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11.
Louisa V. Forbes Tove Sj?gren Fran?oise Auchère David W. Jenkins Bob Thong David Laughton Paul Hemsley Garry Pairaudeau Rufus Turner H?kan Eriksson John F. Unitt Anthony J. Kettle 《The Journal of biological chemistry》2013,288(51):36636-36647
The neutrophil enzyme myeloperoxidase (MPO) promotes oxidative stress in numerous inflammatory pathologies by producing hypohalous acids. Its inadvertent activity is a prime target for pharmacological control. Previously, salicylhydroxamic acid was reported to be a weak reversible inhibitor of MPO. We aimed to identify related hydroxamates that are good inhibitors of the enzyme. We report on three hydroxamates as the first potent reversible inhibitors of MPO. The chlorination activity of purified MPO was inhibited by 50% by a 5 nm concentration of a trifluoromethyl-substituted aromatic hydroxamate, HX1. The hydroxamates were specific for MPO in neutrophils and more potent toward MPO compared with a broad range of redox enzymes and alternative targets. Surface plasmon resonance measurements showed that the strength of binding of hydroxamates to MPO correlated with the degree of enzyme inhibition. The crystal structure of MPO-HX1 revealed that the inhibitor was bound within the active site cavity above the heme and blocked the substrate channel. HX1 was a mixed-type inhibitor of the halogenation activity of MPO with respect to both hydrogen peroxide and halide. Spectral analyses demonstrated that hydroxamates can act variably as substrates for MPO and convert the enzyme to a nitrosyl ferrous intermediate. This property was unrelated to their ability to inhibit MPO. We propose that aromatic hydroxamates bind tightly to the active site of MPO and prevent it from producing hypohalous acids. This mode of reversible inhibition has potential for blocking the activity of MPO and limiting oxidative stress during inflammation. 相似文献
12.
Kalyani Mondal Karin Regnstrom Winse Morishige Robin BarbourGary Probst Ying-Zi XuDean R. Artis Nanhua YaoPaul Beroza Michael P. Bova 《Biochemical and biophysical research communications》2013
Alzheimer’s disease (AD) is a devastating neurodegenerative disease affecting millions of people. β-Secretase-1 (BACE-1), an enzyme involved in the processing of the amyloid precursor protein (APP) to form Aβ, is a well validated target for AD. Herein, the authors characterize 10 randomly selected hydroxyethylamine (HEA) BACE-1 inhibitors in terms of their association and dissociation rate constants and thermodynamics of binding using surface plasmon resonance (SPR). Rate constants of association (ka) measured at 25 °C ranged from a low of 2.42 × 104 M−1 s−1 to the highest value of 8.3 × 105 M−1 s−1. Rate constants of dissociation (kd) ranged from 1.09 × 10−4 s−1 (corresponding to a residence time of close to three hours), to the fastest of 0.028 s−1. Three compounds were selected for further thermodynamic analysis where it was shown that equilibrium binding was enthalpy driven while unfavorable entropy of binding was observed. Structural analysis revealed that upon ligand binding, the BACE-1flap folds down over the bound ligand causing an induced fit. The maximal difference between alpha carbon positions in the open and closed conformations of the flap was over 5 Å. Thus the negative entropy of binding determined using SPR analysis was consistent with an induced fit observed by structural analysis. 相似文献
13.
Chandrasekhar Chanda Angshuman Sarkar Srinivas Sistla Dibakar Chakrabarty 《Biochemical and biophysical research communications》2013
A low molecular weight anti-platelet peptide (6.9 kDa) has been purified from Naja kaouthia venom and was named KT-6.9. MALDI-TOF/TOF mass spectrometry analysis revealed the homology of KT-6.9 peptide sequence with many three finger toxin family members. KT-6.9 inhibited human platelet aggregation process in a dose dependent manner. It has inhibited ADP, thrombin and arachidonic acid induced platelet aggregation process in dose dependent manner, but did not inhibit collagen and ristocetin induced platelet aggregation. Strong inhibition (70%) of the ADP induced platelet aggregation by KT-6.9 suggests competition with ADP for its receptors on platelet surface. Anti-platelet activity of KT-6.9 was found to be 25 times stronger than that of anti-platelet drug clopidogrel. Binding of KT-6.9 to platelet surface was confirmed by surface plasma resonance analysis using BIAcore X100. Binding was also observed by a modified sandwich ELISA method using anti-KT-6.9 antibodies. KT-6.9 is probably the first 3FTx from Indian monocled cobra venom reported as a platelet aggregation inhibitor. 相似文献
14.
