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1.
Kotaro Sakamoto Yoko Kanematsu-Yamaki Yusuke Kamada Masahiro Oka Toshiyuki Ohnishi Masanori Miwa Taiji Asami Hiroshi Inooka 《Biochemistry and Biophysics Reports》2017
ActRIIB (activin receptor type-2B) is an activin receptor subtype constitutively expressed in the whole body, playing a role in cellular proliferation, differentiation, and metabolism. For its various physiological activities, ActRIIB interacts with activin and multiple other ligands including myostatin (MSTN), growth differentiation factor 11 (GDF11), and bone morphogenetic protein 9 (BMP9). Notably, the protein-protein interaction (PPI) between ActRIIB and MSTN negatively controls muscular development. Therefore, this PPI has been targeted for effective treatment of muscle degenerative diseases such as muscular dystrophy and sarcopenia. Here, we report the identification of ligand-selective peptidic ActRIIB-antagonists by phage display technology. Our peptides bound to the extracellular domain of ActRIIB, inhibited PPIs between ActRIIB expressed on the cell surface and its ligands, and subsequently suppressed activation of Smad that serves as the downstream signal of the ActRIIB pathway. Interestingly, these peptidic antagonists displayed different ligand selectivities; the AR2mini peptide inhibited multiple ligands (activin A, MSTN, GDF11, and BMP9), AR9 inhibited MSTN and GDF11, while AR8 selectively inhibited MSTN. This is the first report of artificial peptidic ActRIIB-antagonists possessing ligand-selectivity. 相似文献
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3.
Shanmugam A Suriano R Chaudhuri D Rajoria S George A Mittelman A Tiwari RK 《Peptides》2011,32(6):1097-1102
Prostate cancer (PCa) is one of the most common types of cancer in men in the United States and is the second leading cause of cancer related death in men. Clinically, secreted prostate specific antigen (PSA) has gained recognition because of its proteolytic activity being directly linked to PCa cell proliferation leading to disease initiation and progression. Using phage display technology, we identified four distinct cyclical peptides. These peptides apart from differences in their amino acid sequence, elicited minimal cross reactive antibody responses against each other. One of the four peptides analyzed produced an antibody response that recognizes the PSA protein. We demonstrate that the synthetic PSA peptide mimics identified in our study are immunologically active and produce neutralizing activity and this has relevance and utility for prostate cancer disease progression. 相似文献
4.
There is a constant need to identify novel inhibitors to combat β-lactamase-mediated antibiotic resistance. In this study, we identify three penicillinase-binding peptides, P1 (DHIHRSYRGEFD), P2 (NIYTTPWGSNWS), and P3 (SHSLPASADLRR), using a phage display library. Surface plasmon resonance (SPR) is utilized for quantitative determination and comparison of the binding specificity of selected peptides to penicillinase. An SPR biosensor functionalized with P3-GGGC (SHSLPASADLRRGGGC) is developed for detection of penicillinase with excellent sensitivity (15.8 RU nM−1) and binding affinity (KD = 0.56 nM). To determine if peptides can be good inhibitors for penicillinase, these peptides are mixed with penicillinase and their inhibition efficiency is determined by measuring the hydrolysis of substrate penicillin G using UV–vis spectrophotometry. Peptide P2 (NIYTTPWGSNWS) is found to be a promising penicillinase inhibitor with a Ki of 9.22 μM and a Ki′ of 33.12 μM, suggesting that the inhibition mechanism is a mixed pattern. This peptide inhibitor (P2) can be used as a lead compound to identify more potent small molecule inhibitors for penicillinase. This study offers a potential approach to both detection of β-lactamases and development of novel inhibitors of β-lactamases. 相似文献
5.
Phage display technology could provide a rapid means for the discovery of novel peptides. To find peptide ligands specific for the brain vascular receptors, we performed a modified phage display method. Phages were recovered from mice brain parenchyma after administrated with a random 7-mer peptide library intravenously. A longer circulation time was arranged according to the biodistributive brain/blood ratios of phage particles. Following sequential rounds of isolation, a number of phages were sequenced and a peptide sequence (CTSTSAPYC, denoted as PepC7) was identified. Clone 7-1, which encodes PepC7, exhibited translocation efficiency about 41-fold higher than the random library phage. Immunofluorescence analysis revealed that Clone 7-1 had a significant superiority on transport efficiency into the brain compared with native M13 phage. Clone 7-1 was inhibited from homing to the brain in a dose-dependent fashion when cyclic peptides of the same sequence were present in a competition assay. Interestingly, the linear peptide (ATSTSAPYA, Pep7) and a scrambled control peptide PepSC7 (CSPATSYTC) did not compete with the phage at the same tested concentration (0.2-200?pg). Labeled by Cy5.5, PepC7 exhibited significant brain-targeting capability in in vivo optical imaging analysis. The cyclic conformation of PepC7 formed by disulfide bond, and the correct structure itself play a critical role in maintaining the selectivity and affinity for the brain. In conclusion, PepC7 is a promising brain-target motif never been reported before and it could be applied to targeted drug delivery into the brain. 相似文献
6.
