Cardiovascular disease (CVD) is the leading cause of death and associates with multiple risk factors. Herb medicines have been used to treat CVD long ago in china and several natural products or derivatives (e.g., aspirin and reserpine) are most common drugs all over the world. The objective of this work was to construct a systematic database for drug discovery based on natural products separated from CVD-related medicinal herbs and to research on action mechanism of herb medicines.
The cardiovascular disease herbal database (CVDHD) was designed to be a comprehensive resource for virtual screening and drug discovery from natural products isolated from medicinal herbs for cardiovascular-related diseases. CVDHD comprises 35230 distinct molecules and their identification information (chemical name, CAS registry number, molecular formula, molecular weight, international chemical identifier (InChI) and SMILES), calculated molecular properties (AlogP, number of hydrogen bond acceptor and donors, etc.), docking results between all molecules and 2395 target proteins, cardiovascular-related diseases, pathways and clinical biomarkers. All 3D structures were optimized in the MMFF94 force field and can be freely accessed.
CVDHD integrated medicinal herbs, natural products, CVD-related target proteins, docking results, diseases and clinical biomarkers. By using the methods of virtual screening and network pharmacology, CVDHD will provide a platform to streamline drug/lead discovery from natural products and explore the action mechanism of medicinal herbs. CVDHD is freely available at http://pkuxxj.pku.edu.cn/CVDHD.
Cardiovascular disease; Drug discovery; Network pharmacology; Molecular docking; Virtual screening; Herbal formula; Natural products; Medicinal herbs; Traditional Chinese medicine
Thus far, methods that give quantitative information about lateral interactions in membranes have been restricted peptides or simplified protein constructs studied in detergents, lipid vesicles or bacterial membranes. None of the available methods have been extended to complex or full length membrane proteins. Here we show how free energies of membrane protein dimerization can be measured in mammalian plasma membrane-derived vesicles. The measurements, performed in single vesicles, utilize the Quantitative Imaging FRET (QI-FRET) method. The experiments are described in a step-by-step protocol. The protein characterized is the transmembrane domain of Glycophorin A, the most extensively studied membrane protein, known to form homodimers in hydrophobic environments. The results suggest that molecular crowding in cellular membranes has a dramatic effect on the strength of membrane protein interactions.
The use of plants as natural medicines in the treatment of type II diabetes mellitus (T2DM) has long been of special interest. In this work, we developed a docking score-weighted prediction model based on drug-target network to evaluate the efficacy of medicinal plants for T2DM. High throughput virtual screening from chemical library of natural products was adopted to calculate the binding affinity between natural products contained in medicinal plants and 33 T2DM-related proteins. The drug-target network was constructed according to the strength of the binding affinity if the molecular docking score satisfied the threshold. By linking the medicinal plant with T2DM through drug-target network, the model can predict the efficacy of natural products and medicinal plant for T2DM. Eighteen thousand nine hundred ninety-nine natural products and 1669 medicinal plants were predicted to be potentially bioactive.
High-intensity focused ultrasound (HIFU) is playing an increasingly important role in cancer therapy. Primary synovial sarcomas of the chest wall are extremely rare. We report the first case of noninvasive HIFU therapy for the control of synovial sarcoma. A 51-year-old man was diagnosed with spindle cell sarcoma on the left chest wall through lumpectomy. After four cycles of chemotherapy, local recurrence of the sarcoma was detected. Subsequent extended resection confirmed synovial sarcoma. After five cycles of a new chemotherapy option, the sarcoma relapsed again. Then the patient received five courses of HIFU; this completely ablated the sarcoma without complications. No chemotherapy, radiotherapy, or biological therapy has been applied since. Now the patient is stable and has a high quality of life.
Cancer therapy; High-intensity focused ultrasound; Noninvasive surgery; Synovial sarcoma
The crystal structure of the 11.14 kDa orphan ORF 1382 from Archaeoglobus fulgidus (AF1382) has been determined by sulfur SAD phasing using data collected from a moderately diffracting crystal and 1.9 Å synchrotron X-rays.
