Normal mode analysis as a prerequisite for drug design: application to matrix metalloproteinases inhibitors

We demonstrate the utility of normal mode analysis in correctly predicting the binding modes of inhibitors in the active sites of matrix metalloproteinases (MMPs). We show the accuracy in predicting the positions of MMP-3 inhibitors is strongly dependent on which structure is used as the target, especially when it has been energy minimized. This dependency can be overcome by using intermediate structures generated along one of the normal modes previously calculated for a given target. These results may be of prime importance for further in silico drug discovery.     

Spatial and temporal expression profiles suggest the involvement of gelatinase A and membrane type 1 matrix metalloproteinase in amphibian metamorphosis

The matrix metalloproteinases (MMPs) are a family of proteases capable of degrading various components of the extracellular matrix (ECM). Among them, the membrane type MMP–1 (MT1–MMP) has been shown to participate in the activation of MMP gelatinase A (GelA), suggesting that they may function together in development and pathogenesis. Here, we have investigated the spatiotemporal expression profiles of Xenopus laevis MT1–MMP and GelA genes during thyroid-hormone-dependent metamorphosis. We have focused our studies on two organs: (1) the intestine, which undergoes first the degeneration of the tadpole epithelium through apoptosis and then the development of adult epithelium and other tissues, and (2) the tail, which completely resorbs through programmed cell death. We show that both MT1–MMP and GelA are upregulated in the intestine and tail when both organs undergo metamorphosis. Within the organs, MT1–MMP and GelA are coexpressed in the connective tissues during both natural and thyroid-hormone-induced metamorphosis. In addition, MT1–MMP (but not GelA) is also expressed in the longitudinal muscle cells of the metamorphosing intestine. These results suggest that MT1–MMP and GelA function together in the ECM degradation or remodeling associated with metamorphosis and that MT1–MMP has additional GelA–independent roles in the development of adult longitudinal muscle in the intestine. This research was supported by the Intramural Research Program of the National Institute of Child Health and Human Development, NIH. T. Hasebe and H. Matsuda were supported in part by JSPS (NIH) fellowships.     

Cardosins improve neuronal regeneration after cell disruption: a comparative expression study

The establishment of primary cell cultures is invaluable for studying cell and molecular biological questions. Although primary cell cultures more closely resemble and function like in the native environment, during the culture establishment the cells undergo several changes including the damage sustained during their removal from original tissue. The resultant cells have to rebalance the expression of their processing molecules to ascertain matrix signalling that ensure cell adaptation and consequent proliferation. Hence, we used cardosin, a novel plant enzyme for tissue disaggregation, for isolating and culturing neuronal cells from embryonic rats. The present investigation reports the molecular events, mainly related with matrix metalloproteinases (MMPs)/tissue inhibitor of metalloproteinase (TIMPs) expression, which could substantiate the superior neurite outgrowth and dendritic extension previously described. It was observed that 24 h after primary culture establishment, MMP-2 and MMP-9 messenger RNA (mRNA) are significantly upregulated, while the expression of TIMP-1 and TIMP-2 is unaltered. Regarding the role of laminin in neuronal pathfinding, it was found that the use of anti-laminin antibody and arginine–glycine–aspartate (RGD) peptide exerted inhibitory effects on neurite outgrowth after mechanical lesion where the expression of MMP-9 and TIMP-1 is upregulated under non-permissive conditions in response to mechanical injury.     

Design, synthesis and preliminary evaluation of new cinnamoyl pyrrolidine derivatives as potent gelatinase inhibitors

A series of new cinnamoyl pyrrolidine derivatives have been synthesized based on the l-hydroxyproline scaffold and inhibiting activities on gelatinase (MMP-2 and -9) and APN were tested. Structure–activity relationship studies showed that the side chain with aromatic ring at C4in pyrrolidine ring showed better inhibitory activities on gelatinase than aliphatic side chain. Most compounds exhibited poor activities on APN compared with MMP-2. Within this series, three compounds, A8, B9 and C10, have the good potency (IC₅₀ = 5.2–9.7 nM) and could be used as lead compounds in the future.     

Third generation of matrix metalloprotease inhibitors: Gain in selectivity by targeting the depth of the S1′ cavity

Following the disappointment of clinical trials with early broad-spectrum synthetic inhibitors of matrix metalloproteases (MMPs), the field is now resurging with a new focus on the development of more selective inhibitors. Compounds able to fully discriminate between different members of the MMP family are sorely needed for therapeutic applications. Chemical efforts over the past years have led to very few selective inhibitors of MMPs. The over-exploitation of the hydroxamate function, or other strong zinc-binding groups, might be responsible for this failure. By resorting to weaker zinc-chelating groups, like phosphoryl or carboxylic groups, inhibitors with improved selectivity profiles have been developed. However, the most encouraging results have been obtained with compounds that avoid targeting the zinc but gain their affinity from plunging deeper into the MMP S₁′ cavity. Analyses of the crystal structures of MMP-13 and MMP-8 complexes with such compounds provide novel insights for the design of more selective inhibitors for other members of the MMP family.     

