Molecular dynamics simulation of force-regulated interaction between glycoprotein Ib α and filamin / 生物医学工程学杂志

In resting platelets, the 17 th domain of filamin a (FLNa17) constitutively binds to the platelet membrane glycoprotein Ibα (GPIbα) at its cytoplasmic tail (GPIbα-CT) and inhibits the downstream signal activation, while the binding of ligand and blood shear force can activate platelets. To imitate the pull force transmitted from the extracellular ligand of GPIbα and the lateral tension from platelet cytoskeleton deformation, two pulling modes were applied on the GPIbα-CT/FLNa17 complex, and the molecular dynamics simulation method was used to explore the mechanical regulation on the affinity and mechanical stability of the complex. In this study, at first, nine pairs of key hydrogen bonds on the interface between GPIbα-CT and FLNa17 were identified, which was the basis for maintaining the complex structural stability. Secondly, it was found that these hydrogen bonding networks would be broken down and lead to the dissociation of FLNa17 from GPIbα-CT only under the axial pull force; but, under the lateral tension, the secondary structures at both terminals of FLNa17 would unfold to protect the interface of the GPIbα-CT/FLNa17 complex from mechanical damage. In the range of 0~40 pN, the increase of pull force promoted outward-rotation of the nitrogen atom of the 563 rd phenylalanine (PHE 563-N) at GPIbα-CT and the dissociation of the complex. This study for the first time revealed that the extracellular ligand-transmitted axial force could more effectively relieve the inhibition of FLNa17 on the downstream signal of GPIbα than pure mechanical tension at the atomic level, and would be useful for further understanding the platelet intracellular force-regulated signal pathway.

Semi-rational evolution of ω-transaminase from Aspergillus terreus for enhancing the thermostability / 生物工程学报

ω-transaminase (ω-TA) is a natural biocatalyst that has good application potential in the synthesis of chiral amines. However, the poor stability and low activity of ω-TA in the process of catalyzing unnatural substrates greatly hampers its application. To overcome these shortcomings, the thermostability of (R)-ω-TA (AtTA) from Aspergillus terreus was engineered by combining molecular dynamics simulation assisted computer-aided design with random and combinatorial mutation. An optimal mutant AtTA-E104D/A246V/R266Q (M3) with synchronously enhanced thermostability and activity was obtained. Compared with the wild- type (WT) enzyme, the half-life t1/2 (35 ℃) of M3 was prolonged by 4.8-time (from 17.8 min to 102.7 min), and the half deactivation temperature (T1050) was increased from 38.1 ℃ to 40.3 ℃. The catalytic efficiencies toward pyruvate and 1-(R)-phenylethylamine of M3 were 1.59- and 1.56-fold that of WT. Molecular dynamics simulation and molecular docking showed that the reinforced stability of α-helix caused by the increase of hydrogen bond and hydrophobic interaction in molecules was the main reason for the improvement of enzyme thermostability. The enhanced hydrogen bond of substrate with surrounding amino acid residues and the enlarged substrate binding pocket contributed to the increased catalytic efficiency of M3. Substrate spectrum analysis revealed that the catalytic performance of M3 on 11 aromatic ketones were higher than that of WT, which further showed the application potential of M3 in the synthesis of chiral amines.

Integrated

The drug formulation design of self-emulsifying drug delivery systems (SEDDS) often requires numerous experiments, which are time- and money-consuming. This research aimed to rationally design the SEDDS formulation by the integrated computational and experimental approaches. 4495 SEDDS formulation datasets were collected to predict the pseudo-ternary phase diagram by the machine learning methods. Random forest (RF) showed the best prediction performance with 91.3% for accuracy, 92.0% for sensitivity and 90.7% for specificity in 5-fold cross-validation. The pseudo-ternary phase diagrams of meloxicam SEDDS were experimentally developed to validate the RF prediction model and achieved an excellent prediction accuracy (89.51%). The central composite design (CCD) was used to screen the best ratio of oil-surfactant-cosurfactant. Finally, molecular dynamic (MD) simulation was used to investigate the molecular interaction between excipients and drugs, which revealed the diffusion behavior in water and the role of cosurfactants. In conclusion, this research combined machine learning, central composite design, molecular modeling and experimental approaches for rational SEDDS formulation design. The integrated computer methodology can decrease traditional drug formulation design works and bring new ideas for future drug formulation design.