The crystal structure of the 11.14 kDa orphan ORF 1382 from Archaeoglobus fulgidus (AF1382) has been determined by sulfur SAD phasing using a moderately diffracting crystal and 1.9 Å wavelength synchrotron X-rays. AF1382 was selected as a structural genomics target by the Southeast Collaboratory for Structural Genomics (SECSG) since sequence analyses showed that it did not belong to the Pfam-A database and thus could represent a novel fold. The structure was determined by exploiting longer wavelength X-rays and data redundancy to increase the anomalous signal in the data. AF1382 is a 95-residue protein containing five S atoms associated with four methionine residues and a single cysteine residue that yields a calculated Bijvoet ratio (ΔF
anom/F) of 1.39% for 1.9 Å wavelength X-rays. Coupled with an average Bijvoet redundancy of 25 (two 360° data sets), this produced an excellent electron-density map that allowed 69 of the 95 residues to be automatically fitted. The S-SAD model was then manually completed and refined (R = 23.2%, R
free = 26.8%) to 2.3 Å resolution (PDB entry 3o3k). High-resolution data were subsequently collected from a better diffracting crystal using 0.97 Å wavelength synchrotron X-rays and the S-SAD model was refined (R = 17.9%, R
free = 21.4%) to 1.85 Å resolution (PDB entry 3ov8). AF1382 has a winged-helix–turn–helix structure common to many DNA-binding proteins and most closely resembles the N-terminal domain (residues 1–82) of the Rio2 kinase from A. fulgidus, which has been shown to bind DNA, and a number of MarR-family transcriptional regulators, suggesting a similar DNA-binding function for AF1382. The analysis also points out the advantage gained from carrying out data reduction and structure determination on-site while the crystal is still available for further data collection.
AF1382; orphan ORFs; sulfur SAD; Archaeoglobus fulgidus
The previously reported crystal structures of α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD) show a five-coordinate Zn(II)(His)3(Asp)(OH2) active site. The water ligand is H-bonded to a conserved His228 residue adjacent to the metal center in ACMSD from Pseudomonas fluorescences (PfACMSD). Site directed mutagenesis of His228 to tyrosine and glycine in the present study results in complete or significant loss of activity. Metal analysis shows that H228Y and H228G contain iron rather than zinc, indicating that this residue plays a role in metal selectivity of the protein. As-isolated H228Y displays a blue color, which is not seen in wild-type ACMSD. Quinone staining and resonance Raman analyses indicate that the blue color originates from Fe(III)-tyrosinate ligand-to-metal-charge- transfer (LMCT). Co(II)-substituted H228Y ACMSD is brown in color and exhibits an EPR spectrum showing a high-spin Co(II) center with a well-resolved 59Co (I = 7/2) eight-line hyperfine splitting pattern. The X-ray crystal structures of the as-isolated Fe-H228Y (2.8 Å), Co- (2.4 Å) and Znsubstituted H228Y (2.0 Å resolution) support the spectroscopic assignment of metal ligation of the Tyr228 residue. The crystal structure of Zn-H228G (2.6 Å) was also solved. These four structures show that the water ligand present in WT Zn-ACMSD is either missing (Fe-H228Y, Co-H228Y, and Zn- H228G) or disrupted (Zn-H228Y) in response to His228 mutation. Together, these results highlight the importance of His228 for PfACMSD’s metal specificity as well as maintaining a water molecule as ligand of the metal center. His228 is thus proposed to play a role in activating the metal-bound water ligand for subsequent nucleophilic attack on the substrate.
Natural products have been an important source of lead compounds for drug discovery. How to find and evaluate bioactive natural products is critical to the achievement of drug/lead discovery from natural products.
We collected 19,7201 natural products structures, reported biological activities and virtual screening results. Principal component analysis was employed to explore the chemical space, and we found that there was a large portion of overlap between natural products and FDA-approved drugs in the chemical space, which indicated that natural products had large quantity of potential lead compounds. We also explored the network properties of natural product-target networks and found that polypharmacology was greatly enriched to those compounds with large degree and high betweenness centrality. In order to make up for a lack of experimental data, high throughput virtual screening was employed. All natural products were docked to 332 target proteins of FDA-approved drugs. The most potential natural products for drug discovery and their indications were predicted based on a docking score-weighted prediction model.
Analysis of molecular descriptors, distribution in chemical space and biological activities of natural products was conducted in this article. Natural products have vast chemical diversity, good drug-like properties and can interact with multiple cellular target proteins.
This study aims to address hypothesis that the high mobility group A2 (HMGA2), an oncofetal protein, relates to survivability and serves as a prognostic biomarker for colorectal cancer (CRC).
