Chemical genetics screen

A Forward Chemical Genetic Screen to Identify Inhibitors of... 

In eukaryotes, translation initiation is rate-limiting with much regulation exerted at the ribosome recruitment and ternary complex (elF2 GTP Met-tRNAjMet) formation steps. Although small molecule inhibitors have been extremely useful for chemically dissecting translation, there is a dearth of compounds available to study the initiation phase in vitro and in vivo. In this chapter, we describe reverse and forward chemical genetic screens developed to identify new inhibitors of translation. The ability to manipulate cell extracts biochemically, and to compare the activity of small molecules on translation of mRNA templates that differ in their factor requirements for ribosome recruitment, facilitates identification of the relevant target. 

Characterization of Inhibitors of Translation Identified in Chemical Genetic Screens... 

Dupre, A., Boyer-Chatenet, L., Satder, R.M., Modi, A.P., Lee, J.-H., Nicolette, M.L., Kopelovich, L., Jasin, M., Baer, R, Pauli, T.T., et al, (2008) A Forward Chemical Genetic Screen Reveals an Inhibitor of the Mrell-Rad50-Nbsl Complex. Nat Chem Biol, 4,119. 

Kau, T. R., Schroeder, F., Ramaswamy, S., et al. (2003) A chemical genetic screen identifies inhibitors of regulated nuclear export of a forkhead transcription factor in PTEN deficient tumor cells. Cancer Cell 4, 463-467. 

Table 1 Small molecules and their target proteins identified in forward chemical genetic screening...

Shah K, Shokat KM. A chemical genetic screen for direct v-Src substrates reveals ordered assembly of a retrograde signaling pathway. Chem Biol. 2002 9 35-47. 

To summarize, as shown in Table 6-2, in a forward chemical-genetic screen, scientists start with a phenotype to find the protein or proteins responsible for it, and in a reverse chemical-genetic screen, the starting point is a protein of interest. Completing the trio of arrows in the discovery cycles in both cases provides valuable tools for the dissection of biological systems and mechanisms. 

Phenotypic Assays for Forward Chemical-Genetic Screening... 

Fig. 6-4 Phenotypic assays for chemical genetics, (a) Types of assays that have been used for chemical-genetic screening.

A question raised by chemical-genetic screens is why are some proteins targeted by small molecules more frequent than others. For example, in an antimitotic... 

On testing a set of small molecules in a chemical-genetic screen, it is a natural question to ask how the same small molecules, or ones that are close structural... 

Given a multidimensional matrix of data derived from chemical-genetic screens and computed molecular descriptors (Fig. 6-10), meaningful visual... 

One method of unsupervised learning that has proved useful for analyzing data from chemical-genetic screens is called clustering. This method attempts to cluster objects into sets that are somehow related on the basis of a set... 

Fig. 6-17 Forward chemical-genetic screen compound activity from the initial cell-based for inhibitors of mitosis (data from Ref. 73). and in vitro tubulin polymerization assay.

To expand further the molecular toolbox available for studying intracellular protein acetylation [88], a number of chemical-genetic screens have been performed. To identify probes of the mechanism through which HDAC inhibitors cause cell-cell cycle arrest and affect histone acetylation, a cytoblot cell-based screen was used to identify small-molecule suppressors of the trichostatin A named the ITSAs (for inhibitor of trichostatin A) (Fig. 6-19) [40]. 

Fig. 6-20 Forward chemical-genetic screen for inhibitors of protein deacetylation (data from Ref. 80). (a) Overview of cell-based screens of the 1,3-dioxane-based, diversity-oriented synthesis-derived library using antibodies to measure tubulin and histone acetylation, (b) Relative position of selected active compounds in a three-dimensional principal component model computed from five cell-based assay descriptors. AcTubulin-selective (red),...

Fig. 6-21 Selective inhibitors of a-tubulin (tubacin) and histone deacetylation (histacin) identified by chemical-genetic screening [80].

M. Wessling-Resnick, Chemical genetic screening identifies sulfonamides that raise organellar pH and interfere with membrane traffic, Traffic 2004, 5,478-492. 

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