The Tick-Derived Anticoagulant Madanin Is Processed by Thrombin and Factor Xa

The cysteine-less peptidic anticoagulants madanin-1 and madanin-2 from the bush tick Haemaphysalis longicornis are the founding members of the MEROPS inhibitor family I53. It has been previously suggested that madanins exert their functional activity by competing with physiological substrates for binding to the positively charged exosite I (fibrinogen-binding exosite) of α-thrombin. We hereby demonstrate that competitive inhibition of α-thrombin by madanin-1 or madanin-2 involves binding to the enzyme''s active site. Moreover, the blood coagulation factors IIa and Xa are shown to hydrolyze both inhibitors at different, although partially overlapping cleavage sites. Finally, the three-dimensional structure of the complex formed between human α-thrombin and a proteolytic fragment of madanin-1, determined by X-ray crystallography, elucidates the molecular details of madanin-1 recognition and processing by the proteinase. Taken together, the current findings establish the mechanism of action of madanins, natural anticoagulants that behave as cleavable competitive inhibitors of thrombin.     

Synthesis and Mechanistic Studies of a Novel Homoisoflavanone Inhibitor of Endothelial Cell Growth

Preventing pathological ocular angiogenesis is key to treating retinopathy of prematurity, diabetic retinopathy and age-related macular degeneration. At present there is no small molecule drug on the market to target this process and hence there is a pressing need for developing novel small molecules that can replace or complement the present surgical and biologic therapies for these neovascular eye diseases. Previously, an antiangiogenic homoisoflavanone was isolated from the bulb of a medicinal orchid, Cremastra appendiculata. In this study, we present the synthesis of a novel homoisoflavanone isomer of this compound. Our compound, SH-11052, has antiproliferative activity against human umbilical vein endothelial cells, and also against more ocular disease-relevant human retinal microvascular endothelial cells (HRECs). Tube formation and cell cycle progression of HRECs were inhibited by SH-11052, but the compound did not induce apoptosis at effective concentrations. SH-11052 also decreased TNF-α induced p38 MAPK phosphorylation in these cells. Intriguingly, SH-11052 blocked TNF-α induced IκB-α degradation, and therefore decreased NF-κB nuclear translocation. It decreased the expression of NF-κB target genes and the pro-angiogenic or pro-inflammatory markers VCAM-1, CCL2, IL8, and PTGS2. In addition SH-11052 inhibited VEGF induced activation of Akt but not VEGF receptor autophosphorylation. Based on these results we propose that SH-11052 inhibits inflammation induced angiogenesis by blocking both TNF-α and VEGF mediated pathways, two major pathways involved in pathological angiogenesis. Synthesis of this novel homoisoflavanone opens the door to structure-activity relationship studies of this class of compound and further evaluation of its mechanism and potential to complement existing antiangiogenic drugs.     

含RGD序列的山蛭素突变体的克隆、表达及性质研究

根据LAP（leech antihemostatic protein）理论,在分析山蛭素和decorsin结构特征的基础上,利用重组PCR方法,删除了山蛭素氨基酸序列的33位至35位,同时分别插入了RGDS和来源于decorsin的一段PRGDADP序列构建成为2种含RGD序列的山蛭素突变体,分别命名为HRGD1和HRGD2.这2种突变体在毕赤酵母菌株GS115中得到成功表达.经过超滤、阳离子交换层析和凝胶过滤层析等纯化步骤之后,得到纯度高于95%的目的蛋白.通过以chromozym TH为底物的凝血酶酰胺水解实验和血小板聚集抑制实验证实了其体外生物学活性,HRGD1和HRGD2抑制凝血酶的动力学常数达到10^-13 mol/L水平,抑制血小板的IC₅₀在10^-6 mol/L水平上.     

凝血酶抑制剂TTI的表达及性质研究

将来源于采采蝇的TTI基因序列改造成大肠杆菌偏爱密码子,利用重组PCR方法获得TTI目的基因片段,在大肠杆菌中得到高效表达。经纯化获得了纯度高于98%的融合蛋白,建立了酶活测定方法。实验证明融合蛋白具有抑制凝血酶的活性。当凝血酶浓度为10U/ml,纯化的融合TTI体积为10 l,底物浓度为250 mol/L,融合蛋白对凝血酶的抑制率为73%,确定反应类型为竞争性抑制,Ki为35 mol/L。     

Identification of a Novel Topoisomerase Inhibitor Effective in Cells Overexpressing Drug Efflux Transporters

Background

Natural product structures have high chemical diversity and are attractive as lead structures for discovery of new drugs. One of the disease areas where natural products are most frequently used as therapeutics is oncology.