This is a retro-prospective multiple center study. The HMGA2 expression level was determined by performing immunohistochemistry (IHC) on surgical tissue samples of 89 CRCs from a training set and 191 CRCs from a validation set. The Kaplan-Meier analysis and COX proportional hazard model were employed to analyze survivability.
Multivariate logistic analysis indicated that the expression of HMGA2 significantly correlates with distant metastasis in training set (odd ratio, OR=3.53, 95% CI 1.37-9.70) and validation set (OR=6.38, 95% CI 1.47-43.95). Survival analysis revealed that the overexpression of HMGA2 is significantly associated with poor survival of CRC patients (p < 0.05). The adjusted hazard ratios (HRs) for overall survival were 2.38 (95% CI 1.30-4.34) and 2.14 (95% CI 1.21-3.79) in training and validation sets, respectively. Further investigation revealed that HMGA2 delays the clearance of γ-H2AX in HCT-116 and SW480 cells post γ-irradiation, which supports our finding that CRC patients with HMAG2 positive staining in primary tumors had augmented efficacy of adjuvant radiotherapy (HR=0.18, 95% CI 0.04-0.63).
Overexpression of HMGA2 is associated with metastasis and unequivocally occurred in parallel with reduced survival rates of patients with CRC. Therefore, HMGA2 may potentially serve as a biomarker for predicting aggressive CRC with poor survivability and as an indicator for better response of radiotherapy.
high mobility group A2 protein; colorectum; adenocarcinoma; survival; metastasis
Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCRαβ heterodimer that associates non-covalently with CD3 dimers (the εδ and εγ heterodimers and the ζζ homodimer)1. Upon engagement of the receptor, the CD3 ζ chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2.
The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways.
One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCRβ, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.
Immunology; Issue 61; Gene delivery; primary cells; T cells; fluorescence microscopy; total internal reflection fluorescence; T cell receptor; signaling
Theoretical analysis and experimental validation prove that a multi-dataset data-collection strategy produces better diffraction data. The readiness test is a simple and sensitive method for X-ray data-collection system evaluation and a benchmark.
A multi-dataset (MDS) data-collection strategy is proposed and analyzed for macromolecular crystal diffraction data acquisition. The theoretical analysis indicated that the MDS strategy can reduce the standard deviation (background noise) of diffraction data compared with the commonly used single-dataset strategy for a fixed X-ray dose. In order to validate the hypothesis experimentally, a data-quality evaluation process, termed a readiness test of the X-ray data-collection system, was developed. The anomalous signals of sulfur atoms in zinc-free insulin crystals were used as the probe to differentiate the quality of data collected using different data-collection strategies. The data-collection results using home-laboratory-based rotating-anode X-ray and synchrotron X-ray systems indicate that the diffraction data collected with the MDS strategy contain more accurate anomalous signals from sulfur atoms than the data collected with a regular data-collection strategy. In addition, the MDS strategy offered more advantages with respect to radiation-damage-sensitive crystals and better usage of rotating-anode as well as synchrotron X-rays.
multi-dataset data-collection strategy; readiness test
A-kinase anchoring proteins (AKAPs) regulate cyclic AMP-dependent protein kinase (PKA) signaling in space and time. Dual-specific AKAP 2 (D-AKAP2) binds to the dimerization/docking (D/D) domain of both RI and RII regulatory subunits of PKA with high affinity. Here, we have determined the structures of the RIα D/D domain alone and in complex with D-AKAP2. The D/D domain presents an extensive surface for binding through a well-formed N-termina helix and this surface restricts the diversity of AKAPs that can interact. The structures also underscore the importance of a redox-sensitive disulfide in affecting AKAP binding. An unexpected shift in the helical register of D-AKAP2 compared to the RIIα:D-AKAP2 complex structure makes the mode of binding to RIα novel. Finally, the comparison allows us to deduce a molecular explanation for the sequence and spatial determinants of AKAP specificity.
Traditional virtual screening method pays more attention on predicted binding affinity between drug molecule and target related to a certain disease instead of phenotypic data of drug molecule against disease system, as is often less effective on discovery of the drug which is used to treat many types of complex diseases. Virtual screening against a complex disease by general network estimation has become feasible with the development of network biology and system biology. More effective methods of computational estimation for the whole efficacy of a compound in a complex disease system are needed, given the distinct weightiness of the different target in a biological process and the standpoint that partial inhibition of several targets can be more efficient than the complete inhibition of a single target.
