Contextual compound screening for improved therapeutic discovery.

Cellular behaviors are governed by combinations of systemic and microenvironmental factors; together, these regulate cell signaling responses to growth factors. This contextual microenvironmental influence also determines drug sensitivity. Hence using in vitro systems that model contextual cellular behavior is highly beneficial for effective therapeutic development. Angiogenesis (formation of blood vessels) is driven by a series of dynamic endothelial cell signaling responses to growth factors under the influence of the vascular extracellular matrix and adjacent pericytes. In vitro primary human vascular cell co-cultures self-assemble into capillary-like structures through reciprocal heterotypic interactions that mimic angiogenic context dynamics. By using temporal live-cell imaging-based analysis, unique angiogenic microenvironments can be delineated to quantify the contextual activity of compound inhibitors. We used this in vitro organotypic contextual screening approach to conduct structure-activity relationship analysis on a combretastatin A-4 analogue series to identify novel compounds with potent vascular disrupting activity in vivo.

Combretastatin A-4 and structurally related triazole analogues induce caspase-3 and reactive oxygen species-dependent cell death in PC12 cells.

Cancer cells are more sensitive to oxidative stress due to higher levels of reactive oxygen species. Therefore, the ability of anti-cancer agent combretastatin A-4 (CA-4) and triazole analogues to induce reactive oxygen species may be important for selectivity against cancer cells. The purpose of the present study was to investigate the structural requirements for reactive oxygen species production by CA-4 and the triazole analogues Ana-2, Ana-3 and Ana-4. Ana-2 and Ana-3 mimic the cis configuration in CA-4; Ana-3 lacks the phenolic hydroxyl group, while Ana-4 mimics a trans configuration. The rat pheochromocytoma cancer cell line PC12 was used as model system. CA-4 and Ana-2 were highly toxic; Ana-3 was less toxic, whereas Ana-4 was non-toxic. The probe dihydroethidium detected reactive oxygen species production from CA-4, Ana-2, and Ana-3. CA-4 and Ana-2 also induced oxidation of the reactive oxygen species probe dihydrorhodamine and activation of caspase-3. Thus, the phenolic hydroxyl group in CA-4 and Ana-2 was necessary for dihydrorhodamine oxidation, caspase-3 activation, and increased cytotoxicity.

Synthesis, biological evaluation and molecular modeling of 1,2,3-triazole analogs of combretastatin A-1.

The synthesis, cytotoxicity, inhibition of tubulin polymerization data and anti-angiogenetic effects of seven 1,5-disubstituted 1,2,3-triazole analogs and two 1,4-disubstituted 1,2,3-triazole analogs of combretastatin A-1 (1) are reported herein. The biological studies revealed that the 1,5-disubstituted 1,2,3-triazoles 3-methoxy-6-(1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-5-yl)benzene-1,2-diol (6), 3-methoxy-6-(1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-5-yl)benzene-1,2-diamine (8) and 5-(2,3-difluoro-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazole (9) were the three most active compounds regarding inhibition of both tubulin polymerization and angiogenesis. Molecular modeling studies revealed that combretastatins 1 and 2 and analogs 5-11 could be successfully docked into the colchicine binding site of α,ß-tubulin.

1,2,3-triazole analogs of combretastatin A-4 as potential microtubule-binding agents.

A series of cis-restricted 1,4- and 1,5-disubstituted 1,2,3-triazole analogs of combretastatin A-4 (1) have been prepared. Cytotoxicity and tubulin inhibition studies showed that 2-methoxy-5-((5-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-1-yl)methyl)aniline (5e) and 2-methoxy-5-(1-(3,4,5-trimethoxybenzyl)-1H-1,2,3-triazol-5-yl)aniline (6e) were two of the most active compounds. Molecular modeling studies revealed that the N-2 and N-3 atoms in the triazole rings in 5e and 6e did not form hydrogen bonds with the amino acids in the anticipated pharmacophore.

1,5-Disubstituted 1,2,3-triazoles as cis-restricted analogues of combretastatin A-4: Synthesis, molecular modeling and evaluation as cytotoxic agents and inhibitors of tubulin.

A series of cis-restricted 1,5-disubstituted 1,2,3-triazole analogues of combretastatin A-4 (1) have been prepared. The triazole 12f, 2-methoxy-5-(1-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-5-yl)aniline, displayed potent cytotoxic activity against several cancer cell lines with IC(50) values in the nanomolar range. The ability of triazoles to inhibit tubulin polymerization has been evaluated, and 12f inhibited tubulin polymerization with IC(50)=4.8microM. Molecular modeling experiments involving 12f and the colchicine binding site of alpha,beta-tubulin showed that the triazole moiety interacts with beta-tubulin via hydrogen bonding with several amino acids.