Forward chemical genetics target identification

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. 

The application of forward chemical genetics to studies of translation provides an opportunity to identify small molecules that inhibit or stimulate this process without any underlying assumptions as to which step is most amenable to targeting by the chemical libraries under consideration. The opportunity exists to identify novel factors involved in translation, unravel new activities of known translation initiation factors, or characterize shortlived intermediates that are frozen by the small molecule inhibitor. We have undertaken a forward chemical genetic approach to identify small molecules that inhibit or stimulate translation in extracts prepared from Krebs-2 ascites cells (Novae et al., 2004). These screens have led to the identification of several novel inhibitors of translation initiation and elongation (Bordeleau et al., 2005, 2006 Robert et al., 2006a,b). 

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). 

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... 

A major challenge associated with the forward chemical genetic approach is the identification of the target of the small molecule probe.Affinity chromatography is a common approach to target identification. This approach requires the attachment of the small molecule probe to a solid support the site of the attachment of the linker must be chosen carefully to avoid the ablation of the probe s biological activity. Trapoxin, is a cyclic tetrapeptide whose... 

---