Neurymenolide A, a Novel Mitotic Spindle Poison from the New Caledonian Rhodophyta Phacelocarpus neurymenioides.

The marine α-pyrone macrolide neurymenolide A was previously isolated from the Fijian red macroalga, Neurymenia fraxinifolia, and characterized as an antibacterial agent against antibiotic-resistant strains that also exhibited moderate cytotoxicity in vitro against cancer cell lines. This compound was also shown to exhibit allelopathic effects on Scleractinian corals. However, to date no mechanism of action has been described in the literature. The present study showed, for the first time, the isolation of neurymenolide A from the New Caledonian Rhodophyta, Phacelocarpus neurymenioides. We confirmed the compound's moderate cytotoxicity in vitro against several human cell lines, including solid and hematological malignancies. Furthermore, we combined fluorescence microscopy and flow cytometry to demonstrate that treatment of U-2 OS osteosarcoma human cells with neurymenolide A could block cell division in prometaphase by inhibiting the correct formation of the mitotic spindle, which induced a mitotic catastrophe that led to necrosis and apoptosis. Absolute configuration of the stereogenic center C-17 of neurymenolide A was deduced by comparison of the experimental and theoretical circular dichroism spectra. Since the total synthesis of this compound has already been described, our findings open new avenues in cancer treatment for this class of marine molecules, including a new source for the natural product.

The Anti-Candida albicans Agent 4-AN Inhibits Multiple Protein Kinases.

Small molecules containing quinone and/or oxime moieties have been found as promising anti-fungal agents. One of them is 4-AN, a recently reported potent anti-Candida compound, which inhibits the formation of hyphae, decreases the level of cellular phosphoproteome, and finally shows no toxicity towards human erythrocytes and zebrafish embryos. Here, further research on 4-AN is presented. The results revealed that the compound: (i) Kills Candida clinical isolates, including these with developed antibiotic resistance, (ii) affects mature biofilm, and (iii) moderately disrupts membrane permeability. Atomic force microscopy studies revealed a slight influence of 4-AN on the cell surface architecture. 4-AN was also shown to inhibit multiple various protein kinases, a characteristic shared by most of the ATP-competitive inhibitors. The presented compound can be used in novel strategies in the fight against candidiasis, and reversible protein phosphorylation should be taken into consideration as a target in designing these strategies.

Development of new highly potent imidazo[1,2-b]pyridazines targeting Toxoplasma gondii calcium-dependent protein kinase 1.

Using a structure-based design approach, we have developed a new series of imidazo[1,2-b]pyridazines, targeting the calcium-dependent protein kinase-1 (CDPK1) from Toxoplasma gondii. Twenty derivatives were thus synthesized. Structure-activity relationships and docking studies confirmed the binding mode of these inhibitors within the ATP binding pocket of TgCDPK1. Two lead compounds (16a and 16f) were then identified, which were able to block TgCDPK1 enzymatic activity at low nanomolar concentrations, with a good selectivity profile against a panel of mammalian kinases. The potential of these inhibitors was confirmed in vitro on T. gondii growth, with EC50 values of 100 nM and 70 nM, respectively. These best candidates also displayed low toxicity to mammalian cells and were selected for further in vivo investigations on murine model of acute toxoplasmosis.

Chemical synthesis and biological validation of immobilized protein kinase inhibitory Leucettines.

Leucettines, a family of marine sponge-derived 2-aminoimidazolone alkaloids, are potent inhibitors of DYRKs (dual-specificity, tyrosine phosphorylation regulated kinases) and CLKs (cdc2-like kinases). They constitute promising pharmacological leads for the treatment of several diseases, including Alzheimer's disease and Down syndrome. In order to investigate the scope of potential targets of Leucettine L41, a representative member of the chemical class, we designed an affinity chromatography strategy based on agarose-immobilized leucettines. A synthesis protocol for the attachment of a polyethylene (3 or 4 units) linker to L41 was first established. The linker attachment site on L41 was selected on the basis of the co-crystal structure of L41 with several kinases. L41 was then covalently bound to agarose beads through the primary amine located at the end of the linker. Control, kinase inactive Leucettine was also immobilized, as well as free linker devoid of ligand. Extracts of several mouse tissues revealed a complex pattern of interacting proteins, some of which probably resulting from non-specific, hydrophobic binding, while others representing bona fide Leucettine-interacting proteins. DYRK1A and GSK-3 (glycogen synthase kinase-3) were confirmed as interacting targets by Western blotting in various mouse tissues. The Leucettine affinity chromatography resin constitutes a powerful tool to purify and identify the targets of this new promising therapeutic class of molecules.