《Bioorganic & medicinal chemistry》2016,24(6):1384-1391
Mineralocorticoid receptor (MR) antagonists continue to be a prevalent area of research in the pharmaceutical industry. Herein we report the discovery of various spirooxindole and dibenzoxazepine constructs as potent MR antagonists. SAR analysis of our spirooxindole hit led to highly potent compounds containing polar solubilizing groups, which interact with the helix-11 region of the MR ligand binding domain (LBD). Various dibenzoxazepine moieties were also prepared in an effort to replace a known dibenzoxepane system which interacts with the hydrophobic region of the MR LBD. In addition, an X-ray crystal structure was obtained from a highly potent compound which was shown to exhibit both partial agonist and antagonist modes of action against MR. 相似文献
7.
RON (Recepteur d’Origine Nantais) tyrosine kinase receptor is a promising target for therapeutic intervention in cancer therapy. The aim of this work was identification of RON-binding peptides using phage display and computational modeling their mode of binding. A 12-mer peptide phage library was utilized to perform biopanning against RON. The RON-binding ability of the selected peptide-displaying phage and their possible binding sites were examined by ELISA. Binding modes and affinities were also predicted by docking and molecular dynamics (MD) simulation. The results of ELISA experiment showed that P6 peptide displaying phage has higher affinity for RON compared to others and its binding site is located out of ligand binding site. Docking and MD simulation results also indicated higher affinity of P6 to RON as well as its exosite-binding feature. Taken together, our data suggest a capacity for P6 peptide (FEHSLYKEMTHL) to be utilized as RON binding agent, and hence be used for various purposes, including design of drug delivery systems for transferring cytotoxic agents to RON-positive cancer cells, interfering with RON signaling, peptidomimetics design, and diagnostic imaging. 相似文献
8.
Identification of peptide substrates for human MMP-11 (stromelysin-3) using phage display 总被引:2,自引:0,他引:2
Pan W Arnone M Kendall M Grafstrom RH Seitz SP Wasserman ZR Albright CF 《The Journal of biological chemistry》2003,278(30):27820-27827
The MMP-11 proteinase, also known as stromelysin-3, probably plays an important role in human cancer because MMP-11 is frequently overexpressed in human tumors and MMP-11 levels affect tumorogenesis in mice. Unlike other MMPs, however, human MMP-11 does not cleave extracellular matrix proteins, such as collagen, laminin, fibronectin, and elastin. To help identify physiologic MMP-11 substrates, a phage display library was used to find peptide substrates for MMP-11. One class of peptides containing 26 members had the consensus sequence A(A/Q)(N/A) downward arrow (L/Y)(T/V/M/R)(R/K), where downward arrow denotes the cleavage site. This consensus sequence was similar to that for other MMPs, which also cleave peptides containing Ala in position 3, Ala in position 1, and Leu/Tyr in position 1', but differed from most other MMP substrates in that proline was rarely found in position 3 and Asn was frequently found in position 1. A second class of peptides containing four members had the consensus sequence G(G/A)E downward arrow LR. Although other MMPs also cleave peptides with these residues, other MMPs prefer proline at position 3 in this sequence. In vitro assays with MMP-11 and representative peptides from both classes yielded modest kcat/Km values relative to values found for other MMPs with their preferred peptide substrates. These reactions also showed that peptides with proline in position 3 were poor substrates for MMP-11. A structural basis for the lower kcat/Km values of human MMP-11, relative to other MMPs, and poor cleavage of position 3 proline substrates by MMP-11 is provided. Taken together, these findings explain why MMP-11 does not cleave most other MMP substrates and predict that MMP-11 has unique substrates that may contribute to human cancer. 相似文献
9.
Neel DA Brown ML Lander PA Grese TA Defauw JM Doti RA Fields T Kelley SA Smith S Zimmerman KM Steinberg MI Jadhav PK 《Bioorganic & medicinal chemistry letters》2005,15(10):2553-2557
Syntheses and SAR studies of 3,3-bisaryloxindole analogues provided potent mineralocorticoid receptor (MR) antagonists that were selective over other steroid nuclear hormone receptors. 相似文献
10.
