以重组TACE胞外功能域从噬菌体随机肽库中筛选TACE的抑制肽

肿瘤坏死因子转换酶 (TACE)是加工裂解TNF-α前体的关键酶 ,参与了许多炎症的发生发展过程。为通过肽库筛选得到TACE的抑制肽 ,首先制备筛选靶分子 ,用RT PCR从人外周血单核细胞中分别扩增出TACE的催化区 (T800 )和整个胞外区 (T1300 ) ,然后分别克隆至pET-28a和pET-28c中 ,转化大肠杆菌BL2-1(DE3) ,经IPTG诱导表达出带有His-tag的目的蛋白 ,两者均为包涵体 ,变性复性后过Ni²⁺-NTA亲和层析柱得到纯度达90%的重组蛋白。以纯化的重组T800和T1300分别筛选噬菌体展示随机 15肽库 ,对筛选克隆进行ELISA检测、竞争抑制实验和序列分析。从两个独立的筛选过程中得到一个相同的阳性克隆序列“TRWLVYFSRPYLVAT” ,固相Fmoc法合成该短肽 ,观察其在LPS诱导人单核细胞产生sTNF-α中的作用。结果表明 ,筛选到的短肽可显著抑制TACE的活性 ,减少TNF-α的分泌 ,抑制率可达 60.3% ,为抗炎小分子药物的设计和改造提供线索和依据。     

Implication of TNF-alpha convertase (TACE/ADAM17) in inducible nitric oxide synthase expression and inflammation in an experimental model of colitis

Tumour necrosis factor-alpha (TNF-alpha) is a pro-inflammatory cytokine which is shed in its soluble form by a disintegrin and metalloproteinase (ADAM) called TNF-alpha convertase (TACE; ADAM17). TNF-alpha plays a role in inflammatory bowel disease (IBD) and is involved in the expression of inducible nitric oxide synthase (iNOS) which has also been implicated in IBD. The study was designed to investigate whether colitis induced by trinitrobenzene sulphonic acid (TNBS) in rats produces an increase in TACE activity and/or expression and whether its pharmacological inhibition reduces TNF-alpha levels, iNOS expression and colonic damage in this model. TNBS (30 mg in 0.4 ml of 50% ethanol) was instilled into the colon of female Wistar rats. Saline or TACE inhibitor BB1101 (10 mg/kg/day) was administered intraperitoneally 5 days after TNBS instillation. On day 10, colons were removed and assessed for pathological score, myeloperoxidase (MPO), NO synthase (NOS), TACE enzymatic activity and protein levels, colonic TNF-alpha and NOx- levels. Instillation of TNBS caused an increase in TACE activity and expression and the release of TNF-alpha. TNBS also resulted in iNOS expression and colonic damage. BB1101 blocked TNBS-induced increase in TACE activity, TNF-alpha release and iNOS expression. Concomitantly, BB1101 ameliorated TNBS-induced colonic damage and inflammation. TNBS causes TNF-alpha release by an increase in TACE activity and expression and this results in the expression of iNOS and subsequent inflammation, suggesting that TACE inhibition may prove useful as a therapeutic means in IBD.     

Processing of tumor necrosis factor by the membrane-bound TNF-alpha-converting enzyme, but not its truncated soluble form.

Tumour necrosis factor (TNF)-alpha-converting enzyme (TACE) is a membrane protein belonging to the ADAM (a disintegrin and metalloproteinase) family that cleaves various membrane proteins, including the proform of TNF-alpha. In this study, we constructed expression vectors for the membrane-bound full-length TACE (mTACE) and its truncated soluble form (sTACE). When a human TNF-alpha expression vector was introduced into human 293 cells, processing of TNF-alpha to its mature form was enhanced by coexpressing mTACE, and this processing was inhibited by a metalloproteinase inhibitor. On the other hand, coexpression of sTACE had no effect on the processing of TNF-alpha, although the culture medium of sTACE-transfected cells could cleave a peptide containing the TNF-alpha cleavage site. Fas ligand (FasL)-transfected 293 cells released a considerable amount of soluble FasL, and coexpression of neither mTACE nor sTACE enhanced this shedding. Immunoprecipitation and Western blotting analysis with cells that were cotransfected with TACE and TNF-alpha indicated that both mTACE and sTACE could interact with the proform of TNF-alpha. In the same assay, neither mTACE nor sTACE interacted with FasL. The catalytic domain-lacking TACE mutant, which could also interact TNF-alpha, showed a dominant negative effect on not only TNF-alpha secretion but also FasL secretion. These results suggest that binding of the membrane-anchored but not the soluble form of TACE to TNF-alpha results in efficient ectodomain shedding, and that FasL secretase is a metalloproteinase similar, but not identical, to TACE.     

