Genetic approaches forward chemical genetics

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). 

Like its genetic counterpart, forward chemical genetics relies on a phenotype of interest to guide the selection of biologically active small molecules that modulate a particular biological system or mechanism (Fig. 6-3) [5-7]. Overall, this approach entails a three-step process that... 

Fig. 6-3 Forward versus reverse chemical molecules that can be used to probe the genetics. While forward chemical genetics function of the selected protein. Both relies on a phenotype of interest to guide the approaches require the use of small selection of biologically active small molecules and phenotypic assays but differ...

Forward chemical genetics approaches start with compounds that cause a particular phenotype in a biological system (e.g., ADEPs inhibit cell growth and trigger filamentation). Subsequently, the target of this small molecule and its mechanism of action are elucidated. Hence, the forward chemical genetics workflow is from compounds to gene. Usually, in vivo experiments are carried out ahead of in vitro experiments. 

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... 

In this section, the discovery of small molecules for the interrogation of biological mechanisms will be described. An appropriate small molecule tool may be discovered either on the basis of modulation of the activity of a specific protein (reverse chemical genetics. Section 1.3.1) or its phenotypic effect (forward chemical genetics. Section 1.3.2). Emphasis will be placed on the approaches that may be used to discover useful small molecule tools, and the insights into biological mechanisms that may be accrued. 

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... 

Discovery of Small Molecule Probes Using the Forward Chemical Genetic Approach... 

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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