Small molecule identification

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Despite the complexity of the experiments and the enormous data manipulation necessary, complex biological pathways, as well as new drug targets are being identified by this method. Examples include screens for compounds that arrest cells in mitosis, that block cell migration, and that block the secretory pathway [50], or assays with primary T cells from PLP TCR transgenic mice for their inhibitory activity on the proliferation and secretion of proinflammatory cytokines in PLP-reactive T cells [51], and identification of small-molecule inhibitors of histone acetyltransferase activity [52]. 

Summary The importance of structural studies for the identification of small molecules and for the interpretation of their reactivity is illustrated with examples of silicon-containing compounds. Such compounds are best studied in the gas phase, so that their structures are undistorted by intermolecular interactions, and may be compared with those calculated theoretically. Examples are given of silicon compounds which show major differences between their gas and solid phase structures, even when the intermolecular interactions are quite weak. 

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). 

Although a number of cell lines were shown to be sensitive to inhibition by PatA (Low et al., 2005), we selected RKO cells (IC50 of 0.4 nM in cell proliferation assay) to prepare lysates for the isolation and identification of target protein(s). We often select RKO cells for target identification of small molecules using biotin-conjugates, because they appear to be particularly suitable for target protein isolation. 

Rotational spectroscopy and microwave astronomy are the most accurate way to identify a molecule in space but there are two atmospheric windows for infrared astronomy in the region 1-5 im between the H2O and CO2 absorptions in the atmosphere and in the region 8-20 xrn. Identification of small molecules is possible by IR but this places some requirements on the resolution of the telescope and the spacing of rotational and vibrational levels within the molecule. The best IR telescopes, such as the UK Infrared Telescope on Mauna Kea in Hawaii (Figure 3.13), are dedicated to the 1-30 xm region of the spectrum and have a spatial resolution very close to the diffraction limit at these wavelengths. 

Identification of molecules in space, even small molecules, by IR astronomy requires a rotational progression in the spectrum to be measured and resolved by the telescopes. For the transitions in the simpler molecules such as CO the telescope must be capable of aresolution of 2150/1.93 1114, which is within the resolution limit of the UK Infrared Telescope (3000-5000). However, the rotational constant for CO is rather large and many molecules, especially polyatomic species, will have a rotation constant ten times smaller than this, placing the observation of a resolved rotational progression beyond the resolution of the telescopes. Confidence in the identification of the molecule is then severely dented. The problem is worse for visible astronomy. 

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