Anti-inflammatory Activity of the Protein Z-Dependent Protease Inhibitor.

The protein Z (PZ)-dependent plasma protease inhibitor (ZPI) is a glycoprotein that inhibits factor XIa and, in the presence of PZ, FXa. Recently, ZPI has been shown to be an acute-phase protein (APP). As usually APPs downregulate the harmful effects of inflammation, we tested whether ZPI could modulate the increase of cytokines observed in inflammatory states. We observed that recombinant human ZPI (rhZPI) significantly decreases the levels of interleukin (IL)-1, IL-6, and tumor necrosis factor- α (TNF-α) induced by lipopolysaccharide (LPS) in a whole blood model. This inhibitory effect was unaffected by the presence of PZ or heparin. A ZPI mutant within the reactive loop center ZPI (Y387A), lacking anticoagulant activity, still had an anti-inflammatory activity. Surprisingly, rhZPI did not inhibit the synthesis of IL-6 or TNF-α when purified monocytes were stimulated by LPS, whereas the inhibitory effect was evidenced when lymphocytes were added to monocytes. The requirement of lymphocytes could be due to the synthesis of CCL5 (RANTES), a chemokine mainly produced by activated lymphocytes which is induced by rhZPI, and which can reduce the production of proinflammatory cytokines in whole blood. Lastly, we observed that the intraperitoneal injection of rhZPI significantly decreased LPS-induced IL-6 and TNF-α production in mouse plasma.

Cyclic bridged analogs of isoCA-4: Design, synthesis and biological evaluation.

In this work, a series of cyclic bridged analogs of isocombretastatin A-4 (isoCA-4) with phenyl or pyridine linkers were designed and synthesized. The synthesis of the desired analogs was performed by the formation of nitro-vinyl intermediates, followed by a Cadogan cyclization. Structure activity relationship (SAR) study demonstrates the critical role of the combination of quinaldine as ring A, pyridine as the linker, and indole as ring B in the same molecule, for the cytotoxic activity. Among all tested compounds, compound 42 showed the highest antiproliferative activity against a panel of cancer cell lines with average IC50 values of 5.6 nM. Also, compound 42 showed high antiproliferative activity against the MDR1-overexpressing K562R cell line; thus, it was 1.5- and 12-fold more active than the reference compounds, isoCA-4 and CA-4, respectively. Moreover, 42 displayed a strong antiproliferative activity against the colon-carcinoma cells (HT-29), which are resistant to combretastatin A-4 and isoCA-4, and it was found to be 8000-fold more active than natural CA-4. Compound 42 also effectively inhibited tubulin polymerization both in vitro and in cells, and induced cell cycle arrest in G2/M phase. Next, we demonstrated that compound 42 dose-dependently caused caspase-induced apoptosis of K562 cells through mitochondrial dysfunction. Finally, we evaluated the effect of compound 42 in human no cancer cells compared to the reference compound. We demonstrated that 42 was 73 times less cytotoxic than isoCA-4 in quiescent peripheral blood lymphocytes (PBLs). In summary, these results suggest that compound 42 represents a promising tubulin inhibitor worthy of further investigation.

Design and Synthesis of Tubulin and Histone Deacetylase Inhibitor Based on iso-Combretastatin A-4.

Designing multitarget drugs have raised considerable interest due to their advantages in the treatment of complex diseases such as cancer. Their design constitutes a challenge in antitumor drug discovery. The present study reports a dual inhibition of tubulin polymerization and HDAC activity. On the basis of 1,1-diarylethylenes ( isoCA-4) and belinostat, a series of hybrid molecules was successfully designed and synthesized. In particular compounds, 5f and 5h were proven to be potent inhibitors of both tubulin polymerization and HDAC8 leading to excellent antiproliferative activity.

A fluorine scan of a tubulin polymerization inhibitor isocombretastatin A-4: Design, synthesis, molecular modelling, and biological evaluation.

