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。     

The regulation of TACE catalytic function by its prodomain

Aim To study the function of the prodomain of ADAM17 (TACE) and to develop an approach for interfering with inflammation processes. Method The expression plasmids of the TACE ectodomain (T1300), prodomain (T591), signal peptide and prodomain (T648), full length (T2472), and the turncated TACE without prodomain (T57-T1824) were constructed and designated as pET-28a-T300, pET-28a-T591, pIRES2-EGFP-648, pEGFP-N1-T648, pIRES2-EGFP-T2472, and pIRES2-EGFP-T57-T1824, respectively. After Ni²⁺-NTA resin-affinity chromatography, the recombinant T591 and T1300 proteins were obtained and assayed by western blotting and circular dichroism. The experiment was carried out on THP1 cell lines stimulated by LPS in vitro. The inhibition of recombinant protein T591 to TACE activity was detected by ELISA and immunohistochemical detection. The expression plasmids (pIRES2-EGFP-T648, pIRES2-EGFP-T2472, and pIRES2-EGFP-T57-T1824) were used to transfect the U937 cells. HeLa cells were also transfected with pEGFP-N1-T648. The transfected U937 cells were then stimulated by LPS and the effect of expression plasmids on TNF-α secretion was detected by ELISA and flow cytometry (FCM). Results The recombinant prodomain protein inhibited 57% of the TNF-α secretion and mediated an accumulation of TNF-α on the surface of THP1 cells. An intense green fluorescence was seen in the membranes of HeLa cells transfected with pEGFP-N1-T648. The plasmid pIRES2-EGFP-T648 inhibited TNF-α secretion by 61.09% and mediated an accumulation of mTNF-α on the surface of the U937 cells. The secretion of sTNF-α and the level of the mTNF-α in the pIRES2-EGFP-T57-T1824 transfected cells gave no difference when compared with the pIRES2-EGFP transfected cells. Also the secretion of sTNF-α from the cells transfected by the plasmid pIRES2-EGFP-T2472 increased, while the level of mTNF-α decreased, compared with the pIRES2-EGFP-transfected cells. Conclusion The prodomain has dual effects and might be useful in the molecular design of an anti-inflammatory drug.     

Ectodomain shedding of TNF-alpha is enhanced by nardilysin via activation of ADAM proteases

Tumor necrosis factor-α (TNF-α) is released from cells by proteolytic cleavage of a membrane-anchored precursor. The TNF-α-converting enzyme (TACE/ADAM17) is the major sheddase for ectodomain shedding of TNF-α. At present, however, it is poorly understood how its catalytic activity is regulated. Here, we show that nardilysin (N-arginine dibasic convertase; NRDc) enhanced TNF-α shedding. In a cell-based shedding assay, expression of NRDc synergistically enhanced TACE-induced TNF-α shedding. A peptide cleavage assay in vitro showed that recombinant NRDc enhances the cleavage of TNF-α induced by TACE. Notably, co-incubation of NRDc completely reversed the inhibitory effect of a physiological concentration of salt on TACE’s activity in vitro. Overexpression of NRDc in TACE-deficient fibroblasts resulted in an increase in the amount of TNF-α released. Co-expression of NRDc with ADAM10 promoted the release compared with the sole expression of ADAM10. These results suggested that NRDc enhances TNF-α shedding through activation of not only TACE but ADAM10. Our results indicate the involvement of NRDc in ectodomain shedding of TNF-α, which may be a novel target for anti-inflammatory therapies.     

Definition of peptide inhibitors from a synthetic peptide library by targeting gelatinase B/matrix metalloproteinase-9 (MMP-9) and TNF-α converting enzyme (TACE/ADAM-17)

Gelatinase B/matrix metalloproteinase-9 (MMP-9) is a regulatory and effector metalloproteinase in inflammation. TNF-α is an important proinflammatory cytokine and is released by the action of a Zn²⁺-containing converting enzyme (TACE/ADAM-17). Both metallo-enzymes play important roles during the development of shock syndromes. Combinatorial chemical synthesis and subsequent library deconvolution were previously used to define a peptide inhibitor (Regasepin1) acting, almost to the same degree, on neutrophil collagenase/MMP-8 and MMP-9 in vitro, and protecting mice against lethal endotoxinemia in vivo. We have now extended this approach by incorporating D-form amino acids and residues preferred by TACE. A new peptide library was designed and synthesized, and by deconvolution new peptide inhibitors were defined. These included a TACE-specific inhibitor, an MMP-9- specific inhibitor, and inhibitors for both enzymes.     