噬菌体表面展示技术

噬菌体展示技术(PhageDisplayTechniques,PDT)是一种用于筛选和改造功能性多肽的生物技术。自问世以来,它已被广泛应用于生命科学的许多领域。对噬菌体展示技术的基本原理、噬菌体表面展示系统研究以及噬菌体展示技术的应用、展望等进行了探讨,由此可以看出:噬菌体展示技术的进一步发展,必将为生命科学及相关学科的发展带来深远的影响。     

A widely applicable, high-throughput TR-FRET assay for the measurement of kinase autophosphorylation: VEGFR-2 as a prototype

Homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET) assays represent a highly sensitive and robust high-throughput screening (HTS) method for the quantification of kinase activity. Traditional TR-FRET kinase assays detect the phosphorylation of an exogenous substrate. The authors describe the development and optimization of a TR-FRET technique that measures the autophosphorylation of vascular endothelial growth factor receptor 2 (VEGFR-2) kinase and extend its applicability to a variety of other kinases. The VEGFR-2 assay demonstrated dose-dependent inhibition by compounds known to modulate the catalytic activity of this receptor. In addition, kinetic analysis of a previously characterized VEGFR-2 inhibitor was performed using the method, and results were consistent with those obtained using a different assay format. Because of the known involvement of VEGFR-2 in angiogenesis, this assay should facilitate HTS for antiangiogenic agents. In addition, this general technique should have utility for the screening for inhibitors of kinases as potential therapeutic agents for many other disease indications.     

A fluorescence-based assay for fatty acid amide hydrolase compatible with high-throughput screening

A novel fluorescent assay to continuously monitor fatty acid amide hydrolase (FAAH) activity that is simple, sensitive, and amenable to high-throughput screening (HTS) of compound libraries is described in this article. Stable Chinese hamster ovary (CHO) cell lines expressing either human FAAH or an inactive mutant, FAAH-S241A, were established. Arachidonyl 7-amino, 4-methyl coumarin amide (AAMCA), a novel fluorogenic substrate for FAAH, was designed and synthesized. FAAH catalyzes the hydrolysis of AAMCA to generate arachidonic acid and a highly fluorescent 7-amino, 4-methyl coumarin (AMC). The assay was done at 25 degrees C by incubating whole cell or microsomal preparations from FAAH-expressing cells with AAMCA. Release of AMC was monitored continuously using a fluorometer. Microsomal FAAH catalyzed the hydrolysis of AAMCA with an apparent K(m) of 0.48muM and V(max) of 58pmolmin(-1)mgprotein(-1). The assay is specific for FAAH given that microsomes prepared from cells expressing FAAH-S241A or vector alone had no significant activity against AAMCA. Furthermore, the activity was inhibited by URB-597, an FAAH-specific inhibitor, in a concentration-dependent manner with an IC(50) of 33.5nM. The assay was optimized for HTS and had a Z' value ranging from 0.7 to 0.9. The assay is also compatible with ex vivo analysis of FAAH activity.     

Reactive oxygen species mediate tumor necrosis factor alpha-converting, enzyme-dependent ectodomain shedding induced by phorbol myristate acetate.

