Phage display as a powerful tool to engineer protease inhibitors

Since its introduction by Georges Smith some 25 years ago, phage display has proved to be a powerful molecular technique for selecting proteins with desired biological properties from huge libraries. Early on, various protease inhibitor scaffolds were displayed at the surface of filamentous phages to select new inhibitors with shifted specificities and enhanced affinities towards one or more target protease(s). The past two decades have seen a number of natural protease inhibitors subjected to phage display, mostly to shift and increase their inhibitory specificity, but also to explore the molecular mechanisms by which they interact with their cognate enzymes with low or very high selectivity. This review focuses on the major uses of phage display in the field of protein protease inhibitors. The exquisite molecular mechanisms by which natural protease inhibitors prevent unwanted or excessive proteolysis in cells and tissues are also examined along with some of the general principles underlying the way phage display is applied to these molecules.     

Location of cell cycle regulators cyclin B1, cyclin A, PCNA, Ki67 and cell cycle inhibitors p21, p27 and p57 in human first trimester placenta and deciduas

Although placental development and implantation depend on the coordination of trophoblast proliferation, differentiation and invasion, little is known about the cell cycle regulators that govern the control of these events. The hypothesis that the coordinated expression of cell cycle progression and inhibition factors will determine whether cytotrophoblasts proliferate or undergo cell cycle arrest or cell cycle exit allowing subsequent differentiation was tested. The cell cycle promotors cyclin A, cyclin B1, PCNA, Ki67 and the cell cycle inhibitors p21, p27 and p57 were immunolocalized in tissue sections of first trimester pregnancies (weeks 6 and 9–12). Double staining with cytokeratin 7 allowed unambiguous identification of extravillous cytotrophoblast (EVT) in the decidua. Villous cytotrophoblasts were immunolabelled for Ki67 and cyclin A but only few were stained with anti-cyclin B1. The syncytiotrophoblast was devoid of immunoreactivity for any of the cell cycle progression factors. It expressed especially p21, whereas p27 and p57 were predominantly found in villous cytotrophoblasts. PCNA, Ki67, cyclin A and cyclin B1 were immunolocalized in proximal and distal EVTs of anchoring villi and in EVT which had invaded the upper decidual segments. All EVTs strongly expressed p27 and p57, but not p21. These data clearly suggest different functions for p21, p27 and p57 in placental development with distinct roles for p21 and p57 in syncytiotrophoblast and EVT differentiation, respectively. p27 appears to be involved in both the processes. The results may also challenge the concept of differential mitotic activity in the proximal and distal parts of the first trimester cytotrophoblast cell column, but more functional studies are clearly needed. The presence of p27 and p57 in EVT cells, which invade the deciduas deeply, may account for the loss of mitogenic potential of these cells.     

Synthesis and NMR experiments of (4,5,6-13C)-deoxymannojirimycin. A new entry to 13C-labeled glycosidase inhibitors

The synthesis of (4,5,6-13C)-deoxymannojirimycin is described. The route employed is based on Sharpless asymmetric epoxidation of (1,2,3-13C)(E)-2,4-pentadien-1-ol and uses ring-closing metathesis as a key step. The labeled compound may be easily used for protein-binding experiments using NMR spectroscopic methods.     

<Emphasis Type="Italic">cis</Emphasis>-Cinnamoyl Glucoside as a Major Plant Growth Inhibitor Contained in <Emphasis Type="Italic">Spiraea prunifolia</Emphasis>

Crude extracts of the leaves of Spiraea prunifolia Sieb. showed high plant-growth-inhibiting activity comparable to that of S. thunbergii extracts. To isolate the causal compound in S. prunifolia, we performed bioassay-directed purification by monitoring the biological activity per unit weight of the organism containing the bioactive compound (total activity). We isolated 1-O-cis-cinnamoyl-β-D-glucopyranose (cis-CG) and identified it as the most important growth-inhibiting constituent in the crude extracts. We did not detect 6-O-(4′-hydroxy-2′-methylenebutyroyl)-1-O-cis-cinnamoyl-β-D-glucopyranose (cis-BCG) in S. prunifolia, though it is a major plant growth inhibitor in S. thunbergii together with cis-CG. We estimated the cis-CG content in S. prunifolia to be 3.84 mmol kg⁻¹ F.W. This amount is comparable to the cis-CG plus cis-BCG content in S. thunbergii (3.59 mmol kg⁻¹ F.W.). This indicates that S. prunifolia and S. thunbergii have equally high potential to inhibit plant growth, and cis-CG acts as the most important plant-growth inhibitor in S. prunifolia extracts.     

