Small chemical genetics

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). 

Schreiber, S.L. The small-molecule approach to biology. Chemical genetics and diversity-oriented organic synthesis make possible the systematic exploration of biology. Chem. Eng. News 2003, 81,... 

Kawasumi M, Nghiem P. (2007) Chemical genetics elucidating biological systems with small-molecule compounds. JInvest Dermatol 127, 1577-84. 

Spring, D. R., (2005) Chemical genetics to chemical genomics small molecules offer big insights. Chem. Soc. Rep. 34, 472-482. 

Most recently, we reported small molecule arrays on photoaffinity crosslinker coated gold surfaces (17). The small molecule arrays were fabricated by photoreaction, and then analyzed by SPR imaging technique. The small molecules don t have to be modified chemically for immobilization. The small molecules, which can interact with a target protein, can be screened by this methodology. Therefore, the integration of photoaffinity small molecule array and SPR imaging technique can be the first step of reverse chemical genetics. 

It is critical to this chemical genetic approach to have a library of compounds that have a high probability of being relatively selective otherwise, the ability to interpret the results becomes at least as complex as deciphering highly poly-genetic phenotypes. To address this, diversity-oriented synthesis has been proposed to provide arrays of complex small molecules that are easily synthesized. The natural-product basis for many of the molecules and their complexity are believed to contribute to their cellular potency and selectivity (6). This chemical genetic approach has been applied to identify novel inhibitors of alpha-tubulin and histone deactylation (7). 

Tan DS, Foley MA, Stockwell BR, Shair MD, Schreiber SL, Synthesis and preliminary evaluation of a library of polycyclic small molecules for use in chemical genetic assays, J. Am. Chem. Soc., 121 9073-9087, 1999. 

Tolliday, N. et al. 2006. Small molecules, big players the National Cancer Institute s Initiative for Chemical Genetics. Cancer Res. 66, 8935-8942. 

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