Guttmann RP Day GA Wang X Bottiggi KA 《Biochemical and biophysical research communications》2005,333(4):1087-1092
Calpains are calcium- and thiol-dependent proteases that cleave a variety of intracellular substrates. Overactivation of the calpains has been implicated in a number of diseases and conditions such as ischemic stroke indicating a need for the development of calpain inhibitors. A major problem with current calpain inhibitors has been specific targeting to calpain. To identify highly specific calpain interacting peptides, we developed a peptide-phage library screening method based on the calcium-dependent conformation change associated with calpain activation. A phage-peptide library representing greater than 2 billion expressed 12-mers was incubated with calpain I in the presence of calcium. The calcium-dependent bound phage was then eluted by addition of EGTA. After four rounds of selection we found a conserved 5-mer sequence represented by LSEAL. Synthetic LSEAL inhibited tau-calpain interaction and in vitro proteolysis of tau- and alpha-synuclein by calpains. Deletion of the portion of the tau protein containing a homologous sequence to LSEAL resulted in decreased calpain-mediated tau degradation. These data suggest that these peptides may represent novel calpastatin mimetics. 相似文献
11.
Selection of CC chemokine receptor 5-binding peptide from a phage display peptide library 总被引:2,自引:0,他引:2
Wang FY Zhang TY Luo JX He GA Gu QL Xiao F 《Bioscience, biotechnology, and biochemistry》2006,70(9):2035-2041
Human CC chemokine receptor (CCR) 5 is a G protein-coupled receptor involved in a broad range of human diseases that mediates HIV-1 viral entry into cells. Certain small molecule receptor antagonists to CCR5 have been useful in therapy for these diseases. In this study, CCR5-expressing CHO cells (CHO/CCR5 cells) were used to select CCR5-binding peptides from a phage-displayed 12-mers peptide library. All of the 30 clones selected from the library showed specific binding to CHO/CCR5 cells by enzyme linked immunosorbent assay (ELISA). Seventeen out of the 30 clones shared the amino acid motif AFDWTFVPSLIL. The motif-containing phages and synthetic peptide AFDWTFVPSLIL blocked the binding of mAb 2D7 to CHO/CCR5 cells and competitively inhibited the ability of chemokine regulated on activation normal T cell expressed and secreted (RANTES) binding to CHO/CCR5 cells. Furthermore, the peptide AFDWTFVPSLIL also inhibited RANTES induced increase in the intracellular Ca2+ level in CHO/CCR5 cells. These results suggest that the peptide AFDWTFVPSLIL was specific for CCR5 and that it might become a CCR5 antagonist. 相似文献
12.
Kazusa Nishiyama Yoichi Takakusagi Tomoe Kusayanagi Yuki Matsumoto Shiori Habu Kouji Kuramochi Fumio Sugawara Kengo Sakaguchi Hideyo Takahashi Hideaki Natsugari Susumu Kobayashi 《Bioorganic & medicinal chemistry》2009,17(1):195-202
Here, we report on the identification of trimannoside-recognizing peptide sequences from a T7 phage display screen using a quartz-crystal microbalance (QCM) device. A trimannoside derivative that can form a self-assembled monolayer (SAM) was synthesized and used for immobilization on the gold electrode surface of a QCM sensor chip. After six sets of one-cycle affinity selection, T7 phage particles displaying PSVGLFTH (8-mer) and SVGLGLGFSTVNCF (14-mer) were found to be enriched at a rate of 17/44, 9/44, respectively, suggesting that these peptides specifically recognize trimannoside. Binding checks using the respective single T7 phage and synthetic peptide also confirmed the specific binding of these sequences to the trimannoside-SAM. Subsequent analysis revealed that these sequences correspond to part of the primary amino acid sequence found in many mannose- or hexose-related proteins. Taken together, these results demonstrate the effectiveness of our T7 phage display environment for affinity selection of binding peptides. We anticipate this screening result will also be extremely useful in the development of inhibitors or drug delivery systems targeting polysaccharides as well as further investigations into the function of carbohydrates in vivo. 相似文献
13.