Discovery of human coronaviruses pan-papain-like protease inhibitors using computational approaches / 药物分析学报

The papain-like protease (PLpro) is vital for the replication of coronaviruses (CoVs), as well as for escaping innate-immune responses of the host. Hence, it has emerged as an attractive antiviral drug-target. In this study, computational approaches were employed, mainly the structure-based virtual screening coupled with all-atom molecular dynamics (MD) simulations to computationally identify specific inhibitors of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) PLpro, which can be further developed as potential pan-PLpro based broad-spectrum antiviral drugs. The sequence, structure, and functional con-serveness of most deadly human CoVs PLpro were explored, and it was revealed that functionally important catalytic triad residues are well conserved among SARS-CoV, SARS-CoV-2, and middle east respiratory syndrome coronavirus (MERS-CoV). The subsequent screening of a focused protease in-hibitors database composed of ～7,000 compounds resulted in the identification of three candidate compounds, ADM_13083841, LMG_15521745, and SYN_15517940. These three compounds established conserved interactions which were further explored through MD simulations, free energy calculations, and residual energy contribution estimated by MM-PB(GB)SA method. All these compounds showed stable conformation and interacted well with the active residues of SARS-CoV-2 PLpro, and showed consistent interaction profile with SARS-CoV PLpro and MERS-CoV PLpro as well. Conclusively, the re-ported SARS-CoV-2 PLpro specific compounds could serve as seeds for developing potent pan-PLpro based broad-spectrum antiviral drugs against deadly human coronaviruses. Moreover, the presented infor-mation related to binding site residual energy contribution could lead to further optimization of these compounds.

Mutant P21 Peptides Could Act As An Improved Cyclin A Inhibitors For Cancer Therapy: An In Silico Validation

Objective: The present study delineates the generation of mutant peptide library from a known anticancer peptide, p21 and in silico evaluation for their affinity towards cyclin. A substrate binding groove. Methods: Mutant peptide library was created based on their AntiCP score and was docked with cyclin A using ClusPro2.0 web server. The docked structures were further simulated into an aqueous environment using Gromacs 4.5.6. Visualization was performed using PyMol software and interaction analysis was done using Discovery Studio Visualizer 4.1 Client and LigPlot plus tool. Results: A total of 57 mutant peptides were generated; out of which only 3 namely, K3C (Lys3Cys), K3F (Lys3Phe), and K3W (Lys3Trp) had a greater affinity for cyclin A than WILD p21 peptide (HSKRRLIFS). Molecular dynamic simulation studies showed that the peptides remained docked into the substrate binding groove throughout the run. Among all the peptides, K3C showed a significantly higher negative binding energy with cyclin A as compared to WILD. Conclusion: The overall results suggested that K3C mutant peptide had ~30 % higher affinity towards cyclin A and thus, could further be explored for its anticancer potential. The study also provides an insight into the crucial interactions governing the recognition of substrate binding groove of cyclin A for the development of novel peptide-based anticancer therapeutics.

Structural Study on K-Segment of Rice Dehydrin in Mimetic Membrane / 分析化学

Dehydrins are well associated with the abiotic stress tolerance of the plants, such as dehydration, salt stress and cold stress. They include a highly conserved lysine-rich motif called K-segment, which is believed to play a significant role in dehydrin function. The K-segment shows in vitro antibacterial activity against Gram-positive bacteria like its full-length dehydrin protein. In this study, the structures of the K-segment from rice dehydrin have been investigated by CD spectroscopy, NMR and molecular dynamic simulation. The results reveal that the K-segment is disordered in aqueous solution, but adopts helical structure in mimetic membrane environment, sodium dodecyl sulfate (SDS) micelles. The central region of K-segment forms an a-helix and exhibits amphipathic arrangement,where hydrophobic residues locate on one side and hydrophilic residues are on the other side. The amphipathic feature allows the helix region of the K-segment to insert into the SDS micelles, resulting in stable association with mimetic membrane. To realize the energy minimization,the hydrophobic side of the helix faces to the hydrophobic core of SDS micelles,and the hydrophilic side of the helix faces to the surface of micelles. The precise 3D structure and orientation information of the K-segment obtained in this work might provide new insights in understanding the structure-function relationship of dehydrins.

Investigation of the influence of mechanical signals on the structure of CD44/FERM complex via molecular dynamics simulation / 生物医学工程学杂志

The intracellular domain of clusters of differentiation 44 (CD44) binding to the FERM (protein 4.1-ezrin-radixin-moesin) domain of ERM (ezrin/radixin/moesin) proteins and furthermore triggering the recruitment of spleen tyrosine kinase (Syk) are very important in the process of tumor cell adhesion, migration and proliferation. At first, it was found that CD44/FERM structure was stable by observing CD44/FERM complex conformation and analyzing the interaction of interface residues both in static crystal structure and in equilibrium process. Meanwhile, unconventional immunoreceptor tyrosine-based activation motif (ITAM-like), and phosphorylation sites Y191 and Y205 were buried in FERM domain, which would hinder the phosphorylation of ERM proteins, the recruitment of Syk and subsequent signal transduction. Then, steered molecular dynamics simulation was applied to simulate the interaction between CD44 and FERM domain in the mechanical environment. The results showed that mechanical signal could induce the exposure of the ITAM-like motif and phosphorylation site Y205 by tracking and analyzing CD44/FERM complex conformational changes and the solvent-accessible surface area. This study revealed how the force regulates the activation of downstream signal through CD44 intracellular domain for the first time, and would be useful for further understanding the adhesion and migration pathway of cancer cells and the design of antitumor drugs.

