Discovery and synthesis of chemical inhibitors of proteins required for cell division.
Fig. 5
Primary Screens: quantitative automated high-content microscopy assays to identify inhibitors of cell division. The method involves imaging compound treated cells (stained for actin) at low magnification (4x). Dividing cells adopt a very round morphology. Cells with round shapes are detected and counted using image analysis software to determine the mitotic index.
1) To identify inhibitors of cell division we have used approaches that mimic conventional forward genetics. An efficient primary screen is used to select small molecule chemicals from large libraries (>20,000 compounds) that block cell division. High-throughput automated microscopy allows phenotypes of compound treated cells to be examined and automated digital image analysis of cellular phenotypes (e.g. shape) can be used to compute the fraction of cells blocked in mitosis (mitotic index) (see Fig. 5). Secondary and tertiary screens are then used to select compounds that do not target other cellular processes or proteins for which many inhibitors are currently available (e.g. tubulin inhibitors).
Fig. 6
Strategies for finding targets of bio-active small molecules. Guided by focused combinatorial chemistry and SAR (structure-activity relationships) analysis, HR22C16 was immobilized on affigel matrix. We found the Eg5 in vertebrate cell extracts associated with this matrix. This binding could be competed with soluble HR22C16. For more details, see Marcus et al. JBC (2005).
We use combinatorial synthesis to densely explore the chemical space around a bio-active small molecule we identify from cell-based screens. This can lead to inhibitors with improved potency. Analysis of structure activity relationships (SAR) also allows reagents to be prepared that can be useful for analyzing protein targets and specificity (see Fig. 6). Photo-caged versions of inhibitors can also be designed that allow even faster temporal control and spatial control over target function (Fig. 7).
Please see (Hotha, S. et al. ACIE 2003), (Marcus, A.I. et al. JBC 2005) for more details.
Fig. 7
Strategies for spatially-specific inhibition of target proteins. An HR22C16 analog is shown that has a ‘protecting group’ that can be removed by light.
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