Differential inter- and intra-specific defense induction in Lupinus by Myzus persicae feeding

Experiments were conducted to investigate the potential induction of plant defenses by Myzus persicae Sulzer (Homoptera: Aphididae) feeding on five lupin, Lupinus spp. (Leguminosae), varieties with well‐characterized levels of aphid resistance. Myzus persicae feeding on L. angustifolius and L. luteus varieties induced genotype‐specific changes in their host that were not consistent with the level of aphid resistance or the plant species. The plant responses were systemically detected by apterous and alate forms of the aphids. Chemical assays revealed no induction of oxidizing enzyme (catalase, peroxidase, or polyphenol oxidase) activity, serine or cystein proteinase inhibitors, or soluble phenolics in any of the five varieties tested following 3 days of feeding by 10 or 30 aphids. However, there were significant differences among the five lupin varieties in the levels of peroxidase and polyphenol oxidase activity, proteinase inhibitors, and soluble phenolics.     

Antiproliferative and ultrastructural effects of BPQ-OH, a specific inhibitor of squalene synthase, on Leishmania amazonensis

Parasites of the Leishmania genus require for the growth and viability the de novo synthesis of specific sterols as such as episterol and 5-dehydroepisterol because cholesterol, which is abundant in their mammalian hosts, does not fulfill the parasite sterol requirements. Squalene synthase catalyzes the first committed step in the sterol biosynthesis and has been studied as a possible target for the treatment of high cholesterol levels in humans. In this work we investigated the antiproliferative and ultrastructural effects induced by 3-(biphenyl-4-yl)-3-hydroxyquinuclidine (BPQ-OH), a specific inhibitor of squalene synthase, on promastigote and amastigote forms of Leishmania amazonensis. BPQ-OH had a potent dose-dependent growth inhibitory effect against promastigotes and amastigotes, with IC(50) values 0.85 and 0.11 microM, respectively. Ultrastructural analysis of the treated parasites revealed several changes in the morphology of promastigote forms. The main ultrastructural change was found in the plasma membrane, which showed signs of disorganization, with the concomitant formation of elaborated structures. We also observed alterations in the mitochondrion-kinetoplast complex such as mitochondrial swelling, rupture of its internal membrane and an abnormal compaction of the kinetoplast. Other alterations included the appearance of multivesicular bodies, myelin-like figures, alterations of the flagellar membrane and presence of parasites with two or more nuclei and kinetoplasts. We conclude that the BPQ-OH was a potent growth inhibitor of L. amazonensis, which led to profound changes of the parasite's ultrastructure and might be a valuable lead compound for the development of novel anti-Leishmania agents.     

Apoptosis in anititumor strategies: Modulation of cell cycle or differentiation

There is a strong evidence that administration of antitumor drugs triggers apoptotic death of target cells. A characteristic feature of appotosis is active participation of the affected cell in its demise. Attempts have been made, therefore, to potentiate the cytotoxicity of a variety of agents by modulating the propensity of cells to respond by apoptosis. Several strategies to enhance apoptosis that involve modulation of the cell cycle or differentiation are discussed. Loss of control of the G₁ checkpoint in tumor cells allows one to design treatments that arrest normal cells at the checkpoint and attempt to selectively kill tumor cells with S phase specific drugs. The possibility of a restoration of the apoptosis triggering function of the tumor suppressor gene p53 when the G₁ checkpoint function is abolished is expected to increase tumor cells' sensitivity to S phase poisons. Because induction of apoptosis by many antitumor drugs is cell cycle phase specific, drug combinations that preferentially trigger apoptosis at different phases of the cycle, or recruitment of cells to the sensitive phase, offer another antitumor strategy. There is also evidence that apoptosis is potentiated when cell differentiation is triggered follwing DNA damage. This observation suggests that strategies which combine DNA damaging and differentiating drugs, under conditions where the latter are administered following DNA damage caused by the former, may be successful.