The Antibiotic Negamycin Crosses the Bacterial Cytoplasmic Membrane by Multiple Routes.

Negamycin is a natural pseudodipeptide antibiotic with promising activity against Gram-negative and Gram-positive bacteria, including Enterobacteriaceae, Pseudomonas aeruginosa, and Staphylococcus aureus, and good efficacy in infection models. It binds to ribosomes with a novel binding mode, stimulating miscoding and inhibiting ribosome translocation. We were particularly interested in studying how the small, positively charged natural product reaches its cytoplasmic target in Escherichia coli Negamycin crosses the cytoplasmic membrane by multiple routes depending on environmental conditions. In a peptide-free medium, negamycin uses endogenous peptide transporters for active translocation, preferentially the dipeptide permease Dpp. However, in the absence of functional Dpp or in the presence of outcompeting nutrient peptides, negamycin can still enter the cytoplasm. We observed a contribution of the DppA homologs SapA and OppA, as well as of the proton-dependent oligopeptide transporter DtpD. Calcium strongly improves the activity of negamycin against both Gram-negative and Gram-positive bacteria, especially at concentrations around 2.5 mM, reflecting human blood levels. Calcium forms a complex with negamycin and facilitates its interaction with negatively charged phospholipids in bacterial membranes. Moreover, decreased activity at acidic pH and under anaerobic conditions points to a role of the membrane potential in negamycin uptake. Accordingly, improved activity at alkaline pH could be linked to increased uptake of [3H]negamycin. The diversity of options for membrane translocation is reflected by low resistance rates. The example of negamycin demonstrates that membrane passage of antibiotics can be multifaceted and that for cytoplasmic anti-Gram-negative drugs, understanding of permeation and target interaction are equally important.

The new sulindac derivative IND 12 reverses Ras-induced cell transformation.

The nonsteroidal anti-inflammatory drug Sulindac has chemopreventive and antitumorigenic properties. Its metabolites induce apoptosis and inhibit signaling pathways critical for malignant transformation, including the Ras pathway. Here we show that the new Sulindac derivative IND 12 reverses the phenotype of Ras-transformed MDCK-f3 cells and restores an untransformed epithelioid morphology characterized by growth in monolayers with regular cell-cell adhesions. Moreover, IND 12 treatment induces the expression at membranes of the cell adhesion protein E-cadherin and increases the level of the E-cadherin-bound beta-catenin. As a consequence, IND 12-treated MDCK-f3 cells lose their invasion capacity and regain the ability to aggregate. In the presence of IND 12, MDCK-f3 cells show regenerated expression and activity ratios of the small GTPases Rac and Rho normally found in untransformed MDCK cells. Strikingly, IND 12 treatment decreases the levels of phosphorylated mitogen-activated protein kinases, which are downstream substrates of the Ras-regulated Raf/mitogen-activated protein kinase pathway, and the level of Ras-induced activation of gene expression. Our findings identify a novel drug with high potential in cancer therapy by targeting Ras-induced cell transformation.

New indene-derivatives with anti-proliferative properties.

Metabolites of the non-steroidal anti-inflammatory drug Sulindac inhibit cell proliferation by affecting several intracellular signaling pathways including the tumorigenic Ras/Raf/MAPK pathway. Here, we report the synthesis of eight new indene derivatives derived from the Sulindac structure, and present data on their anti-proliferative properties and their effects on the p21ras protein.