内毒素结合肽模拟肽P11的体外活性研究

目的对从噬菌体展示随机肽库筛选获得的内毒素结合肽模拟肽进行体外拈抗内毒素活性鉴定。方法采用FMOC固相合成法化学合成内毒素结合肽模拟肽P11,并进行拮抗内毒素活性和细胞毒性测定。结果亲和ELISA检测显示P11与LPS有较高的亲和力,通过生长曲线和流式细胞学分析细胞周期显示P11对人U937细胞生长无明显影响。流式细胞检测显示P11呈剂量依赖性抑制FITC—LPS与人外周血单核细胞（PBMC）结合。细胞因子生成抑制实验显示10μg/mlP11可显著抑制LPS诱导PBMC和U937细胞TNF—αmRNA转录和蛋白表达。结论体外活性鉴定结果表明化学合成的模拟肽P11可抑制LPS诱导的炎性反应。     

Current perspective of TACE inhibitors: A review

Rheumatoid Arthritis (RA) is one of the most common autoimmune inflammatory conditions, affecting approximately 1% of the adult population worldwide. TNF-α is a pleitropic, pro-inflammatory cytokine which plays a pivotal role in the origin and progression of RA and other immune mediated disorders. The success of anti-TNF-α biological agents proved that inhibition of TNF-α could result in effective control of RA. Since the discovery of anti-TNF-α biologicals, much efforts have gone into developing an orally bioavailable small size TNF-α antagonist. One of the ways to block TNF-α in biological fluids is to inhibit TNF-α converting enzyme (TACE). This target has been validated in preclinical trials using TACE inhibitors. But, even after more than a decade no single TACE inhibitor has passed the Phase II clinical trials. Very recently, it has been shown that TACE inhibitors could also be used for inhibition of pathogenic EGFR signaling in cancer. Hence, TACE inhibitors could perform a dual role, in curing not only RA but also certain cancerous conditions. Developments in the field have prompted us to review the research work on TACE inhibitors, especially their structure activity relationships and molecular modeling studies.     

P-糖蛋白的表达及其结合肽的筛选

为获得P 糖蛋白胞外段 ,构建了高效表达载体pGEX Pgp ,转化大肠杆菌DH5α ,进行表达、鉴定及纯化 ,以获得的融合蛋白为靶蛋白 ,筛选噬菌体随机 12肽库 ,免疫细胞化学方法进行鉴定 .SDS PAGE分析 ,表达出约 30kD大小的蛋白 ;从噬菌体随机肽库中筛选获得了与P 糖蛋白特异性结合的噬菌体阳性克隆 ,测序获得了其特异性结合肽序列 :NDGLLFTWQPSP .免疫细胞化学结果显示 :筛选得到的噬菌体阳性克隆可与耐药细胞BIU 87 ADM结合 ,而与敏感细胞BIU 87不结合 .结果表明 ,筛选获得的结合肽可与耐药的肿瘤细胞结合 ,表现出一定的肿瘤特异性 .P 糖蛋白结合肽的筛选 ,为进行人膀胱癌多药耐药的靶向治疗等工作奠定了基础 .     