Ectodomain shedding of cell surface membrane-anchoring proteins is an important process in a wide variety of physiological events(1, 2). Tumor necrosis factor alpha (TNF-alpha) converting enzyme (TACE) is the first discovered mammalian sheddase responsible for cleavage of several important surface proteins, including TNF-alpha, TNF p75 receptor, L-selectin, and transforming growth factor-a. Phorbol myristate acetate (PMA) has long been known as a potent agent to enhance ectodomain shedding. However, it is not fully understood how PMA activates TACE and induces ectodomain shedding. Here, we demonstrate that PMA induces both reactive oxygen species (ROS) generation and TNF p75 receptor shedding in Mono Mac 6 cells, a human monocytic cell line, and l-selectin shedding in Jurkat T-cells. ROS scavengers significantly attenuated PMA-induced TNF p75 receptor shedding. Exogenous H2O2 mimicked PMA-induced enhancement of ectodomain shedding, and H2O2-induced shedding was blocked by TAPI, a TACE inhibitor. Furthermore, both PMA and H2O2 failed to cause ectodomain shedding in a cell line that lacks TACE activity. By use of an in vitro TACE cleavage assay, H2O2 activated TACE that had been rendered inactive by the addition of the TACE inhibitory pro-domain sequence. We presume that the mechanism of TACE activation by H2O2 is due to an oxidative attack of the pro-domain thiol group and disruption of its inhibitory coordination with the Zn++ in the catalytic domain of TACE. These results demonstrate that ROS production is involved in PMA-induced ectodomain shedding and implicate a role for ROS in other shedding processes.     

Phage antibody display libraries: a powerful antibody discovery platform for immunotherapy

Phage display technology (PDT), a combinatorial screening approach, provides a molecular diversity tool for creating libraries of peptides/proteins and discovery of new recombinant therapeutics. Expression of proteins such as monoclonal antibodies (mAbs) on the surface of filamentous phage can permit the selection of high affinity and specificity therapeutic mAbs against virtually any target antigen. Using a number of diverse selection platforms (e.g. solid phase, solution phase, whole cell and in vivo biopannings), phage antibody libraries (PALs) from the start point provides great potential for the isolation of functional mAb fragments with diagnostic and/or therapeutic purposes. Given the pivotal role of PDT in the discovery of novel therapeutic/diagnostic mAbs, in the current review, we provide an overview on PALs and discuss their impact in the advancement of engineered mAbs.     

从噬菌体文库中筛选潜在的GMCSF的拮抗剂(英文)

以粒细胞巨噬细胞集落刺激因子（ＧＭＣＳＦ） 为筛选文库的靶分子, 通过高效筛选（Ｈｉｇｈ ｔｈｒｏｕｇｈｐｕｔｓｃｒｅｅｎｉｎｇ, ＨＴＳ） 方法来筛选多种多肽噬菌体文库, 在一个以噬菌体主要蛋白质为载体的多肽噬菌体文库中筛选到了一些与ＧＭＣＳＦ结合的多肽, 并通过了ＥＬＩＳＡ和微淘选（ｍｉｃｒｏｐａｎｎｉｎｇ） 实验的证实。这些多肽先导化合物经过进一步的优化, 可能成为ＧＭＣＳＦ细胞因子的拮抗剂     

Reactive site mutations in tissue inhibitor of metalloproteinase-3 disrupt inhibition of matrix metalloproteinases but not tumor necrosis factor-alpha-converting enzyme

Tissue inhibitor of metalloproteinase-3 (TIMP-3) is a dual inhibitor of the matrix metalloproteinases (MMPs) and some adamalysins, two families of extracellular and cell surface metalloproteinases that function in extracellular matrix turnover and the shedding of cell surface proteins. The mechanism of inhibition of MMPs by TIMPs has been well characterized, and since the catalytic domains of MMPs and adamalysins are homologous, it was assumed that the interaction of TIMP-3 with adamalysins is closely similar. Here we report that the inhibition of the extracellular region of ADAM-17 (tumor necrosis factor alpha-converting enzyme (TACE)) by the inhibitory domain of TIMP-3 (N-TIMP-3) shows positive cooperativity. Also, mutations in the core of the MMP interaction surface of N-TIMP-3 dramatically reduce the binding affinity for MMPs but have little effect on the inhibitory activity for TACE. These results suggest that the mechanism of inhibition of ADAM-17 by TIMP-3 may be distinct from that for MMPs. The mutant proteins are also effective inhibitors of tumor necrosis factor alpha (TNF-alpha) release from phorbol ester-stimulated cells, indicating that they provide a lead for engineering TACE-specific inhibitors that may reduce side effects arising from MMP inhibition and are possibly useful for treatment of diseases associated with excessive TNF-alpha levels such as rheumatoid arthritis.     

