A cytotoxic survey on 2-amino-1H-imidazol based synthetic marine sponge alkaloid analogues.

Here, we describe the synthesis and biologic activity evaluation of 20 novel synthetic marine sponge alkaloid analogues with 2-amino-1H-imidazol (2-AI) core. Cytotoxicity was tested on murine 4T1 breast cancer, A549 human lung cancer, and HL-60 human myeloid leukemia cells by the resazurin assay. A total of 18 of 20 compounds showed cytotoxic effect on the cancer cell lines with different potential. Viability of healthy human fibroblasts and peripheral blood mononuclear cells upon treatment was less hampered compared to cancer cell lines supporting tumor cell specific cytotoxicity of our compounds. The most cytotoxic compounds resulted the following IC50 values 28: 2.91 µM on HL-60 cells, and 29: 3.1 µM on 4T1 cells. The A549 cells were less sensitive to the treatments with IC50 15 µM for both 28 and 29. Flow cytometry demonstrated the apoptotic effect of the most active seven compounds inducing phosphatidylserine exposure and sub-G1 fragmentation of nuclear DNA. Cell cycle arrest was also observed. Four compounds caused depolarization of the mitochondrial membrane potential as an early event of apoptosis. Two lead compounds inhibited tumor growth in vivo in the 4T1 triple negative breast cancer and A549 human lung adenocarcinoma xenograft models. Novel marine sponge alkaloid analogues are demonstrated as potential anticancer agents for further development.

PMDTA-catalyzed multicomponent synthesis and biological activity of 2-amino-4H-chromenes containing a phosphonate or phosphine oxide moiety.

A new approach for the preparation of (2-amino-3-cyano-4H-chromen-4-yl)phosphonate derivatives is described. The multicomponent reaction of salicylaldehydes, malononitrile and dialkyl phosphites catalyzed by pentamethyldiethylenetriamine (PMDTA) provided the bicyclic derivatives in high yields. The method developed did not require chromatographic separation, since the products could be recovered from the reaction mixture by simple filtration. Our approach made also possible condensation with secondary phosphine oxides, and this reaction has not been previously reported in the literature. The crystal structures of five derivatives were studied by single-crystal XRD analysis. The in vitro cytotoxicity on different cell lines and the antibacterial activity of the (2-amino-4H-chromen-4-yl)phosphonates synthesized were also explored. According to the IC50 values determined, several derivatives showed moderate or promising activity against mouse fibroblast (NIH/3T3) and human promyelocytic leukemia (HL-60) cells. Furthermore, three (2-amino-3-cyano-4H-chromen-4-yl)phosphine oxides were active against selected Gram-positive bacteria.

Synthesis and In Vitro Cytotoxicity and Antibacterial Activity of Novel 1,2,3-Triazol-5-yl-phosphonates.

Novel 1,2,3-triazol-5-yl-phosphonates were prepared by the copper(I)-catalyzed domino reaction of phenylacetylene, organic azides and dialkyl phosphites. The process was optimized on the synthesis of the dibutyl (1-benzyl-4-phenyl-1H-1,2,3-triazol-5-yl)phosphonate in respect of the catalyst, the base and the solvent, as well as of the reaction parameters (molar ratio of the starting materials, atmosphere, temperature and reaction time). The method elaborated could be applied to a range of organic azides and dialkyl phosphites, which confirmed the large scope and the functional group tolerance. The in vitro cytotoxicity on different cell lines and the antibacterial activity of the synthesized 1,2,3-triazol-5-yl-phosphonates was explored. According to the IC50 values determined, only modest antibacterial effect was detected, while some derivatives showed moderate activity against human promyelocytic leukemia HL-60 cells.

The Anti-Inflammatory Role of Mannich Curcuminoids; Special Focus on Colitis.

The incidence of inflammatory bowel disease (IBD) increases gradually in Western countries with high need for novel therapeutic interventions. Mannich curcuminoids, C142 or C150 synthetized in our laboratory, have been tested for anti-inflammatory activity in a rat model of TNBS (2,4,6-trinitrobenzenesulphonic acid) induced colitis. Treatment with C142 or C150 reduced leukocyte infiltration to the submucosa and muscular propria of the inflamed gut. C142 or C150 rescued the loss of body weight and C150 decreased the weight of standard colon preparations proportional with 20% less tissue oedema. Both C142 and C150 curcumin analogues caused 25% decrease in the severity of colonic inflammation and haemorrhagic lesion size. Colonic MPO (myeloperoxidase) enzyme activity as an indicator of intense neutrophil infiltration was 50% decreased either by C142 or C150 Mannich curcuminoids. Lipopolysaccharide (LPS) co-treatment with Mannich curcuminoids inhibited NF-κB (nuclear factor kappa B) activity on a concentration-dependent manner in an NF-κB-driven luciferase expressing reporter cell line. Co-treatment with LPS and curcuminoids, C142 or C150, resulted in NF-κB inhibition with 3.57 µM or 1.6 µM half maximal effective concentration (EC50) values, respectively. C150 exerted a profound inhibition of the expression of inflammatory cytokines, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-4 (IL-4) in human PBMCs (peripheral blood mononuclear cells) upon LPS stimulus. Mannich curcuminoids reported herein possess a powerful anti-inflammatory activity.