Selection of substrate recognition sequence of protein kinase with ferric chelation affinity chromatography

Protein kinase substrate phage (PKS phage) was constructed by fusing the substrate recognition consensus sequence of cAMP-dependent protein kinase (cAPK) with bacteriophage minor coat protein g3p and by dis-playing it on the surface of filamentous bacteriophage fd. Phosphorylation in vitro by cAPK showed a unique labelled band of approximately 60 ku, which was consistent with the molecular weight of the PKS-g3p fusion protein. Some weakly phosphorylated bands for both PKS phage and wild-type phage were also observed. Phage display random 15-mer peptide library phosphorylated by cAPK was selected with ferric (Fe3 ) chelalion affinity resin. After 4 rounds of screening, phage clones were picked out to determine the displayed peptide sequences by DNA sequencing. The results showed that 5 of 14 sequenced phages displayed the cAPK recognition sequence motif (R)RXS/T. Their in vitro phosphorylation analyses revealed the specific labelled bands corresponding to the positive PKS phages with and without the typ     

细胞间粘附分子1特异结合肽的筛选及其生物功能

采用两种方法对噬菌体展示随机十五肽库进行亲和淘选 .ELISA法筛选特异结合高亲和力的阳性噬菌体单克隆 ,测序 ,得到 6个与人细胞间粘附分子 1(ICAM 1)高亲和力的噬菌体展示十五肽单克隆 .再经ELISA法从这 6个噬菌体单克隆中选择与ICAM 1亲和力最高的单克隆 ,同时利用蛋白空间结构位象模拟技术对小肽与ICAM 1的亲和力进行模拟研究 .最终获取目的小肽的氨基酸序列为GRGEFRGRDNSVSVV .目的单克隆噬菌体与ICAM - 1的亲和常数Ka 为 7 87× 10 7L mol .体外合成、纯化并标记目的小肽 .ELISA法验证目的小肽与人ICAM 1的结合呈浓度依赖性 ,抗ICAM 1多抗不能拮抗目的小肽与ICAM 1的结合 .采用免疫组化方法证实 ,此目的小肽具有与炎症组织中高表达的ICAM 1特异性结合的功能 .在动物体内 ,荧光标记的目的小肽具有向高表达ICAM 1的炎症部位特异性聚集的功能 .说明此目的肽可尝试作为以ICAM 1为靶的“肽导向药物”的前导肽 .     

Definition of peptide inhibitors from a synthetic peptide library by targeting gelatinase B/matrix metalloproteinase-9 (MMP-9) and TNF-α converting enzyme (TACE/ADAM-17)

Gelatinase B/matrix metalloproteinase-9 (MMP-9) is a regulatory and effector metalloproteinase in inflammation. TNF-α is an important proinflammatory cytokine and is released by the action of a Zn(2+)-containing converting enzyme (TACE/ADAM-17). Both metallo-enzymes play important roles during the development of shock syndromes. Combinatorial chemical synthesis and subsequent library deconvolution were previously used to define a peptide inhibitor (Regasepin1) acting, almost to the same degree, on neutrophil collagenase/MMP-8 and MMP-9 in vitro, and protecting mice against lethal endotoxinemia in vivo. We have now extended this approach by incorporating D-form amino acids and residues preferred by TACE. A new peptide library was designed and synthesized, and by deconvolution new peptide inhibitors were defined. These included a TACE-specific inhibitor, an MMP-9- specific inhibitor, and inhibitors for both enzymes.     

跨膜型肿瘤坏死因子α稳定表达细胞株的构建

目的：在体内,相对分子质量为26×103的跨膜型肿瘤坏死因子α（mTNF-α）可被TNF转化酶（TACE）酶解成相对分子质量为17×103的游离型TNF-α（sTNF-α）。为评价新研发的TNF-α全人源单克隆抗体与mTNF-α的结合能力,以及该单抗的ADCC作用,须构建只能稳定表达mTNF-α而不受TACE影响的Sp2/0细胞株。方法：PCR扩增缺失了TACE酶切位点的人TNF-α野生型基因序列,构建pcDNA3.1（＋）-dTNF-α重组质粒并测序鉴定;重组质粒转染Sp2/0细胞,构建只能稳定表达mTNF-α的细胞株,用流式细胞仪检测其表达情况。结果：pcDNA3.1（＋）-dTNF-α重组质粒测序结果与设计缺失基因的碱基序列完全一致,重组质粒构建成功;转染的阳性细胞株经流式细胞仪检测有表达,其中Clone11、15、16的表达量最高。结论：跨膜型TNF-α稳定表达细胞株构建成功,可用于TNF-α全人源单克隆抗体体外药效的评价。     

