptors. In this work, we propose a novel strategy by integrating pharmacophore and membrane molecular dynamics (MD) simulations to improve homology modeling of G protein‐coupled receptors with ligand selectivity. To validate this integrated strategy, the A_2A adenosine receptor (A_2AAR), whose structures in both active and inactive states have been established, has been chosen as an example. We performed blind predictions of the active‐state A_2AAR structure based on the inactive‐state structure and compared the performance of different refinement strategies. The blind prediction model combined with the integrated strategy identified ligand–receptor interactions and conformational changes of key structural elements related to the activation of A_2AAR, including (i) the movements of intracellular ends of TM3 and TM5/TM6; (ii) the opening of ionic lock; (iii) the movements of binding site residues. The integrated strategy of pharmacophore with molecular dynamics simulations can aid in the optimization in the identification of side chain conformations in receptor models. This strategy can be further investigated in homology modeling and expand its applicability to other G protein‐coupled receptor modeling, which should aid in the discovery of more effective and selective G protein‐coupled receptor ligands.     

Biological and conformational studies of [Val4]morphiceptin and [D-Val4]morphiceptin analogs incorporating cis-2-aminocyclopentane carboxylic acid as a peptidomimetic for proline

We report the synthesis, biological activity, and conformational analysis of tetrapeptide analogs related to [Val⁴] morphiceptin and [d -Val⁴]morphiceptin in which the proline at the second position has been replaced with cw-2-aminocyclopentane carboxylic acid (cis-2-Ac⁵c). Since the cis-2-Ac⁵c residue contains a normal amide, only the trans form has been observed about the amide bond between the first and second residues. The cis-2-Ac⁵c is a β amino acid with two chiral centers resulting in two possible configurational isomers, namely (1s, 2R) and (1R, 2S) forms. The analogs containing the (1s, 2R)-Ac⁵c residue show activity at the μ-receptor but are inactive at the δ-receptor, resulting in a high selectivity for the μ-receptor. The (1R, 2S)-Ac⁵c containing analogs are completely inactive at both the μ- and δ-receptors. The conformational analysis indicates that the separation of the aromatic rings of the tyrosine and phenylalanine residues, as expressed by the center-to-center distance, is 10.1-12.7 Å for the preferred conformations of the bioactive analogs containing the (1S, 2R)-Ac⁵c residue while a range of 4.8-7.0 Å is observed for the preferred conformations of the inactive analogs with the (1R, 2S)-Ac⁵c residue. A comparison of the findings from the conformational analysis and biological assays establishes the fact that a relatively large separation of the two aromatic side chains is required for the μ-opioid receptor activity of these molecules. Since the tetrapeptide amides studied in this investigation show similar biological profiles to those of the morphiceptin-related analogs, we have compared the preferred conformations estimated for the cis-2-Ac⁵c containing analogs with those of morphiceptin. One of the low energy conformations calculated for morphiceptin with the cis form about the tyrosine and proline residues has considerable topological similarity with the bioactive analogs containing the (1S, 2R)-Ac⁵c residue, indicating that the cis from about these two residues is required for the biological activity of the morphiceptin-related analogs containing the proline at the second position.     

The glycan‐mediated mechanism on the interactions of gp120 with CD4 and antibody: Insights from molecular dynamics simulation

N‐linked glycans such as 234 and 276 gp₁₂₀ glycans are vital components of HIV evasion from humoral immunity and important for HIV‐1 neutralization of many broadly neutralizing antibodies (bNAbs). However, it is unknown the action mechanism of two glycans. To investigate the roles of the glycans on the interactions of gp120 with CD4 and antibody, molecular dynamic simulations based on gp120‐CD4‐8ANC195 complex with 234 and 276 gp₁₂₀ glycans, 234 gp₁₂₀ glycan, 276 gp₁₂₀ glycan, and without glycan were performed. Our results reveal that 276 gp₁₂₀ glycan can enhance gp120‐CD4 and gp120‐antibody interactions through the formation of hydrogen bonds of the glycan with CD4 and antibody and make the binding interface of gp120, CD4 and antibody stable; 234 gp₁₂₀ glycan primarily reinforces gp120‐antibody interactions and weakly affects gp120‐CD4 interactions as it mainly lies between gp120 and antibody. The co‐operating of two glycans can enhance gp120‐CD4 and gp120‐antibody associations. Through the structural analysis, it can be seen that 234 gp₁₂₀ glycan leads to moving upward of two glycans and the variable region of heavy chain, which is favorable for the interactions of gp120 with CD4 and antibody. The information obtained in this study can provide the guidance for design vaccines and small molecule inhibitors.     

