Centrocountins

The focused library of around 60 indoloquinolizines was subjected to the phenotypic assay for mitosis modulators at a concentration of 30 pM and this led to the identification of 24a (centrocountin 1, Scheme 16.2a) as a mitotic inhibitor. 

After treatment with centrocountin 1, a dose-dependent increase (from 1 to 25 pM, Box 16.5) in the number of mitotic cells with misaligned chromosomes was detected (Figure 16.3b). At higher concentrations of 25-50 pM, the number of mitotic cells with multipolar mitotic spindles also increased with concentration (Figure 16.3c). Treatment with centrocountin 1 caused fivefold prolonged mitosis compared to the control, which is indicative of an activated spindle-assembly checkpoint and mitotic arrest. An increase in apoptosis was observed as a consequence of this arrest. 

Scheme 16.2 (a) Chemicai structure of centrocountin 1 (24a). (b) Structure-activity relationship for centrocountin derivatives, (c) Chemical structures of the employed probes (26-28). 

Several potential proteins could be excluded as targets of centrocountin 1. Characterization of the compound revealed no inhibition of various kinases involved in mitosis regulation. As most of the mitotic inhibitors target tubulin/... 

To confirm NPM and/or Crml as targets of centrocountin 1, proteins enriched during the pulldown were subjected to immunoblotting to detect NPM and Crm 1... 

Crmi as targets of centrocountins. (a) Detection of NPM and Crmi by means of immunobiotting after enrichment by affinity chromatography using 26 in the absence or presence of different concentrations of centrocountin 1 ((fi)-24a) (competition approach), (b) Binding of 28 to NPM or... 

The synthesis of the natural-product-inspired indoloquinolizine compound collection, the HCS for mitotic inhibitors and the elucidation of the mode of action of centrocountin 1 is a demonstrative example of the forward-chemical genetics approach. It illustrates the workflow for the identification of biologically active small molecules in cells and target deconvolution. Centrocountin 1 was the most potent hit compound in a screen for mitotic inhibition and impairs the proper chromosome congression in cells. This results in chromosomal misalignment and... 

Synthesis of Natural-Product-lnspired Centrocountins Using Domino Reactions... 

Scheme 14.30 Waldmann and Kumar s synthetic approach to centrocountins (200), showing key intermediates of the proposed 12-step domino sequence.

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