We developed a novel approach by integrating the affinity predictions from multi-target docking studies with biological network efficiency analysis to estimate the anticoagulant activities of compounds. From results of network efficiency calculation for human clotting cascade, factor Xa and thrombin were identified as the two most fragile enzymes, while the catalytic reaction mediated by complex IXa:VIIIa and the formation of the complex VIIIa:IXa were recognized as the two most fragile biological matter in the human clotting cascade system. Furthermore, the method which combined network efficiency with molecular docking scores was applied to estimate the anticoagulant activities of a serial of argatroban intermediates and eight natural products respectively. The better correlation (r = 0.671) between the experimental data and the decrease of the network deficiency suggests that the approach could be a promising computational systems biology tool to aid identification of anticoagulant activities of compounds in drug discovery.
This article proposes a network-based multi-target computational estimation method for anticoagulant activities of compounds by combining network efficiency analysis with scoring function from molecular docking.
Dysregulation of ligand-independent receptor tyrosine kinase (RTK) dimerization, which is the first step in RTK activation, leads to pathologies. A mechanistic understanding of the dimerization process is lacking, and this lack of basic knowledge is one bottleneck in developing effective RTK-targeted therapies. For instance, the roles and the relative contributions of the different RTK domains to RTK dimerization are unknown. Here we use quantitative imaging Förster resonance energy transfer (QI-FRET) to determine the contribution of the extracellular (EC) domain of fibroblast growth factor receptor 3 (FGFR3) to FGFR3 dimerization. We provide the first direct experimental evidence that the contribution of FGFR3 EC domain to dimerization is repulsive in the absence of ligand, and on the order of 1 kcal/mole. The magnitude of this repulsive contribution is similar to the dimer over-stabilization that can occur due to pathogenic single amino acid mutations, and therefore significant for biological function.
Malignant peripheral nerve sheath tumors are rare tumor entities that originate from peripheral nerve sheaths and have an unfavorable prognosis. Common sites include deeper soft tissues, usually in the proximity of a nerve trunk. Breast is an absolutely rare location of this lesion, and presentation as a breast lump in the male breast is even rarer.
A 65-year-old man presented with a 6-month history of a painless mass of the left breast. Tissue biopsy was performed. Histopathology revealed a malignant spindle cell tumor which was confirmed to be a melanocytic malignant peripheral nerve sheath tumor on the basis of immunopositivity for HMB45 and S-100.
There are no generally accepted guidelines for the treatment of malignant peripheral nerve sheath tumors in the male breast. The patient was referred for radiation therapy after simple mastectomy.
Malignant peripheral nerve sheath tumor; Breast; Melanocytic
The crystal structure of the minimized α/β-hydrolase fold protein encoded by the gene TTHA1544 from T. thermophilus HB8 has been determined at 2.0 Å resolution.
The gene encoding TTHA1544 is a singleton found in the Thermus thermophilus HB8 genome and encodes a 131-amino-acid protein. The crystal structure of TTHA1544 has been determined at 2.0 Å resolution by the single-wavelength anomalous dispersion method in order to elucidate its function. There are two molecules in the asymmetric unit. Each molecule consists of four α-helices and six β-strands, with the β-strands composing a central β-sheet. A structural homology search revealed that the overall structure of TTHA1544 resembles the α/β-hydrolase fold, although TTHA1544 lacks the catalytic residues of a hydrolase. These results suggest that TTHA1544 represents the minimized α/β-hydrolase fold and that an additional component would be required for its activity.
T. thermophilus HB8; TTHA1544; α/β-hydrolase fold; singleton
The energetics of transmembrane (TM) helix dimerization in membranes, and the thermodynamic principles behind receptor tyrosine kinase (RTK) TM domain interactions during signal transduction, can be studied using Forster resonance energy transfer (FRET). For instance, FRET studies have yielded the stabilities of wild-type fibroblast growth factor receptor 3 (FGFR3) TM domains and two FGFR3 pathogenic mutants, Ala391Glu and Gly380Arg, in the native bilayer environment. To further our understanding of the molecular mechanisms of deregulated FGFR3 signaling underlying different pathologies, here we determine the effect of the Gly382Asp FGFR3 mutation, identified in a multiple myeloma cell line, on the energetics of FGFR3 TM domain dimerization. We measure dimerization energetics using a novel FRET acquisition and processing method, termed “excitation-emission FRET (EE-FRET)”, which improves the accuracy of thermodynamic measurements of TM helix association. The EE-FRET method, verified here by analyzing previously published data for wild-type FGFR3 TM domain, should have broad utility in studies of protein interactions, particularly in cases when fluorophore concentrations cannot be controlled.