Discovery and SAR of hydantoin TACE inhibitors

We disclose inhibitors of TNF-α converting enzyme (TACE) designed around a hydantoin zinc binding moiety. Crystal structures of inhibitors bound to TACE revealed monodentate coordination of the hydantoin to the zinc. SAR, X-ray, and modeling designs are described. To our knowledge, these are the first reported X-ray structures of TACE with a hydantoin zinc ligand.     

A novel vascular-targeting peptide for gastric cancer delivers low-dose TNFα to normalize the blood vessels and improve the anti-cancer efficiency of 5-fluorouracil

Various vascular-targeted agents fused with tumor necrosis factor α (TNFα) have been shown to improve drug absorption into tumor tissues and enhance tumor vascular function. TCP-1 is a peptide selected through in vivo phage library biopanning against a mouse orthotopic colorectal cancer model and is a promising agent for drug delivery. This study further investigated the targeting ability of TCP-1 phage and peptide to blood vessels in an orthotopic gastric cancer model in mice and assessed the synergistic anti-cancer effect of 5-fluorouracil (5-FU) with subnanogram TNFα targeted delivered by TCP-1 peptide. In vivo phage targeting assay and in vivo colocalization analysis were carried out to test the targeting ability of TCP-1 phage/peptide. A targeted therapy for improvement of the therapeutic efficacy of 5-FU and vascular function was performed through administration of TCP-1/TNFα fusion protein in this model. TCP-1 phage exhibited strong homing ability to the orthotopic gastric cancer after phage injection. Immunohistochemical staining suggested that and TCP-1 phage/TCP-1 peptide could colocalize with tumor vascular endothelial cells. TCP-1/TNFα combined with 5-FU was found to synergistically inhibit tumor growth, induce apoptosis and reduce cell proliferation without evident toxicity. Simultaneously, subnanogram TCP-1/TNFα treatment normalized tumor blood vessels. Targeted delivery of low-dose TNFα by TCP-1 peptide can potentially modulate the vascular function of gastric cancer and increase the drug delivery of chemotherapeutic drugs.     

Multiple shRNA-mediated knockdown of TACE reduces the malignancy of HeLa cells

Tumor necrosis factor-alpha (TNF-α) converting enzyme (TACE) is a key enzyme involved in the proteolytic shedding of the ectodomain of several membrane-bound growth factors, cytokines and receptors. Here, we constructed a multiple short hairpin RNA (shRNA) expression vector containing four shRNAs against TACE. We found that in HeLa cells our multiple shRNAs vector produced a higher level of TACE knockdown than any single shRNA vector containing only one TACE shRNA. Silencing TACE expression in HeLa cells decreased their malignancy by decreasing the proliferation, adhesion and migration, as well as inducing apoptosis in these cells. Furthermore, our data suggest that the effects of TACE on the malignancy of HeLa cells may be mediated via activation of the EGFR (epidermal growth factor receptor) signaling pathway. Our findings suggest that using a combination of shRNAs within one vector to silence the expression of TACE might be a potential therapeutic strategy for tumors.     

Discovery of novel spirocyclopropyl hydroxamate and carboxylate compounds as TACE inhibitors

We have discovered nanomolar inhibitors of TNF-α convertase (TACE) comprised of a novel spirocyclic scaffold and either a carboxylate or hydroxamate zinc binding moiety. X-ray crystal structures and computer models of selected compounds binding to TACE explain the observed SAR. We report the first TACE X-ray crystal structure for an inhibitor with a carboxylate zinc ligand.     