The actin-binding ERM protein Moesin directly regulates spindle assembly and function during mitosis.

Ezrin-Radixin-Moesin proteins are highly conserved, actin-binding cytoskeletal proteins that play an essential role in microvilli formation, T-cell activation, and tumor metastasis by linking actin filaments to the plasma membrane. Recent studies demonstrated that the only Ezrin-Radixin-Moesin protein of Drosophila melanogaster, Moesin, is involved in mitotic spindle function through stabilizing cell shape and microtubules at the cell cortex. We previously observed that Moesin localizes to the mitotic spindle; hence, we tested for the biological significance of this surprising localization and investigated whether it plays a direct role in spindle function. To separate the cortical and spindle functions of Moesin during mitosis we combined cell biological and genetic methods. We used early Drosophila embryos, in which mitosis occurs in the absence of a cell cortex, and found in vivo evidence for the direct requirement of Moesin in mitotic spindle assembly and function. We also found that the accumulation of Moesin precedes the construction of the microtubule spindle, and the fusiform structure formed by Moesin persists even after the microtubules have disassembled.

Immunoprofiling of Drosophila Hemocytes by Single-cell Mass Cytometry.

Single-cell mass cytometry (SCMC) combines features of traditional flow cytometry (i.e., fluorescence-activated cell sorting) with mass spectrometry, making it possible to measure several parameters at the single-cell level for a complex analysis of biological regulatory mechanisms. In this study, weoptimizedSCMC to analyze hemocytes of the Drosophila innate immune system. We used metal-conjugated antibodies (against cell surface antigens H2, H3, H18, L1, L4, and P1, and intracellular antigens 3A5 and L2) and anti-IgM (against cell surface antigen L6) to detect the levels of antigens, while anti-GFP was used to detect crystal cells in the immune-induced samples. We investigated the antigen expression profile of single cells and hemocyte populations in naive states, in immune-induced states, in tumorous mutants bearing a driver mutation in the Drosophila homologue of Janus kinase (hopTum) and carrying a deficiency of the tumor suppressor gene lethal(3)malignant blood neoplasm-1  [l(3)mbn1], as well as in stem cell maintenance-defective hdcΔ84 mutant larvae. Multidimensional analysis enabled the discrimination of the functionally different major hemocyte subsets for lamellocytes, plasmatocytes, and crystal cells, anddelineated the unique immunophenotype of Drosophila mutants. We have identified subpopulations of L2+/P1+ and L2+/L4+/P1+ transitional phenotype cells in the tumorous strains l(3)mbn1 and hopTum, respectively, and a subpopulation of L4+/P1+ cells upon immune induction. Our results demonstrated for the first time that SCMC, combined with multidimensional bioinformatic analysis, represents a versatile and powerful tool to deeply analyze the regulation of cell-mediated immunity of Drosophila.

The raspberry Gene Is Involved in the Regulation of the Cellular Immune Response in Drosophila melanogaster.

Drosophila is an extremely useful model organism for understanding how innate immune mechanisms defend against microbes and parasitoids. Large foreign objects trigger a potent cellular immune response in Drosophila larva. In the case of endoparasitoid wasp eggs, this response includes hemocyte proliferation, lamellocyte differentiation and eventual encapsulation of the egg. The encapsulation reaction involves the attachment and spreading of hemocytes around the egg, which requires cytoskeletal rearrangements, changes in adhesion properties and cell shape, as well as melanization of the capsule. Guanine nucleotide metabolism has an essential role in the regulation of pathways necessary for this encapsulation response. Here, we show that the Drosophila inosine 5'-monophosphate dehydrogenase (IMPDH), encoded by raspberry (ras), is centrally important for a proper cellular immune response against eggs from the parasitoid wasp Leptopilina boulardi. Notably, hemocyte attachment to the egg and subsequent melanization of the capsule are deficient in hypomorphic ras mutant larvae, which results in a compromised cellular immune response and increased survival of the parasitoid.

A novel method for the identification of factors involved in host-pathogen interactions in Drosophila melanogaster.

A new method was established, standardized and validated for screening factors involved in the response to septic injury in Drosophila melanogaster. The method, based on inducing lesion by removing the tarsal segments of the first pair of legs of Drosophila adults and exposing them to different bacteria, imitates injury that often occurs in the natural habitat. The method is easy to perform, highly reproducible and suitable for large-scale genetic screens with the aim of identifying factors involved in host-pathogen interactions. The technique was validated by using mutant variations of different components of the immune response, blood clotting as well as the involvement of a number of genes known to be instrumental in the humoral and cell-mediated immune responses of Drosophila was confirmed. Moreover, the combination of the present method with antibiotic treatment allows the screening of potential antimicrobial drugs in vivo.