Method and Findings

A library of natural products (NCI Natural Product set) was screened for compounds that induce apoptosis of HCT116 colon carcinoma cells using an assay that measures an endogenous caspase-cleavage product. One of the apoptosis-inducing compounds identified in the screen was thaspine (taspine), an alkaloid from the South American tree Croton lechleri. The cortex of this tree is used for medicinal purposes by tribes in the Amazonas basin. Thaspine was found to induce conformational activation of the pro-apoptotic proteins Bak and Bax, mitochondrial cytochrome c release and mitochondrial membrane permeabilization in HCT116 cells. Analysis of the gene expression signature of thaspine-treated cells suggested that thaspine is a topoisomerase inhibitor. Inhibition of both topoisomerase I and II was observed using in vitro assays, and thaspine was found to have a reduced cytotoxic effect on a cell line with a mutated topoisomerase II enzyme. Interestingly, in contrast to the topoisomerase II inhibitors doxorubicin, etoposide and mitoxantrone, thaspine was cytotoxic to cell lines overexpressing the PgP or MRP drug efflux transporters. We finally show that thaspine induces wide-spread apoptosis in colon carcinoma multicellular spheroids and that apoptosis is induced in two xenograft mouse models in vivo.

Conclusions

The alkaloid thaspine from the cortex of Croton lechleri is a dual topoisomerase inhibitor effective in cells overexpressing drug efflux transporters and induces wide-spread apoptosis in multicellular spheroids.     

In Vitro and In Vivo Analysis of RTK Inhibitor Efficacy and Identification of Its Novel Targets in Glioblastomas

Treatment for glioblastoma consists of radiotherapy and temozolomide-based chemotherapy. However, virtually all patients recur, leading to a fatal outcome. Receptor tyrosine kinase (RTK)-targeted therapy has been the focus of attention in novel treatment options for these patients. Here, we compared the efficacy of imatinib, sunitinib, and cediranib in glioblastoma models. In the present work, the biologic effect of the drugs was screened by viability, cell cycle, apoptosis, migration, and invasion in vitro assays or in vivo by chick chorioallantoic membrane assay. Intracellular signaling was assessed by Western blot and the RTK targets were identified using phospho-RTK arrays. The amplified status of KIT, PDGFRA, and VEGFR2 genes was assessed by quantitative polymerase chain reaction. In a panel of 10 glioblastoma cell lines, we showed that cediranib was the most potent. In addition, cediranib and sunitinib synergistically sensitize the cells to temozolomide. Cediranib efficacy was shown to associate with higher cytostatic and unique cytotoxic effects in vitro and both antitumoral and antiangiogenic activity in vivo, which could associate with its great capacity to inhibit mitogen-activated protein kinase (MAPK) and AKT pathways. The molecular status of KIT, PDGFRA, and VEGFR2 did not predict glioblastoma cell responsiveness to any of the RTK inhibitors. Importantly, phospho-RTK arrays revealed novel targets for cediranib and sunitinib therapy. In conclusion, the novel targets found may be of value as future biomarkers for therapy response in glioblastoma and lead to the rational selection of patients for effective molecular targeted treatment.     

中华硬蜱凝血酶抑制剂的分离纯化与活性

通过凝胶过滤和反相高压液相层析。从200只半饱吸血的中华硬蜱(Ixodide sinesis)唾液腺中分离纯化得到一凝血酶抑制剂。用飞行质谱测定其分子量为6．356kDa,与其他蜱类来源的凝血酶抑制剂分子量不同,提示为一新蛋白分子。该抑制剂对凝血酶有强烈的抑制活性,对激活的第X因子和胰蛋白酶有微弱的抑制活性。其发现将为发展疫苗、生物控制中华硬蜱提供资料和目标抗原。     

Identification of a Novel HIV-1 Inhibitor Targeting Vif-dependent Degradation of Human APOBEC3G Protein

APOBEC3G (A3G) is a cellular cytidine deaminase that restricts HIV-1 replication by inducing G-to-A hypermutation in viral DNA and by deamination-independent mechanisms. HIV-1 Vif binds to A3G, resulting in its degradation via the 26 S proteasome. Therefore, this interaction represents a potential therapeutic target. To identify compounds that inhibit interaction between A3G and HIV-1 Vif in a high throughput format, we developed a homogeneous time-resolved fluorescence resonance energy transfer assay. A 307,520 compound library from the NIH Molecular Libraries Small Molecule Repository was screened. Secondary screens to evaluate dose-response performance and off-target effects, cell-based assays to identify compounds that attenuate Vif-dependent degradation of A3G, and assays testing antiviral activity in peripheral blood mononuclear cells and T cells were employed. One compound, N.41, showed potent antiviral activity in A3G(+) but not in A3G(−) T cells and had an IC₅₀ as low as 8.4 μm and a TC₅₀ of >100 μm when tested against HIV-1_Ba-L replication in peripheral blood mononuclear cells. N.41 inhibited the Vif-A3G interaction and increased cellular A3G levels and incorporation of A3G into virions, thereby attenuating virus infectivity in a Vif-dependent manner. N.41 activity was also species- and Vif-dependent. Preliminary structure-activity relationship studies suggest that a hydroxyl moiety located at a phenylamino group is critical for N.41 anti-HIV activity and identified N.41 analogs with better potency (IC₅₀ as low as 4.2 μm). These findings identify a new lead compound that attenuates HIV replication by liberating A3G from Vif regulation and increasing its innate antiviral activity.     