Takakusagi Y Kuroiwa Y Sugawara F Sakaguchi K 《Bioorganic & medicinal chemistry》2008,16(15):7410-7414
An 11-mer unique peptide sequence SIFPLCNSGAL was identified as a methotrexate (MTX)-binding peptide from a T7 phage display screen using a quartz-crystal microbalance (QCM) biosensor. The synthetic peptide displayed weak interaction with MTX (K(D) 2.23x10(-5) M) using surface plasmon resonance (SPR). Interestingly, analysis of the primary amino acid sequence of the peptide identified similarities to the MTX-binding site of dihydrofolate reductase (DHFR). Our results highlight the importance of this primary sequence for the recognition of the MTX molecule. 相似文献
14.
Hydrolysis of beta-lactam antibiotics by beta-lactamase enzymes is the most common mechanism of bacterial resistance to these agents. Several small-molecule, mechanism-based inhibitors of beta-lactamases such as clavulanic acid are clinically available although resistance to these inhibitors has been increasing in bacterial populations. In addition, these inhibitors act only on class A beta-lactamases. Here we utilized phage display to identify peptides that bind to the class A beta-lactamase, TEM-1. The binding affinity of one of these peptides was further optimized by the synthesis of peptide arrays using SPOT synthesis technology. After two rounds of optimization, a linear 6-mer peptide with the sequence RRGHYY was obtained. A soluble version of this peptide was synthesized and found to inhibit TEM-1 beta-lactamase with a K(i) of 136 micro M. Surprisingly, the peptide inhibits the class A Bacillus anthracis Bla1 beta-lactamase with a K(i) of 42 micro M and the class C beta-lactamase, P99, with a K(i) of 140 micro M, despite the fact that it was not optimized to bind these enzymes. This peptide may be a useful starting point for the design of non-beta-lactam, broad-spectrum peptidomimetic inhibitors of beta-lactamases. 相似文献
15.
Rosander A Frykberg L Ausmees N Müller P 《Molecular plant-microbe interactions : MPMI》2003,16(8):727-737
A novel gene bank of Bradyrhizobium japonicum USDA110spc4 was constructed using pG3DSS, a phagemid vector designed for detecting genes encoding secreted proteins. In this phagemid, the phage protein III lacks its indigenous signal peptide required for protein secretion, thus recombinant fusion proteins are displayed on the phage surface only if a functional signal peptide is provided by an inserted DNA fragment. In addition, the N-terminal half of protein III has been replaced by a short linker region (the E-tag) that is recognized by a monoclonal antibody, which enables isolation of phages displaying a fusion protein. The expression library described here, therefore, provides a powerful means to affinity select for B. japonicum genes encoding extracytoplasmic proteins. In total, 182 DNA sequences were analyzed, among which 132 different putative extracytoplasmic proteins could be identified. The function of most proteins could be predicted and support an extracytoplasmic localization. In addition, genes encoding novel extracytoplasmic proteins were found. In particular, a novel family of small proteins has been identified that is characterized by a conserved pattern of four cysteine residues. 相似文献
16.
Identification of keratinocyte-specific markers using phage display and mass spectrometry 总被引:1,自引:0,他引:1
Jensen KB Jensen ON Ravn P Clark BF Kristensen P 《Molecular & cellular proteomics : MCP》2003,2(2):61-69
Specific molecular markers for various normal and pathogenic cell states and cell types provide knowledge of basic biological systems and have a direct application in targeted therapy. We describe a proteomic method based on the combination of new and improved phage display antibody technologies and mass spectrometry that allows identification of cell type-specific protein markers. The most important features of the method are (i) reduction of experimental noise originating from background binding of phage particles and (ii) isolation of affinity binders after a single round of selection, which assures a high diversity of binders. The method demonstrates, for the first time, the ability to detect, identify, and analyze both secreted and membrane-associated extracellular proteins as well as a variety of different cellular structures including proteins and carbohydrates. The optimized phage display method was applied to analysis of human skin keratinocytes resulting in the isolation of a panel of antibodies. Fourteen of these antibodies were further characterized, half of which predominantly recognized keratinocytes in a screen of a range of different cell types. Three cognate keratinocyte antigens were subsequently identified by mass spectrometry as laminin-5, plectin, and fibronectin. The combination of phage display technology with mass spectrometry methods for protein identification is a general and promising approach for proteomic analysis of cell surface complexity. 相似文献
17.
Chagnaud J. L. Moynet D. Londos-Gagliardi D. Bezian J. H. Vincendeau P. Fleury H. Guillemain B. 《International journal of peptide research and therapeutics》2001,8(2):95-106
Summary Phage peptide libraries constitute powerful tools for the mapping of epitopes recognized by monoclonal antibodies (mAbs).
