Forward chemical genetics screening

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. 

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. 

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

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. 6-17 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.

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),...

Forward and Reverse Chemical Genetics 308 Phenotypic Assays for Forward Chemical-Genetic Screening 311... 

A forward chemical genetic screen is used to identify compounds on the basis of a phenotype that is assumed to correlate with an underlying cellular process. Once a hit... 

In a pioneering forward chemical genetic screen (whole-cell mitotic arrest assay detected by fluorescence microscopy), a cell-permeable small molecule, monastrol (Figure 1.9), was identified, as it caused inhibition of the normal mitotic spindle formation but did not affect normal tubulin formation. In subsequent studies, testing the inhibition of the formation of the mutant phenotype led to the identification of the primary molecular target in the signaling cascade, a molecular motor protein, kinesin, Eg5. Monastrol treatment showed a phenotype identical to the blocking of Eg5 function by microinjection of Eg5-specific antibodies (Kapoor et al., 2000). 

As forward chemical genetic screens are commonly performed in the context of a human disease hypothesis, a blurred line between probe development and drug discovery can potentially create incentives to limit the use of discovered compounds through patent protection. While the intent of chemical biology is not to stifle therapeutic development but conversely to enable future... 

With these guidelines in mind, specific approaches to probe development can be implemented to transition forward chemical genetic screening hits into validated probes of biological processes, and these strategies are highlighted in Subheading 3. 

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