Identification of Novel Mutations Responsible for Resistance to MK-2048, a Second-Generation HIV-1 Integrase Inhibitor

MK-2048 represents a prototype second-generation integrase strand transfer inhibitor (INSTI) developed with the goal of retaining activity against viruses containing mutations associated with resistance to first-generation INSTIs, raltegravir (RAL) and elvitegravir (EVG). Here, we report the identification of mutations (G118R and E138K) which confer resistance to MK-2048 and not to RAL or EVG. These mutations were selected in vitro and confirmed by site-specific mutagenesis. G118R, which appeared first in cell culture, conferred low levels of resistance to MK-2048. G118R also reduced viral replication capacity to approximately 1% that of the isogenic wild-type (wt) virus. The subsequent selection of E138K partially restored replication capacity to ≈13% of wt levels and increased resistance to MK-2048 to ≈8-fold. Viruses containing G118R and E138K remained largely susceptible to both RAL and EVG, suggesting a unique interaction between this second-generation INSTI and the enzyme may be defined by these residues as a potential basis for the increased intrinsic affinity and longer “off” rate of MK-2048. In silico structural analysis suggests that the introduction of a positively charged arginine at position 118, near the catalytic amino acid 116, might decrease Mg²⁺ binding, compromising enzyme function and thus leading to the significant reduction in both integration and viral replication capacity observed with these mutations.Selective pressure exerted by antiretroviral drugs, in conjunction with high viral mutation rates, promotes the inevitable emergence of drug-resistant HIV-1 variants. This necessitates an ongoing search for novel antiretroviral compounds that either have novel mechanisms and inhibit different stages of viral replication or inhibit targets that have acquired resistance to existing drugs. In the latter case, such newer next-generation agents should ideally display resistance profiles which are distinct and nonoverlapping with those of the first-generation drugs.Integration of viral cDNA into the host cell genome is a distinct feature of retroviral replication, and inhibitors of HIV-1 integrase have recently been added to the arsenal of clinically approved antiretroviral drugs. Raltegravir (RAL) was the first integrase strand transfer inhibitor (INSTI) to be approved by the U.S. Food and Drug Administration (FDA) after clinical trials showed that this drug promoted a rapid and sustained antiviral effect (13). Elvitegravir (EVG), another integrase inhibitor, is currently in phase III clinical trials (27). Resistance mutations common to both of these first-generation integrase inhibitors have been reported and can result in high levels of drug resistance (26). Mutations which engender cross-resistance between RAL and EVG have been reported in clinical trials, cell culture studies, and biochemical assays (9, 26). This has prompted the search for second-generation integrase inhibitors that might display novel patterns of resistance, allowing their use in patients who have failed therapy with RAL or EVG. MK-2048 (28) is a prototype second-generation INSTI that retains potency against viruses containing common single and double mutations observed in the clinic with first-generation agents with a 95% inhibitory concentration (IC₉₅) in the nM range. MK-2048 has been previously reported to be active against viruses resistant to RAL and EVG (28, 29). Given common mechanisms of action among INSTIs and a lack of structural information on integrase inhibitor complexes with resistance mutations, an understanding of resistance to second-generation agents such as MK-2048 is important.This article describes the selection of resistance to MK-2048 in tissue culture and the characterization of mutations associated with such resistance, G118R and E138K. The identification of distinct mutations which appear to confer resistance to MK-2048 and not to either RAL or EVG has potential implications for understanding the structural basis for the second-generation profile of this compound as well as future drug discovery and development efforts focused on this mechanism.     

一种水蛭素类融合蛋白与凝血酶作用的动力学模拟

通过将凝血酶抑制剂水蛭素的C端20肽片段嫁接到血小板结合蛋白AnnexinⅤ上,可以期望获得既具有抗凝血活性,同时又具有导向性的新型工程蛋白质分子.利用计算机辅助分子设计手段模拟了该融合蛋白的分子结构,并对该融合蛋白对凝血酶的抑制作用进行了分子动力学模拟,得到了支持上述想法的结果.     