A novel series of tubulin polymerization inhibitors, based on fluorinated derivatives of isocombretastatin A-4 was synthesized with the goal of evaluating the effect of these compounds on the proliferative activity. The introduction of fluorine atom was performed on the phenyl ring or at the linker between the two aromatic rings. The modification of isoCA-4 by introduction of difluoromethoxy group at the para-position (3i) and substitution of the two protons of the linker by two fluorine atoms (3m), produced the most active compounds in the series, with IC50 values of 0.15-2.2 nM (3i) and 0.1-2 nM (3m) respectively, against a panel of six cancer cell lines. Compounds 3i and 3m had greater antiproliferative activity in comparison with references CA-4 or isoCA-4, the presence of fluorine group leads to a significant enhancement of the antiproliferative activity. Molecular docking studies indicated that compounds 3i and 3m occupy the colchicine binding site of tubulin. Evaluation of cytotoxicity in Human noncancer cells indicated that the compounds 3i and 3m were practically ineffective in quiescent peripheral blood lymphocytes, and may have a selective antiproliferative activity against cancer cells. Analyses of cell cycle distribution, and morphological microtubules organization showed that compound 3m induced G2/M phase arrest and, dramatically disrupted the microtubule network.

Gas6 deficiency in recipient mice of allogeneic transplantation alleviates hepatic graft-versus-host disease.

Growth arrest-specific gene 6 (Gas6) is expressed in antigen-presenting cells and endothelial cells (ECs) but not in T cells. When wild-type (WT) or Gas6(-/-) mice received allogeneic non-T cell-depleted bone marrow cells, hepatic graft-versus-host disease (GVHD) was alleviated in Gas6(-/-) recipients regardless of donor genotype, but not in WT recipients. T-cell infiltration was more prominent and diffuse in WT than in Gas6(-/-) recipients' liver. When mice received 0.5 x 10(6) allogeneic T cells with T cell-depleted allogeneic bone marrow, clinical signs indicated that GVHD was less severe in Gas6(-/-) than in WT recipients, as shown by a significant improvement of the survival and reduced liver GVHD. These data demonstrate that donor cells were not involved in the protection mechanism. In addition, lack of Gas6 in antigen-presenting cells did not affect WT or Gas6(-/-) T-cell proliferation. We therefore assessed the response of WT or Gas6(-/-) ECs to tumor necrosis factor-alpha. Lymphocyte transmigration was less extensive through Gas6(-/-) than WT ECs and was not accompanied by increases in adhesion molecule levels. Thus, the lack of Gas6 in ECs impaired donor T-cell transmigration into the liver, providing a rationale for considering Gas6 pathway as a potential nonimmunosuppressive target to minimize GVHD in patients receiving allogeneic hematopoietic stem cell transplantation.

Role of the growth arrest-specific gene 6 (gas6) product in thrombus stabilization.

Growth arrest-specific gene 6 (gas6) product enhances the formation of stable platelet macroaggregates in response to various agonists. To determine whether Gas6 amplifies the response to known platelet agonists through one or more of its receptor tyrosine kinases of the Tyro3 family, mice deficient in any one of the Gas6 receptors (Gas6-Rs: Tyro3, Axl, or Mer) were submitted to thrombosis challenge and their platelet function. The loss of any one of the Gas6-Rs protects mice against thromboembolism induced by collagen-epinephrine and stasis-induced thrombosis. Importantly, these mice do not suffer spontaneous bleeding and have a normal bleeding time but a tendency to repetitively re-bleed after transient hemostasis. Re-bleeding in mice lacking any one of the Gas6-Rs is not due to thrombocytopenia or coagulopathy but to a platelet dysfunction characterized by a lack of the second wave of platelet aggregation and an impaired clot retraction, at least in part by reducing outside-in alpha(IIb)beta(3) signaling and platelet granule secretion. The early release of Gas6 by agonists perpetuates platelet activation through its three receptors, reinforcing outside-in alpha(IIb)beta(3) signaling by activation of PI3K and Akt signaling and stimulation of tyrosine phosphorylation of the beta(3) integrin. Furthermore, "trapping" Gas6 prevents pathological thrombosis, which indicates that blocking this novel cross-talk between the Gas6-Rs and alpha(IIb)beta(3) integrin may constitute a novel target for antithrombotic therapy.