High-throughput determination of mode of inhibition in lead identification and optimization

After finishing the primary high-throughput screening, the screening team is often faced with thousands of hits to be evaluated further. Effective filtering of these hits is crucial in identifying leads. Mode of inhibition (MOI) study is extremely useful in validating whether the observed compound activity is specific to the biological target. In this article, the authors describe a high-throughput MOI determination method for evaluating thousands of compounds using an existing screening infrastructure. Based on enzyme or receptor kinetics theory, the authors developed the method by measuring the ratio of IC(50) or percent inhibition at 2 carefully chosen substrate or ligand concentrations to define an inhibitor as competitive, uncompetitive, or noncompetitive. This not only facilitates binning of HTS hits according to their MOI but also greatly expands HTS utility in support of the medicinal chemistry team's lead optimization practice. Three case studies are presented to demonstrate how the method was applied successfully in 3 discovery programs targeting either an enzyme or a G-protein-coupled receptor.     

Loss of ectodomain shedding due to mutations in the metalloprotease and cysteine-rich/disintegrin domains of the tumor necrosis factor-alpha converting enzyme (TACE)

Tumor necrosis factor-alpha converting enzyme (TACE), a multidomain protease essential for development and disease, releases the ectodomains from many transmembrane proteins in a regulated fashion. To understand the mechanism underlying the regulation of TACE activity, we sought to identify the cause of ectodomain shedding deficiencies in two mutated CHO sublines designated M1 and M2. Transfection of expression vectors for human and mouse TACE restored ectodomain shedding of TNF-alpha and TGF-alpha, suggesting that defects in the TACE gene contribute to the phenotype of M1 and M2 cells. The overall levels of endogenous TACE forms in M1 cells were significantly lower than those found in their parental cells, whereas only TACE zymogen, but not its mature form, was detectable in M2 cells. Molecular analyses suggested that M1 cells contained only one expressible TACE allele encoding an M435I point mutation in the catalytic center of the protease, and M2 cells produced two TACE variants with distinct point mutations in the catalytic domain (C225Y) and the cysteinerich/disintegrin domain (C600Y). Overexpression of the C225Y and C600Y TACE by transient transfection largely compensated for maturation defects in the variants but failed to restore TNF-alpha and TGF-alpha release in the shedding-defective CHO cell lines and fibroblasts derived from TACE-null mouse embryo. Further mutagenesis and functional analyses demonstrated that Cys(600) was absolutely essential for ectodomain shedding, suggesting that Cys(600), similar to Cys(225), participates in disulfide bonding, which is critical for both the processing and catalysis of TACE.     

Display of active subtilisin 309 on phage: analysis of parameters influencing the selection of subtilisin variants with changed substrate specificity from libraries using phosphonylating inhibitors