多细胞趋磁原核生物研究进展

多细胞趋磁原核生物指一类由含有磁小体的革兰氏阴性原核细胞聚集而成的球形或者卵球形细胞聚集体,一般由745个细胞组成,直径在223μm之间,它们可在地磁场或外加磁场中沿磁力线定位并做定向运动。通过对巴西潟湖、美国盐湖、德国和法国海岸带花瓣型MMPs以及最近对中国青岛海域潮间带菠萝型MMPs研究结果的总结,分别从MMPs的生物学特征、细胞内容物以及生态学分布及分类地位等方面的研究进行综述,并对未来研究方向进行一定的展望。     

T N F-α、IL-10 、基质金属蛋白酶与MO D S 的研究进展

多器官功能障碍综合征（multiple organ dysfunction syndrome,MODS）已成为临床死亡的主要原因,是目前国际医学界研究热点之一,本文主要介绍了TNF-α、IL-10、基质金属蛋白酶的生物学作用以及它们与MODS的关系,为MODS的治疗和研究提供一些参考。     

Expression of matrix metalloproteinase 8 (MMP-8) and tyrosinase-related protein-1 (TYRP-1) correlates with the absence of metastasis in an isogenic human breast cancer model

The multi-step nature of metastasis poses difficulties in both design and interpretation of experiments to unveil the mechanisms causing the process. In order to facilitate such studies, we have previously derived a pair of breast tumor cell lines that originate from the same breast tumor but which have diametrically opposite metastatic capabilities. In this system, the monoclonal cell line M-4A4 is metastatic to the lungs of athymic mice, whereas clone NM-2C5 is equally tumorigenic but non-metastatic. Here, we report that representational difference analysis (RDA) of cDNA obtained from the two clonal populations revealed an increased expression of tyrosinase-related protein-1 (TYRP-1) and the matrix metalloproteinase-8 (MMP-8) genes in the non-metastatic cell line. RNA and protein analyses in cultured cells and in primary xenograft tissues confirmed that the non-metastatic cell line expresses TYRP-1 and MMP-8 at levels that are at least 20-fold higher than the metastatic counterpart. Other members of the MMP family (MMP-9 and MMP-2) and the tissue inhibitor of metalloproteinase-2 (TIMP-2) were found to be expressed at similar levels in both populations. The effects of MMP-8 and TYRP-1 on in vitro invasion and migration were assessed in cells whose expression of these genes was altered by stable transduction with sense and antisense constructs. Specific down-regulation of MMP-8 in non-metastatic NM-2C5 cells resulted in a 2.5-fold increased capacity to invade through Matrigel. Unlike other members of the matrix metalloproteinase family, MMP-8 has not previously been implicated in the processes of tumorigenesis or metastasis. The successful identification of two proteins that are differentially expressed in these matched clonal cell lines and the tumors that they produce demonstrates the feasibility of using this approach to search for genes that are associated with aberrant differentiation toward metastatic behavior.     

Gelatinolytic and fibrinolytic activity in fresh-frozen plasma

Fresh-frozen plasma (FFP) was evaluated for gelatinolytic and fibrinolytic activity. Gelatin zymography revealed that gelatinase A (MMP-2) was constitutively present in FFP whereas gelatinase B (MMP-9) was present at variable levels. The presence of MMP-9 likely represents differential release from neutrophils during FFP collection or processing. Although fibrin matrices generated from FFP or freshly prepared plasma contained characteristic crosslinked - dimers and -monomers, matrices generated from FFP were resistant to spontaneous plasmin-dependent fibrinolysis. This observation likely stems from the plasminogen activator instability and could potentially lead to a hypofibrinolytic state. The impact of these in vitro findings to protease balance in patients receiving multiple FFP doses remains to be determined.     

uPA/plasmin system-mediated MMP-9 activation is implicated in bronchial epithelial cell migration

To examine the effects of the uPA/plasmin system on cell migration in relation to the activation of MMP-9, we used ex vivo and in vitro wound-repair models of human bronchial epithelial cells and videomicroscopy techniques that make possible cell tracking and quantification of cell migration speeds. We observed that uPA was only detected in migrating cells at the wound edges and located at crucial sites for cell/extracellular matrix interactions. The implication of uPA in human bronchial epithelial cell migration was studied by incubating cultures with a monoclonal antibody raised against uPA and these experiments led to a 70% reduction in cell velocity. To examine the effects of the plasmin system on cell migration, we incubated cultures with increasing concentrations of plasmin or activated MMP-9. We observed a significant dose-dependent increase in cell migration velocity with plasmin (P < 0.001) and MMP-9 (P < 0.001). Moreover, addition of exogenous plasmin led to a twofold increase of activated MMP-9 in migrating cells. We also demonstrated that the addition of anti-uPA IgG led to an inhibition of 43% of activated MMP-9. In conclusion, these results show that uPA is involved in human bronchial epithelial cells migration. This action is mediated by the generation of plasmin, which in turn activates MMP-9, thus making possible cell migration.