Feng-Jung Chen Kuan-Wei Lee Chun-Chieh Lai Sue-Ping Lee Hsiao-Hsuian Shen Shu-Ping Tsai Bang-Hung Liu Ling-Mei Wang Gunn-Guang Liou 《Biochemical and biophysical research communications》2013
Receptor tyrosine kinases (RTKs) regulate many cellular processes, and Sprouty2 (Spry2) is known as an important regulator of RTK signaling pathways. Therefore, it is worth investigating the properties of Spry2 in more detail. In this study, we found that Spry2 is able to self-assemble into oligomers with a high-affinity KD value of approximately 16 nM, as determined through BIAcore surface plasmon resonance analysis. The three-dimensional (3D) structure of Spry2 was resolved using an electron microscopy (EM) single-particle reconstruction approach, which revealed that Spry2 is donut-shaped with two lip-cover domains. Furthermore, the method of energy dispersive spectrum obtained through EM was analyzed to determine the elements carried by Spry2, and the results demonstrated that Spry2 is a silicon- and iron-containing protein. The silicon may contribute to the electroconductivity of Spry2, and this property exhibits a concentration-dependent feature. This study provides the first report of a silicon- and iron-containing protein, and its 3D structure may allow us (1) to study the potential mechanism through the signal transduction is controlled by switching the electronic transfer on or off and (2) to develop a new type of conductor or even semiconductor using biological or half-biological hybrid materials in the future. 相似文献
15.
Zheng Zhang Sriram Jakkaraju Joy Blain Kenneth Gogol Lei Zhao Robert C. Hartley Courtney A. Karlsson Bart L. Staker Thomas E. Edwards Lance J. Stewart Peter J. Myler Michael Clare Darren W. Begley James R. Horn Timothy J. Hagen 《Bioorganic & medicinal chemistry letters》2013,23(24):6860-6863
Published biological data suggest that the methyl erythritol phosphate (MEP) pathway, a non-mevalonate isoprenoid biosynthetic pathway, is essential for certain bacteria and other infectious disease organisms. One highly conserved enzyme in the MEP pathway is 2C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (IspF). Fragment-bound complexes of IspF from Burkholderia pseudomallei were used to design and synthesize a series of molecules linking the cytidine moiety to different zinc pocket fragment binders. Testing by surface plasmon resonance (SPR) found one molecule in the series to possess binding affinity equal to that of cytidine diphosphate, despite lacking any metal-coordinating phosphate groups. Close inspection of the SPR data suggest different binding stoichiometries between IspF and test compounds. Crystallographic analysis shows important variations between the binding mode of one synthesized compound and the pose of the bound fragment from which it was designed. The binding modes of these molecules add to our structural knowledge base for IspF and suggest future refinements in this compound series. 相似文献
16.
Feng Liu Yalu Cui Fang Yang Zhijue Xu Lin-Tai Da Yan Zhang 《Bioorganic & medicinal chemistry》2019,27(15):3372-3382
Ellagitannin-derived ellagic acid (EA) and colonic metabolite urolithins are functional dietary ingredients for cancer prevention, but the underlying mechanism need elucidation. Mucin-type O-glycosylation, initiated by polypeptide N-acetyl-α-galactosaminyltransferases (ppGalNAc-Ts), fine-tunes multiple biological processes and is closely associated with cancer progression. Herein, we aim to explore how specific tannin-based polyphenols affect tumor behavior of colorectal cancer cells (CRC) by modulating O-glycosylation. Utilizing HPLC-based enzyme assay, we find urolithin D (UroD), EA and gallic acid (GA) potently inhibit ppGalNAc-Ts. In particular, UroD inhibits ppGalNAc-T2 through a peptide/protein-competitive manner with nanomolar affinity. Computational simulations combined with site-directed mutagenesis further support the inhibitors’ mode of action. Moreover, lectin analysis and metabolic labelling reveal that UroD can reduce cell O-glycans but not N-glycans. Transwell experiments prove that UroD inhibits migration and invasion of CRC cells. Our work proves that specific tannin-based polyphenols can potently inhibit ppGalNAc-Ts activity to reduce cell O-glycosylation and lead to lowering the migration and invasion of CRC cells, suggesting that disturbance of mucin-type O-glycosylation is an important mechanism for the function of dietary polyphenols. 相似文献
17.