Many attempts have been made to endow enzymes with new catalytic activities. One general strategy involves the creation of random combinatorial libraries of mutants associated with an efficient screening or selection scheme. Phage display has been shown to greatly facilitate the selection of polypeptides with desired properties by establishing a close link between the polypeptide and the gene that encodes it. Selection of phage displayed enzymes for new catalytic activities remains a challenge. The aim of this study was to display the serine protease subtilisin 309 (savinase) from Bacillus lentus on the surface of filamentous fd phage and to develop selection schemes that allow the extraction of subtilisin variants with a changed substrate specificity from libraries. Subtilisins are produced as secreted preproenzyme that mature in active enzyme autocatalytically. They have a broad substrate specificity but exhibit a significant preference for hydrophobic residues and very limited reactivity toward charged residues at the P4 site in the substrate. Here, we show that savinase can be functionally displayed on phage in the presence of the proteic inhibitor CI2. The free enzyme is released from its complex with CI2 upon addition of the anionic detergent LAS. The phage-enzyme can be panned on streptavidin beads after labelling by reaction with (biotin-N-epsilon-aminocaproyl-cystamine-N'-glutaryl)-l-Ala-l-Ala-l-P ro-Phe(P)-diphenyl ester. Reactions of libraries, in which residues 104 and 107 forming part of the S4 pocket have been randomised, with (biotin-N-epsilon-aminocaproyl-cystamine-N'-glutaryl)-alpha-l-Lys-l-A la-l-Pro-Phe(P)-diphenylester allowed us to select enzymes with increased specific activity for a substrate containing a lysine in P4. Parameters influencing the selection as for instance the efficiency of maturation of mutant enzymes in libraries have been investigated.     

Redox cycling compounds generate H2O2 in HTS buffers containing strong reducing reagents--real hits or promiscuous artifacts?

Redox cycling compounds (RCCs) generate μM concentrations of hydrogen peroxide (H(2)O(2)) in the presence of strong reducing agents, common buffer components used to maintain the catalytic activity and/or folding of target proteins for high throughput screening (HTS) assays. H(2)O(2) generated by RCCs can indirectly inhibit the catalytic activity of proteins by oxidizing accessible cysteine, tryptophan, methionine, histidine, or selenocysteine residues, and indeed several important classes of protein targets are susceptible to H(2)O(2)-mediated inactivation; protein tyrosine phosphatases, cysteine proteases, and metalloenzymes. The main sources of H(2)O(2) in cells are the Nox enzyme/SOD systems, peroxisome metabolism, and the autoxidation of reactive chemicals by enzyme mediated redox cycling at both the microsomal and mitochondrial sites of electron transport. Given the role of H(2)O(2) as a second messenger involved in the regulation of many signaling pathways it is hardly surprising that compounds that can generate intracellular H(2)O(2) by enzyme mediated redox cycling would have pleiotropic effects. RCCs can therefore have serious negative consequences for the probe and/or lead generation process: primary HTS assay hit rates may be inflated by RCC false positives; crucial resources will be diverted to develop and implement follow up assays to distinguish RCCs from real hits; and screening databases will become annotated with the promiscuous activity of RCCs. In an attempt to mitigate the serious impact of RCCs on probe and lead generation, two groups have independently developed assays to indentify RCCs.     

Phage Display of the Serpin Alpha-1 Proteinase Inhibitor Randomized at Consecutive Residues in the Reactive Centre Loop and Biopanned with or without Thrombin

In spite of the power of phage display technology to identify variant proteins with novel properties in large libraries, it has only been previously applied to one member of the serpin superfamily. Here we describe phage display of human alpha-1 proteinase inhibitor (API) in a T7 bacteriophage system. API M358R fused to the C-terminus of T7 capsid protein 10B was directly shown to form denaturation-resistant complexes with thrombin by electrophoresis and immunoblotting following exposure of intact phages to thrombin. We therefore developed a biopanning protocol in which thrombin-reactive phages were selected using biotinylated anti-thrombin antibodies and streptavidin-coated magnetic beads. A library consisting of displayed API randomized at residues 357 and 358 (P2–P1) yielded predominantly Pro-Arg at these positions after five rounds of thrombin selection; in contrast the same degree of mock selection yielded only non-functional variants. A more diverse library of API M358R randomized at residues 352–356 (P7–P3) was also probed, yielding numerous variants fitting a loose consensus of DLTVS as judged by sequencing of the inserts of plaque-purified phages. The thrombin-selected sequences were transferred en masse into bacterial expression plasmids, and lysates from individual colonies were screening for API-thrombin complexing. The most active candidates from this sixth round of screening contained DITMA and AAFVS at P7–P3 and inhibited thrombin 2.1-fold more rapidly than API M358R with no change in reaction stoichiometry. Deep sequencing using the Ion Torrent platform confirmed that over 800 sequences were significantly enriched in the thrombin-panned versus naïve phage display library, including some detected using the combined phage display/bacterial lysate screening approach. Our results show that API joins Plasminogen Activator Inhibitor-1 (PAI-1) as a serpin amenable to phage display and suggest the utility of this approach for the selection of “designer serpins” with novel reactivity and/or specificity.     