The crystal structure of the hypothetical protein PF0899 from P. furiosus has been determined to 1.85 Å resolution.
The hypothetical protein PF0899 is a 95-residue peptide from the hyperthermophilic archaeon Pyrococcus furiosus that represents a gene family with six members. P. furiosus ORF PF0899 has been cloned, expressed and crystallized and its structure has been determined by the Southeast Collaboratory for Structural Genomics (http://www.secsg.org). The structure was solved using the SCA2Structure pipeline from multiple data sets and has been refined to 1.85 Å against the highest resolution data set collected (a presumed gold derivative), with a crystallographic R factor of 21.0% and R
free of 24.0%. The refined structure shows some structural similarity to a wedge-shaped domain observed in the structure of the major capsid protein from bacteriophage HK97, suggesting that PF0899 may be a structural protein.
structural genomics; SECSG; Pfu-871755; PF0899; high-throughput structure
The molybdenum-cofactor biosynthesis protein C from T. thermophilus has been crystallized in two different space groups, P21 and R32; the crystals diffracted to 1.9 and 1.75 Å resolution, respectively.
The Gram-negative aerobic eubacterium Thermus thermophilus is an extremely important thermophilic microorganism that was originally isolated from a thermal vent environment in Japan. The molybdenum cofactor in this organism is considered to be an essential component required by enzymes that catalyze diverse key reactions in the global metabolism of carbon, nitrogen and sulfur. The molybdenum-cofactor biosynthesis protein C derived from T. thermophilus was crystallized in two different space groups. Crystals obtained using the first crystallization condition belong to the monoclinic space group P21, with unit-cell parameters a = 64.81, b = 109.84, c = 115.19 Å, β = 104.9°; the crystal diffracted to a resolution of 1.9 Å. The other crystal form belonged to space group R32, with unit-cell parameters a = b = 106.57, c = 59.25 Å, and diffracted to 1.75 Å resolution. Preliminary calculations reveal that the asymmetric unit contains 12 monomers and one monomer for the crystals belonging to space group P21 and R32, respectively.
thermophilic microorganisms; Thermus thermophilus; molybdenum-cofactor biosynthesis; MoaC
Strongly diffracting crystals of a methanol-induced corrinoid protein from M. thermoacetica have been obtained.
A corrinoid protein was induced and overexpressed in methanol-grown cells of the thermophilic anaerobic bacterium Moorella thermoacetica. The protein was purified from cytosolic extracts. After screening for crystallization conditions and optimization, crystals were obtained that diffracted strongly on a rotating-anode X-ray source. A diffraction data set was collected and processed including reflections to 1.9 Å resolution. Reflections were indexed in a primitive orthorhombic cell with unit-cell parameters a = 55.69, b = 62.74, c = 34.54 Å. N-terminal amino-acid sequencing indicates that the crystals contain a C-terminal fragment of the protein.
Severe acute respiratory syndrome coronavirus (SARS-CoV) is the pathogen of SARS, which caused a global panic in 2003. We describe here the screening of Chinese herbal medicine-based, novel small molecules that bind avidly with the surface spike protein of SARS-CoV and thus can interfere with the entry of the virus to its host cells. We achieved this by using a two-step screening method consisting of frontal affinity chromatography-mass spectrometry coupled with a viral infection assay based on a human immunodeficiency virus (HIV)-luc/SARS pseudotyped virus. Two small molecules, tetra-O-galloyl-β-d-glucose (TGG) and luteolin, were identified, whose anti-SARS-CoV activities were confirmed by using a wild-type SARS-CoV infection system. TGG exhibits prominent anti-SARS-CoV activity with a 50% effective concentration of 4.5 μM and a selective index of 240.0. The two-step screening method described here yielded several small molecules that can be used for developing new classes of anti-SARS-CoV drugs and is potentially useful for the high-throughput screening of drugs inhibiting the entry of HIV, hepatitis C virus, and other insidious viruses into their host cells.