Structure based virtual screening, docking and molecular dynamic simulation studies to identify potent mdm2-p53 inhibitors: Future implications for cancer therapy

Objective: To identify the p53 binding pocket as well as active residues in mdm2 protein, and search for similar (or) better compounds to inhibit mdm2-p53 interactions in comparison to FDA approved drug (Nutlin) by ligand and structure based virtual screening methods, docking, and molecular dynamic simulation studies. Methods: A structure and ligand based virtual screening for mdm2 protein; targeting the key residues involved in their active binding of p53 peptide was conducted after obtaining structurally suitable compounds similar to Nutlin from ZINC database. These compounds are virtually screened onto the mdm2 protein targeting its active binding site where p53 binds. The best compound with highest binding affinity was taken up for further analysis using molecular dynamic simulations for further validating the docking studies and to reveal interactions during the conformational changes Results: We discovered several compounds that are potentially able to block the interaction between active residues of mdm2 and p53 complex, suggesting their capability to act as anti-cancer agents. As proven by our structure based virtual screening studies coupled with semi-flexible and flexible docking studies, compound ZINC59256947 was found to be the strongest inhibitors for mdm2 protein amongst all of those isolates from ZINC database. Conclusion: Our results suggest that, a simple, selective and reliable inhibition assay can be performed to search for novel inhibitors of p53-mdm2 interaction. Therefore this study provides a rationalization to the ability of a ZINC59256947, an isolate from ZINC database with strong binding affinity towards mdm2 protein, for future implications as anti-cancer agent.

Analyzing resistance pattern of non-small cell lung cancer to crizotinib using molecular dynamic approaches.

Crizotinib is the potential anticancer drug used for the treatment of non-small cell lung cancer (NSCLC) approved by FDA in 2011. The main target for the crizotinib is anaplastic lymphoma kinase (ALK). Evidences available indicate that double mutant ALK (L1196M and G1269A) confers resistance to crizotinib. However, how mutation confers drug resistance is not well-understood. Hence, in the present study, molecular dynamic (MD) simulation approach was employed to study the impact of crizotinib binding efficacy with ALK structures at a molecular level. Docking results indicated that ALK double mutant (L1196M and G1269A) significantly affected the binding affinity for crizotinib. Furthermore, MD studies revealed that mutant ALK-crizotinib complex showed higher deviation, higher fluctuation and decreased number of intermolecular H-bonds, when compared to the native ALK-crizotinib complex. These results may be immense importance for the molecular level understanding of the crizotinib resistance pattern and also for designing potential drug molecule for the treatment of lung cancer.

Construction of an engineered alpha 1-antitrypsin with inhibitory activity based on theoretical studies

Background: The elastase inhibitor alpha-1-antitrypsin (AAT), is a member of the serpin superfamily of protease inhibitors. AAT has a characteristic secondary structure of three-beta-sheets, nine-alpha-helices and a reactive central loop (RCL). This protein inhibits target proteases by forming a stable complex in which the cleaved RCL is inserted into beta-sheet-A of the serpin, leading to a conformational change in the AAT protein. Spontaneous polymerization and instability of AAT are challenges with regard to producing drugs against AAT-deficient diseases. Therefore, the purpose of many investigations currently is to produce drugs with lower degrees of polymerization and higher stabilities. In order to investigate the effect of the N-terminal segment (residues 1-43) on AAT structure, molecular dynamic (MD) simulation was used to study structural properties including Root-mean-square deviation (RMSD), internal motions, intramolecular non-bonded interactions and the total accessible surface area (ASA) of native and reduced AAT. These properties were compared in native and truncated AAT. Results: Theoretical studies showed no noticeable differences in the dynamic and structural properties of the two structures. These findings provided the basis for the experimental phase of the study in which sequences from the two AAT constructs were inserted into the expression vector pGAPZ and transformed into Pichia pastoris. Results showed no differences in the activities and polymerization of the two AAT constructs. Conclusions: As small-scale medicines are preferred by lung drug delivery systems, in this study AAT was designed and constructed by decreasing the number of amino acids at the N-terminal region.

Taxifolin acts as type I inhibitor for VEGFR-2 kinase: Stability evaluation by molecular dynamic simulation.

The VEGFR-2 kinase specific intracellular signalling cascades leading to proliferation, migration, survival of endothelial cells and increased permeability of vessels which contributes to angiogenesis. ATP is essentially required by VEGFR-2 to perform phosphorylation of specific proteins and to maintain cascade downstream. Taxifolin (plant polyphenol) inhibit the VEGFR-2 kinase by binding at ATP-binding pocket revealed by molecular docking study. Further, stability of VEGFR-2 kinase-taxifolin complex is validated by molecular dynamic simulation. RMSD analysis for 3800 ps confirmed the stability of complex. Furthermore, thermodynamic stability was evidenced by stable total energy, potential energy, and, temperature and pressure profile. After MD simulation taxifolin was found to stably interact with pocket residues Cys 917 and Lys 1053 along with water molecules. These results suggest that therapeutic inhibition of VEGFR-2 by taxifolin as a type I inhibitor may be a promising ways to retard signaling cascade of specific proteins which play crucial role in cancer proliferation and also in development of second generation type II inhibitors.