Using screening of phage displayed random peptide libraries we have characterized the binding epitopes of three mAbs directed
against the surface envelope glycoprotein (gp46) of the human T-cell leukemia virus type I (HTLV-I). Two phage libraries,
displaying random heptapeptides with or without flanking cysteine residues, were screened for binding to mAbs 7G5D8, DB4 and
4F5F6. The SSSSTPL consensus sequence isolated from constrained heptapeptide library defines the epitope recognized by DB4
mAb and corresponds to the exact region 249–252 of the virus sequence. The APPMLPH consensus sequence isolated from non constrained
heptapeptide library defines the epitope recognized by 7G5D8 mAb and corresponds to the region 187–193 with a single amino
acid substitution, methionine to leucine at position 190. The third consensus sequence LYWPHD isolated from constrained heptapeptide
library defines the epitope recognized by 4F5F6 mAb. It corresponds to an epitope without direct equivalence with the virus
sequence. The data presented here showed that 7G5D8 and DB4 mAbs are raised against linear epitopes while 4F5F6 mAb recognized
a continoous topographic epitope. 相似文献
18.
J. L. Chagnaud D. Moynet D. Londos-Gagliardi J. H. Bezian P. Vincendeau H. Fleury B. Guillemain 《Letters in Peptide Science》2001,8(2):95-106
Phage peptide libraries constitute powerful tools for themapping of epitopes recognized by monoclonal antibodies (mAbs).Using screening of phage displayed random peptide libraries wehave characterized the binding epitopes of three mAbs directedagainst the surface envelope glycoprotein (gp46) of the humanT-cell leukemia virus type I (HTLV-I). Two phage libraries,displaying random heptapeptides with or without flankingcysteine residues, were screened for binding to mAbs 7G5D8, DB4and 4F5F6. The SSSSTPL consensus sequence isolated fromconstrained heptapeptide library defines the epitope recognizedby DB4 mAb and corresponds to the exact region 249–252 of thevirus sequence. The APPMLPH consensus sequence isolated fromnon constrained heptapeptide library defines the epitoperecognized by 7G5D8 mAb and corresponds to the region 187–193with a single amino acid substitution, methionine to leucine atposition 190. The third consensus sequence LYWPHD isolated fromconstrained heptapeptide library defines the epitope recognizedby 4F5F6 mAb. It corresponds to an epitope without directequivalence with the virus sequence. The data presented hereshowed that 7G5D8 and DB4 mAbs are raised against linearepitopes while 4F5F6 mAb recognized a continuous topographic epitope. 相似文献
19.
Phage display biopanning has been used for a number of applications including ligand generation for targeted drug delivery, targeting gene therapy vectors and identification of protein-protein interaction sites. In this study, a random phage display library was used to isolate peptide ligands to the endothelial protein C receptor (EPCR), identifying 74 different peptide sequences and several motifs. Binding to EPCR was characterized by a solid phase binding assay, demonstrating that 95% of isolated peptides were specific for EPCR. Several homologies with potential relevance to EPCR biology were identified, the most notable being leukolysin (MT-MMP6) and cerastocytin. 相似文献
20.
Isolation of high-affinity peptide antagonists of 14-3-3 proteins by phage display. 总被引:11,自引:0,他引:11
The 14-3-3 proteins interact with diverse cellular molecules involved in various signal transduction pathways controlling cell proliferation, transformation, and apoptosis. To aid our investigation of the biological function of 14-3-3 proteins, we have set out to identify high-affinity antagonists. By screening phage display libraries, we have identified a set of peptides which bind 14-3-3 proteins. One of these peptides, termed R18, exhibited a high affinity for different isoforms of 14-3-3 with estimated K(D) values of 7-9 x 10(-)(8) M. Recognition of multiple isoforms of 14-3-3 suggests the targeting of R18 to a structure that is common among 14-3-3 proteins, such as the conserved ligand-binding groove. Indeed, mutations that alter critical residues in the ligand-binding site of 14-3-3 drastically decreased the level of 14-3-3-R18 association. R18 efficiently blocked the binding of 14-3-3 to the kinase Raf-1, a physiological ligand of 14-3-3, and effectively abolished the protective role of 14-3-3 against phosphatase-induced inactivation of Raf-1. The cocrystal structure of R18 in complex with 14-3-3zeta revealed the occupancy of the general binding groove of 14-3-3zeta by R18, explaining the potent inhibitory effect of R18 on 14-3-3-ligand interactions. Such a well-defined peptide will be an effective tool for probing the role of 14-3-3 in various signaling pathways, and may lead to the development of 14-3-3 antagonists with pharmacological applications. 相似文献