Antimicrobial activity of arginine‐ and tryptophan‐rich hexapeptides: the effects of aromatic clusters,d‐amino acid substitution and cyclization

Abstract: Many antimicrobial peptides bear arginine (R)‐ and tryptophan (W)‐rich sequence motifs. Based on the sequence Ac‐RRWWRF‐NH₂, sets of linear and cyclic peptides were generated by changes in the amino acid sequence, l ‐d ‐amino acid exchange and naphthylalanine substituted for tryptophan. Linear RW‐peptides displayed moderate activity towards Gram‐positive Bacillus subtilis (15 < MIC < 31 μm ) and were inactive against Gram‐negative Escherichia coli at peptide concentrations <100 μm . Cyclization induced high antimicrobial activity. The effect of cyclization was most pronounced for peptides with three adjacent aromatic residues. Incorporation of d ‐amino acid residues had minor influence on the biological activity. The haemolytic activity of all RW‐peptides at 100 μm concentration was low (<7% lysis for linear R/W‐rich peptides and <28% for the cyclic analogues). Introduction of naphthylalanine enhanced the biological activities of both the linear and cyclic peptides. All peptides induced permeabilization of large unilamellar vesicles (LUVs) composed of lipids of the membrane of B. subtilis and erythrocytes, but surprisingly had no effect on LUVs composed of lipids of the E. coli inner membrane. The profiles of peptide activity against B. subtilis and red blood cells correlated with the permeabilizing effects on the corresponding model membranes and were related to hydrophobicity parameters as derived from reversed phase high‐performance liquid chromatography (HPLC). The results underlined the importance of amphipathicity as a driving force for cell lytic activity and suggest that conformational constraints and an appropriate position of aromatic residues allowing the formation of hydrophobic clusters are highly favourable for antimicrobial activity and selectivity.     

Network pharmacology and experimental evidence reveal the protective mechanism of Yi-Qi Cong-Ming decoction on age-related hearing loss

ContextYi-Qi Cong-Ming (YQCM) decoction has been widely used to prevent age-related hearing loss (ARHL), the most prevalent neurodegenerative disease in the elderly.ObjectiveTo explore the mechanism of YQCM decoction in the treatment of ARHL.Materials and methodsThe chemical constituents of YQCM were screened from the Traditional Chinese Medicine Systems Pharmacology Database. Potential targets of YQCM against ARHL were predicted by DrugBank, GeneCards, and OMIM database. Protein-protein network and enrichment analysis were used for exploring possible molecular mechanisms. Molecular docking and an in vitro model of ARHL by exposing auditory cells with 100 μM H₂O₂ for 3 h were applied. Cell viability and mitochondrial membrane potential (ΔΨM) were detected by CCK-8 and high-content analysis. γH2AX and cleaved caspase-3 were detected by Western blot.ResultsThe main compounds have good affinities with hub targets, especially AKT1, PTGS2, and CASP3. GO and KEGG analysis showed that the main biological process and key targets were related to negative regulation of the apoptotic process. H₂O₂ treatment could reduce the cell viability by 68% and impaired ΔΨM, while 90 μg/mL YQCM pre-treatment could restore the cell viability by 97.45% and increase ΔΨM (2-fold higher). YQCM pre-treatment also reduced γH2AX and cleaved caspase-3 protein levels.ConclusionsOur study suggested that YQCM prevents ARHL by modulating the apoptosis process in auditory hair cells. Moreover, this study proved that bioinformatics analysis combined with molecular docking and cell model is a promising method to explore other possible pharmacological interventions of ARHL.     

Computer simulations of transport through membranes: passive diffusion, pores, channels and transporters

A key function of biological membranes is to provide mechanisms for the controlled transport of ions, nutrients, metabolites, peptides and proteins between a cell and its environment. We are using computer simulations to study several processes involved in transport. In model membranes, the distribution of small molecules can be accurately calculated; we are making progress towards understanding the factors that determine the partitioning behaviour in the inhomogeneous lipid environment, with implications for drug distribution, membrane protein folding and the energetics of voltage gating. Lipid bilayers can be simulated at a scale that is sufficiently large to study significant defects, such as those caused by electroporation. Computer simulations of complex membrane proteins, such as potassium channels and ATP-binding cassette (ABC) transporters, can give detailed information about the atomistic dynamics that form the basis of ion transport, selectivity, conformational change and the molecular mechanism of ATP-driven transport. This is illustrated in the present review with recent simulation studies of the voltage-gated potassium channel KvAP and the ABC transporter BtuCD.     

Exploring binding mechanisms of VEGFR2 with three drugs lenvatinib,sorafenib, and sunitinib by molecular dynamics simulation and free energy calculation

Lenvatinib (LEN), sorafenib (SOR), and sunitinib (SUN) are drugs targeting vascular endothelial growth factor receptor 2 (VEGFR2). Despite sharing similar chemical structures and bioactivities, LEN and SOR bind to different functional states of VEGFR2, viz. DFG‐in and DFG‐out state, respectively. SUN binds to the DFG‐out state of VEGFR2 just like SOR but with less potency. Thus, detail binding mechanisms between VEGFR2 and these drugs, especially dynamic interaction, are valuable for future drug design. In the present work, molecular dynamics simulation, essential dynamic analysis, and molecular mechanics/generalized born surface area were performed to these VEGFR2–drugs systems. Rank of calculated binding affinities is in accordance with the experimental data. The binding free energy calculation suggests that van der Waals interaction plays a vital role in the binding. Per‐residue free energy decomposition indicates that residues L840, V848, A866, E885, L889, V899, V916, F918, C919, L1035, C1045, D1046, and F1047 play an important role in the binding between VEGFR2 and LEN/SOR. While residues L840, V848, E917, F918, C919, G922, L1035, and F1047 contribute the major hydrophobic interaction for SUN binding to the receptor. Our results also reveal that residue E885/D1046 plays a vital role in binding via forming hydrogen bonds with drugs.