Secreted human amyloid precursor protein binds semaphorin 3a and prevents semaphorin-induced growth cone collapse

The amyloid precursor protein (APP) is well known for giving rise to the amyloid-β peptide and for its role in Alzheimer's disease. Much less is known, however, on the physiological roles of APP in the development and plasticity of the central nervous system. We have used phage display of a peptide library to identify high-affinity ligands of purified recombinant human sAPPα(695) (the soluble, secreted ectodomain from the main neuronal APP isoform). Two peptides thus selected exhibited significant homologies with the conserved extracellular domain of several members of the semaphorin (Sema) family of axon guidance proteins. We show that sAPPα(695) binds both purified recombinant Sema3A and Sema3A secreted by transfected HEK293 cells. Interestingly, sAPPα(695) inhibited the collapse of embryonic chicken (Gallus gallus domesticus) dorsal root ganglia growth cones promoted by Sema3A (K(d)≤8·10(-9) M). Two Sema3A-derived peptides homologous to the peptides isolated by phage display blocked sAPPα binding and its inhibitory action on Sema3A function. These two peptides are comprised within a domain previously shown to be involved in binding of Sema3A to its cellular receptor, suggesting a competitive mechanism by which sAPPα modulates the biological action of semaphorins.     

内皮抑素30肽基因真核表达载体的构建及实验研究

目的构建改构内皮抑素抗肿瘤相关肽（30肽）的真核表达载体pVAX1,检测该重组载体的生物学活性。方法在30肽基因的5′端加入胶原蛋白ⅩⅧ信号肽编码序列,通过PCR扩增获得目的基因30肽,并连接到质粒pVAX1中,构建表达分泌型内皮抑素的重组质粒pVAX1-30E,然后将重组质粒pVAX1-30E直接注入小鼠肿瘤组织。通过ELISA小鼠体内抑瘤实验检测目的基因的表达及其活性。结果ELISA实验表明构建的分泌型内皮抑素重组质粒pVAX1-30E能在肿瘤细胞中表达30肽,免疫组化结果表明瘤组织中表达的30肽能抑制肿瘤微血管的新生,而体内抑瘤实验表明在肿瘤部位直接注射重组质粒能抑制肿瘤生长,抑瘤率为28.19%。结论通过向瘤组织中直接注射分泌型内皮抑素重组质粒pVAX1-30E可以抑制小鼠体内肿瘤微血管新生和肿瘤生长而实现其抗肿瘤活性。     

TACE is required for fetal murine cardiac development and modeling

Tumor necrosis factor-alpha converting enzyme (TACE) is a membrane-anchored, Zn-dependent metalloprotease, which belongs to the ADAM (a disintegrin and metalloprotease) family. TACE functions as a membrane sheddase to release the ectodomain portions of many transmembrane proteins, including the precursors of TNFalpha, TGFalpha, several other cytokines, as well as the receptors for TNFalpha, and neuregulin (ErbB4). Mice with TACE(DeltaZn/DeltaZn) null mutation die at birth with phenotypic changes, including failure of eyelid fusion, hair and skin defects, and abnormalities of lung development. Abnormal fetal heart development was not previously described. Herein, we report that TACE(DeltaZn/DeltaZn) null mutant mice by late gestation exhibit markedly enlarged fetal hearts with increased myocardial trabeculation and reduced cell compaction, mimicking the pathological changes of noncompaction of ventricular myocardium. In addition, larger cardiomyocyte cell size and increased cell proliferation were observed in ventricles of TACE(DeltaZn/DeltaZn) knockout mouse hearts. At the molecular level, reduced expression of epidermal growth factor receptor, attenuated protein cleavage of ErbB4, and changes in MAPK activation were also detected in TACE(DeltaZn/DeltaZn) knockout heart tissues. The data suggest that TACE-mediated cell surface protein ectodomain shedding plays an essential and a novel regulatory role during cardiac development and modeling.     

Biaryl substituted hydantoin compounds as TACE inhibitors

We disclose further optimization of hydantoin TNF-α convertase enzyme (TACE) inhibitors. SAR with respect to the non-prime region of TACE active site was explored. A series of biaryl substituted hydantoin compounds was shown to have sub-nanomolar K_i, good rat PK, and good selectivity versus MMP-1, -2, -3, -7, -9, and -13.