Discovery of Bile Salt Hydrolase Inhibitors Using an Efficient High-Throughput Screening System

The global trend of restricting the use of antibiotic growth promoters (AGP) in animal production necessitates the need to develop valid alternatives to maintain productivity and sustainability of food animals. Previous studies suggest inhibition of bile salt hydrolase (BSH), an intestinal bacteria-produced enzyme that exerts negative impact on host fat digestion and utilization, is a promising approach to promote animal growth performance. To achieve the long term goal of developing novel alternatives to AGPs, in this study, a rapid and convenient high-throughput screening (HTS) system was developed and successfully used for identification of BSH inhibitors. With the aid of a high-purity BSH from a chicken Lactobacillus salivarius strain, we optimized various screening conditions (e.g. BSH concentration, reaction buffer pH, incubation temperature and length, substrate type and concentration) and establish a precipitation-based screening approach to identify BSH inhibitors using 96-well or 384-well microplates. A pilot HTS was performed using a small compound library comprised of 2,240 biologically active and structurally diverse compounds. Among the 107 hits, several promising and potent BSH inhibitors (e.g. riboflavin and phenethyl caffeate) were selected and validated by standard BSH activity assay. Interestingly, the HTS also identified a panel of antibiotics as BSH inhibitor; in particular, various tetracycline antibiotics and roxarsone, the widely used AGP, have been demonstrated to display potent inhibitory effect on BSH. Together, this study developed an efficient HTS system and identified several BSH inhibitors with potential as alternatives to AGP. In addition, the findings from this study also suggest a new mode of action of AGP for promoting animal growth.     

Gelastatins and their hydroxamates as dual functional inhibitors for TNF-alpha converting enzyme and matrix metalloproteinases: synthesis, biological evaluation, and mechanism studies

The hydroxamic acid analogues (2) of the natural product gelastatins (1) were prepared by 1 step conversion reaction. The synthetic analogues (2) showed potent enzymatic inhibitory activities against MMP-2, MMP-9, and TACE IC50's of 6, 23, and 28 nM, respectively. In addition, 2 were able to inhibit TNF-alpha production effectively in mice as well as in a macrophage cell line, RAW 264.7. The protective effect of 2 also was examined on LPS-induced acute septic shock model. The mechanism of TNF-alpha inhibition was examined by RT-PCR and Western blot analyses. The relation of TACE and alpha-secretase was examined using cellular alpha-secretase assays on IMR-32 and SH-SY5Y cell lines. The docking mode of 2 with the catalytic domain of TACE was illustrated to analyze the binding mode for the further analogue design.     

Cutting edge: a dominant negative form of TNF-alpha converting enzyme inhibits proTNF and TNFRII secretion.

TNF-alpha converting enzyme (TACE) is the protease responsible for processing proTNF from the 26-kDa membrane-anchored precursor to the secreted 17-kDa TNF-alpha. We show here that a deletion mutant of TACE (dTACE), lacking the pro and catalytic domains of the protease, acts as a dominant negative for proTNF processing in transfected HEK293 cells. We used the same system to test the effect of dTACE on TNFRII processing. Overexpression of dTACE with TNFRII resulted in >80% inhibition of TNFRII shedding. Although significant inhibition of TNF-alpha and TNFRII shedding was achieved with dTACE, we could not detect a cell surface accumulation of the noncleaved substrates above that observed in the absence of dTACE. Our results suggest that TNFRII is a substrate for TACE, and that dTACE is capable of interfering with the function of endogenous TACE, either by binding and sequestering TACE substrates via the disintegrin domain, transmembrane domain, or cytoplasmic tail, or by some other mechanism that has yet to be determined.