Potent antiproliferative cembrenoids accumulate in tobacco upon infection with Rhodococcus fascians and trigger unusual microtubule dynamics in human glioblastoma cells.

AIMS: Though plant metabolic changes are known to occur during interactions with bacteria, these were rarely challenged for pharmacologically active compounds suitable for further drug development. Here, the occurrence of specific chemicals with antiproliferative activity against human cancer cell lines was evidenced in hyperplasia (leafy galls) induced when plants interact with particular phytopathogens, such as the Actinomycete Rhodococcus fascians. METHODS: We examined leafy galls fraction F3.1.1 on cell proliferation, cell division and cytoskeletal disorganization of human cancer cell lines using time-lapse videomicroscopy imaging, combined with flow cytometry and immunofluorescence analysis. We determined the F3.1.1-fraction composition by gas chromatography coupled to mass spectrometry. RESULTS: The leafy galls induced on tobacco by R. fascians yielded fraction F3.1.1 which inhibited proliferation of glioblastoma U373 cells with an IC50 of 4.5 µg/mL, F.3.1.1 was shown to increase cell division duration, cause nuclear morphological deformations and cell enlargement, and, at higher concentrations, karyokinesis defects leading to polyploidization and apoptosis. F3.1.1 consisted of a mixture of isomers belonging to the cembrenoids. The cellular defects induced by F3.1.1 were caused by a peculiar cytoskeletal disorganization, with the occurrence of fragmented tubulin and strongly organized microtubule aggregates within the same cell. Colchicine, paclitaxel, and cembrene also affected U373 cell proliferation and karyokinesis, but the induced microtubule rearrangement was very different from that provoked by F3.1.1. Altogether our data indicate that the cembrenoid isomers in F3.1.1 have a unique mode of action and are able to simultaneously modulate microtubule polymerization and stability.

A phenotypic assay to identify Chikungunya virus inhibitors targeting the nonstructural protein nsP2.

Chikungunya virus (CHIKV) is a mosquito-transmitted pathogen responsible for an acute infection of abrupt onset, characterized by high fever, polyarthralgia, myalgia, headaches, chills, and rash. In 2006, CHIKV was responsible for an epidemic outbreak of unprecedented magnitude in the Indian Ocean, stressing the need for therapeutic approaches. Since then, we have acquired a better understanding of CHIKV biology, but we are still missing active molecules against this reemerging pathogen. We recently reported that the nonstructural nsP2 protein of CHIKV induces a transcriptional shutoff that allows the virus to block cellular antiviral response. This was demonstrated using various luciferase-based reporter gene assays, including a trans-reporter system where Gal4 DNA binding domain is fused to Fos transcription factor. Here, we turned this assay into a high-throughput screening system to identify small molecules targeting nsP2-mediated shutoff. Among 3040 molecules tested, we identified one natural compound that partially blocks nsP2 activity and inhibits CHIKV replication in vitro. This proof of concept suggests that similar functional assays could be developed to target other viral proteins mediating a cellular shutoff and identify innovative therapeutic molecules.

Discovery of isoerianin analogues as promising anticancer agents.

The cytotoxic activity of a series of 23 new isoerianin derivatives with modifications on both the A and B rings was studied. Several compounds exhibited excellent antiproliferative activity at nanomolar concentrations against a panel of human cancer cell lines. The most cytotoxic compound, isoerianin (3), strongly inhibits tubulin polymerization in the micromolar range. Moreover, isoerianin leads to G2/M phase cell-cycle arrest in H1299 and K562 cancer cells, and strongly induces apoptosis. Isoerianin also disrupts the vessel-like structures formed by human umbilical vein endothelial cells (HUVECs) in vitro, suggesting that this compound may act as a vascular disrupting agent. It clearly appears that in this compound series, the 1,1-ethane bridge encountered in isoerianin derivatives can replace the 1,2-ethane bridge of natural erianin with no loss of activity. This reinforces the bioisosteric replacement approach in the combretastatin series previously reported by our research group.

Synthesis of antiproliferative flavones from calycopterin, major flavonoid of Calycopteris floribunda Lamk.

Eighteen new analogues of 5,3'-dihydroxy-3,6,7,8,4'-pentamethoxy-flavone, a potent natural cytotoxic and antimitotic flavone, were synthesized from calycopterin, the major flavonoid of Calycopteris floribunda Lamk., a traditional Asian medicinal plant. One of them, the 3'-amino substituted analogue, displayed almost the same activity as the reference compound. Pharmacomodulation at C-3' on the B-ring, and at C-5,6,7 and 8 on the A-ring allowed to refine structure-activity relationships within the cytotoxic flavones series.

Synthesis and biological evaluation of cis-locked vinylogous combretastatin-A4 analogues: derivatives with a cyclopropyl-vinyl or a cyclopropyl-amide bridge.

A series of novel combretastatin A4 analogues, in which the cis-olefinic bridge is replaced by a cyclopropyl-vinyl or a cyclopropyl-amide moiety, were synthesized and evaluated for inhibition of tubulin polymerization and antiproliferative activity. The derivative 9a with a (cis,E)-cyclopropyl-vinyl unit is the most promising compound. As expected, molecular docking of 9a has shown that only one of the cis-cyclopropyl enantiomers is a good ligand for tubulin.

Synthesis and biological evaluation of a series of tangeretin-derived chalcones.

A series of chalcones polyoxygenated on the ring A (with pentamethoxy or 2'-hydroxy-3',4',5',6'-tetramethoxy substitution patterns) was synthesized from tangeretin, a natural Citrus flavonoid. These chalcones were evaluated for their antiproliferative, activation of apoptosis, inhibition of tubulin assembly and antileishmanial activities. Comparison with the reference analogous 3',4',5'-trimethoxylated chalcones showed that such peroxygenated substitution patterns on the ring A were less beneficial to these activities.