Identification of a Novel Triterpenoid Saponin from Pisum sativum as a Specific Inhibitor of the Diguanylate Cyclase of Acetobacter xylinum

A specific and highly potent inhibitor of diguanylate cyclase,the key regulatory enzyme of the cellulose synthesizing apparatusin the bacterium Acetobacter xylinum, was isolated from extractsof etiolated pea shoots (Pisum sativum). The inhibitor has beenpurified by a multistep procedure, and sufficient amounts ofhighly purified compound (3-8 mg) for spectral analysis wereobtained. The structure of this compound was established as3-O-a-L-rhamnopyranosyl-(l     

Identification of Novel Compounds Inhibiting Chikungunya Virus-Induced Cell Death by High Throughput Screening of a Kinase Inhibitor Library

Chikungunya virus (CHIKV) is a mosquito-borne arthrogenic alphavirus that causes acute febrile illness in humans accompanied by joint pains and in many cases, persistent arthralgia lasting weeks to years. The re-emergence of CHIKV has resulted in numerous outbreaks in the eastern hemisphere, and threatens to expand in the foreseeable future. Unfortunately, no effective treatment is currently available. The present study reports the use of resazurin in a cell-based high-throughput assay, and an image-based high-content assay to identify and characterize inhibitors of CHIKV-infection in vitro. CHIKV is a highly cytopathic virus that rapidly kills infected cells. Thus, cell viability of HuH-7 cells infected with CHIKV in the presence of compounds was determined by measuring metabolic reduction of resazurin to identify inhibitors of CHIKV-associated cell death. A kinase inhibitor library of 4,000 compounds was screened against CHIKV infection of HuH-7 cells using the resazurin reduction assay, and the cell toxicity was also measured in non-infected cells. Seventy-two compounds showing ≥50% inhibition property against CHIKV at 10 µM were selected as primary hits. Four compounds having a benzofuran core scaffold (CND0335, CND0364, CND0366 and CND0415), one pyrrolopyridine (CND0545) and one thiazol-carboxamide (CND3514) inhibited CHIKV-associated cell death in a dose-dependent manner, with EC₅₀ values between 2.2 µM and 7.1 µM. Based on image analysis, these 6 hit compounds did not inhibit CHIKV replication in the host cell. However, CHIKV-infected cells manifested less prominent apoptotic blebs typical of CHIKV cytopathic effect compared with the control infection. Moreover, treatment with these compounds reduced viral titers in the medium of CHIKV-infected cells by up to 100-fold. In conclusion, this cell-based high-throughput screening assay using resazurin, combined with the image-based high content assay approach identified compounds against CHIKV having a novel antiviral activity - inhibition of virus-induced CPE - likely by targeting kinases involved in apoptosis.     

一株嗜盐菌新种的分离与鉴定

从舟山册子岛船舶压载水泥样中分离到一株细菌S3-22,其与已知细菌的16S rDNA序列相似性低于97%,G+C mol%为54.9 mol%,主要脂肪酸iso-C17:1ω9c(24.99%),细胞醌型为甲基萘醌MK-5。革兰氏染色阴性,最适生长条件为30～37℃、pH7、3%NaCl。嗜盐,氧化酶、接触酶、淀粉酶、酯酶呈阳性,可还原硝酸盐。依据其16S rDNA序列相似性、系统发育学分析及细胞与分子水平的鉴定表明,该菌是Kordiimonas属的一个新种,菌株S3-22的16S rDNA序列登陆号为FJ847942。     

Proteolysis of Human Thrombin Generates Novel Host Defense Peptides

The coagulation system is characterized by the sequential and highly localized activation of a series of serine proteases, culminating in the conversion of fibrinogen into fibrin, and formation of a fibrin clot. Here we show that C-terminal peptides of thrombin, a key enzyme in the coagulation cascade, constitute a novel class of host defense peptides, released upon proteolysis of thrombin in vitro, and detected in human wounds in vivo. Under physiological conditions, these peptides exert antimicrobial effects against Gram-positive and Gram-negative bacteria, mediated by membrane lysis, as well as immunomodulatory functions, by inhibiting macrophage responses to bacterial lipopolysaccharide. In mice, they are protective against P. aeruginosa sepsis, as well as lipopolysaccharide-induced shock. Moreover, the thrombin-derived peptides exhibit helical structures upon binding to lipopolysaccharide and can also permeabilize liposomes, features typical of “classical” helical antimicrobial peptides. These findings provide a novel link between the coagulation system and host-defense peptides, two fundamental biological systems activated in response to injury and microbial invasion.