Identification and validation of protein targets of bioactive small molecules
ABSTRACT Identification and validation of protein targets of bioactive small molecules is an important problem in chemical biology and drug discovery. Currently, no single method is satisfactory for this task. Here, we provide an overview of common methods for target identification and validation that historically were most successful. We have classified for the first time the existing methods into two distinct and complementary types, the 'top-down' and 'bottom-up' approaches. In a typical top-down approach, the cellular phenotype is used as a starting point and the molecular target is approached through systematic narrowing down of possibilities by taking advantage of the detailed existing knowledge of cellular pathways and processes. In contrast, the bottom-up approach entails the direct detection and identification of the molecular targets using affinity-based or genetic methods. A special emphasis is placed on target validation, including correlation analysis and genetic methods, as this area is often ignored despite its importance.
- SourceAvailable from: Gabriel Fenteany
[Show abstract] [Hide abstract]
- "Consequently, this approach has the potential to work in model systems that do not permit forward genetics and, unlike reverse genetics, does not require any starting information about likely players in the process being studied. The macromolecular target of any small molecule identified from a library can be identified by biochemical approaches [e.g., Lomenick et al., 2011; Titov and Liu, 2012]. Even prior to such identification , specific inhibitors can be extremely useful with respect to ordering hierarchies of cell biological events. "
ABSTRACT: Screening of small molecule libraries offers the potential to identify compounds that inhibit specific biological processes and, ultimately, to identify macromolecules that are important players in such processes. To date, however, most screens of small molecule libraries have focused on identification of compounds that inhibit known proteins or particular steps in a given process, and have emphasized automated primary screens. Here we have used "low tech" in vivo primary screens to identify small molecules that inhibit both cytokinesis and single cell wound repair, two complex cellular processes that possess many common features. The "diversity set", an ordered array of 1990 compounds available from the National Cancer Institute, was screened in parallel to identify compounds that inhibit cytokinesis in Dendraster excentricus (sand dollar) embryos and single cell wound repair in Xenopus laevis (frog) oocytes. Two small molecules were thus identified: Sph1 and Sph2. Sph1 reduces Rho activation in wound repair and suppresses formation of the spindle midzone during cytokinesis. Sph2 also reduces Rho activation in wound repair and may inhibit cytokinesis by blocking membrane fusion. The results identify two small molecules of interest for analysis of wound repair and cytokinesis, reveal that these processes are more similar than often realized and reveal the potential power of low tech screens of small molecule libraries for analysis of complex cellular processe. © 2012 Wiley Periodicals, Inc.Cytoskeleton 11/2012; 69(11). DOI:10.1002/cm.21085 · 3.01 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: The fluorescein derivants (Fluorescein: (2-(6-Hydroxy-3-oxo-(3H)-xanthen-9-yl) benzoic acid), Fluorescein-DA: (Bis [N,N-bis (carboxymethyl) aminomethyl] fluorescein) and Fluorescein-DAFe(III): (Bis [N,N-bis (carboxymethyl) aminomethyl] fluoresceinFerrous(III)) with a tricyclic plane structure were used to study the interaction and sonodynamic damage to bovine serum albumin (BSA) under ultrasonic irradiation through fluorospectrometry and UV-vis spectrophotometry. Besides, because of the existence of Fe(III) ion in Fluorescein-DAFe(III), under ultrasonic irradiation the sonocatalytic activity in the damage of BSA molecules was also found. Three-dimensional fluorescence spectra and three-dimensional fluorescence contour profile spectra were mentioned to determine the fluorescence quenching and the conformation change of BSA in the absence and presence of these fluorescein derivants. As judged from the experimental results, the fluorescence quenching of BSA in aqueous solution caused by these fluorescein derivants were all attributed to static quenching process. The damage degree and mode were related to some factors such as ultrasonic irradiation time, fluorescein derivant concentration and ionic strength. Finally, several quenchers were used to determine the amount and kind of generated reactive oxygen species (ROS) during sonodynamic and sonocatalytic reaction processes. It suggests that these fluorescein derivants induce protein damage via various ROS, at least, including singlet oxygen ((1)O2) and hydroxyl radicals (OH). Perhaps, this paper may offer some important subjects for broadening the application of these fluorescein derivants in sonodynamic therapy (SDT) and sonocatalytic therapy (SCT) technologies for tumor treatment.Spectrochimica Acta Part A Molecular and Biomolecular Spectroscopy 03/2013; 110C:364-376. DOI:10.1016/j.saa.2013.03.073 · 2.13 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Phenotypic screens, in which chemical libraries are assayed on cells with the aim to isolate compounds that interfere with a given cell function, are a risky but powerful strategy to discover, in the same approach, new therapeutic targets and the compounds able to regulate them. With a strong experience of nearly 10 years in the field, we present the advantages of such an approach, the possible troubles and technical solutions. We also present in this paper a french network which has been recently built and that gather all the competencies needed for screening approaches.Medecine sciences: M/S 10/2013; 29(10):897-905. DOI:10.1051/medsci/20132910018 · 0.52 Impact Factor