I. N. Sokotun O. V. Gnedenko A. V. Leychenko M. M. Monastyrnaya E. P. Kozlovskaya A. A. Molnar A. S. Ivanov 《Biochemistry (Moscow) Supplemental Series B: Biomedical Chemistry》2007,1(2):139-142
The interaction of the inhibitor VJ (InhVJ), isolated from sea anemone R. macrodactylus, with different proteases was investigated using the method of biosensor analysis. The following enzymes were tested: serine proteases (trypsin, α-chymotrypsin, plasmin, thrombin, kallikrein), cysteina protease (papain) and aspartic protease (pepsin). In the rage of the concentrations studied (10–400 nM) inhibitor VJ interacted only with trypsin and α-chymotrypsin. The intermolecular complexes formation between inhibitor VJ and each of these enzymes was characterized by the following kinetic and thermodynamics parameters: KD = 7.38 × 10?8 M and 9.93 × 10?7 M for pairs InhVJ/trypsin and InhVJ/α-chymotrypsin, respectively. 相似文献
18.
《Journal of molecular biology》2019,431(24):4817-4833
Factor XI (FXI), the zymogen of activated FXI (FXIa), is an attractive target for novel anticoagulants because FXI inhibition offers the potential to reduce thrombosis risk while minimizing the risk of bleeding. BAY 1213790, a novel anti-FXIa antibody, was generated using phage display technology. Crystal structure analysis of the FXIa–BAY 1213790 complex demonstrated that the tyrosine-rich complementarity-determining region 3 loop of the heavy chain of BAY 1213790 penetrated deepest into the FXIa binding epitope, forming a network of favorable interactions including a direct hydrogen bond from Tyr102 to the Gln451 sidechain (2.9 Å). The newly discovered binding epitope caused a structural rearrangement of the FXIa active site, revealing a novel allosteric mechanism of FXIa inhibition by BAY 1213790. BAY 1213790 specifically inhibited FXIa with a binding affinity of 2.4 nM, and in human plasma, prolonged activated partial thromboplastin time and inhibited thrombin generation in a concentration-dependent manner. 相似文献
19.
Wangxiao He Pietro Mazzuca Weirong Yuan Kristen Varney Antonella Bugatti Alfredo Cagnotto Cinzia Giagulli Marco Rusnati Stefania Marsico Luisa Diomede Mario Salmona Arnaldo Caruso Wuyuan Lu Francesca Caccuri 《Biochimica et Biophysica Acta (BBA)/General Subjects》2019,1863(1):13-24
Background
HIV-1 matrix protein p17 variants (vp17s) detected in HIV-1-infected patients with non-Hodgkin's lymphoma (HIV-NHL) display, differently from the wild-type protein (refp17), B cell growth-promoting activity. Biophysical analysis revealed that vp17s are destabilized as compared to refp17, motivating us to explore structure-function relationships.Methods
We used: biophysical techniques (circular dichroism (CD), nuclear magnetic resonance (NMR) and thermal/GuHCL denaturation) to study protein conformation and stability; Surface plasmon resonance (SPR) to study interactions; Western blot to investigate signaling pathways; and Colony Formation and Soft Agar assays to study B cell proliferation and clonogenicity.Results
By forcing the formation of a disulfide bridge between Cys residues at positions 57 and 87 we obtained a destabilized p17 capable of promoting B cell proliferation. This finding prompted us to dissect refp17 to identify the functional epitope. A synthetic peptide (F1) spanning from amino acid (aa) 2 to 21 was found to activate Akt and promote B cell proliferation and clonogenicity. Three positively charged aa (Arg15, Lys18 and Arg20) proved critical for sustaining the proliferative activity of both F1 and HIV-NHL-derived vp17s. Lack of any interaction of F1 with the known refp17 receptors suggests an alternate one involved in cell proliferation.Conclusions
The molecular reasons for the proliferative activity of vp17s, compared to refp17, relies on the exposure of a functional epitope capable of activating Akt.General significance
Our findings pave the way for identifying the receptor(s) responsible for B cell proliferation and offer new opportunities to identify novel treatment strategies in combating HIV-related NHL. 相似文献20.
Kantham L Kerr-Bayles L Godde N Quick M Webb R Sunderland T Bond J Walder K Augert G Collier G 《Biochemical and biophysical research communications》2003,304(1):125-129
Previously we found elevated beacon gene expression in the hypothalamus of obese Psammomys obesus. Beacon administration into the lateral ventricle of P. obesus stimulated food intake and body weight gain. In the current study we used yeast two-hybrid technology to screen for proteins in the human brain that interact with beacon. CLK4, an isoform of cdc2/cdc28-like kinase family of proteins, was identified as a strong interacting partner for beacon. Using active recombinant proteins and a surface plasmon resonance based detection technique, we demonstrated that the three members of this subfamily of kinases (CLK1, 2, and 4) all interact with beacon. Based on the known sequence and functional properties of beacon and CLKs, we speculate that beacon could either modulate the function of key regulatory molecules such as PTP1B or control the expression patterns of specific genes involved in the central regulation of energy metabolism. 相似文献