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The natural product (−)-pironetin is a structurally simple small molecule microtubule-perturbing agent whose biological activities appear to be exquisitely dependent on defined stereochemistry and the presence of an eletrophilic α,β-unsaturated lactone moiety. We used alkaloid-catalyzed acyl halide-aldehyde cyclocondensation reactions in asymmetric total syntheses of (−)-pironetin and three synthetic analogs, and evaluated their biological activities by high-content analysis in cell culture and in a zebrafish model. Synthetic (−)-pironetin and 2,3-dihydro-3-hydroxypironetin caused mitotic arrest and programmed cell death in human lung cancer cells but not in normal lung fibroblasts, had nanomolar growth inhibitory activity in multi-drug resistant cells, and inhibited neovascularization in zebrafish embryos. Synthetic (−)-pironetin delayed the onset but increased the extent of tubulin assembly in vitro. The data illustrate the power of acyl halide-aldehyde cyclocondensation to generate biologically active synthetic analogs of stereochemically complex targets and suggest that (−)-pironetin and 2,3-dihydro-3-hydroxypironetin possess unique properties that may bestow them with advantages over existing microtubule-perturbing agents in the context of a whole organism or under conditions of multi-drug resistance. [ABSTRACT FROM AUTHOR]

Presently, the zebrafish is the only vertebrate model compatible with contemporary paradigms of drug discovery. Zebrafish embryos are amenable to automation necessary for high-throughput chemical screens, and optical transparency makes them potentially suited for image-based screening. However, the lack of tools for automated analysis of complex images presents an obstacle to using the zebrafish as a high-throughput screening model. We have developed an automated system for imaging and analyzing zebrafish embryos in multi-well plates regardless of embryo orientation and without user intervention. Images of fluorescent embryos were acquired on a high-content reader and analyzed using an artificial intelligence-based image analysis method termed Cognition Network Technology (CNT). CNT reliably detected transgenic fluorescent embryos ( Tg(fli1:EGFP) y1) arrayed in 96-well plates and quantified intersegmental blood vessel development in embryos treated with small molecule inhibitors of anigiogenesis. The results demonstrate it is feasible to adapt image-based high-content screening methodology to measure complex whole organism phenotypes. Developmental Dynamics 238:656-663, 2009. © 2009 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

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