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Odell, Anahí V.

Microbiology and Molecular Genetics

2013

PhD

Chemical genetics approaches allow interrogation of a variety of cellular processes using organic small molecules to modulate these events. Similar to introducing modifications in the genome to study their effect on the phenotype, the use of small molecules that affect the activity of the targets they interact with translates into a particular phenotype. One avenue to pursue this kind of study is forward chemical genetics which involves selecting for a desired phenotype in large scale screens with small molecules, analogous to a forward classical genetics approach. The main disadvantage of this method is the lack of information on the potential target/s once effective small molecules are identified. Thus, to accelerate the lead optimization programme and learn new biology around those small molecules and their targets, reliable drug target identification methodologies are required.
The Yeast Three-Hybrid (Y3H) system is suitable for this purpose given its simplicity, independence of natural protein target abundance in the cell and high throughput capabilities. This system is a modified version of Yeast Two-Hybrid analysis in which the readout of the reporter genes relies on the interaction of two proteins (an anchor and a target of the compound of interest) with a bridging molecule or Chemical Inducer of Dimerization (CID), thereby dimerizing the two functional domains of a transcription factor. izing the two functional domains of a transcription factor. During the course of this study we investigated the effect of modifying different features of a CID using a compound-target pair that shows strong interaction in Y3H.
We proposed a modular design of the CID that enabled fast and efficient CID synthesis and used it to generate a CID series with varying linker chemistries, length and point of attachment of the linker on the compound of interest. Using the Y3H system we also explored the target profile of an anti-parasitic drug and identified a novel target of this compound, TgBRADIN. We found this protein to be invilved in the process of tachyzoite-to-bradyzoite differentiation, a process we also showed to be enhanced by the treatment with the drug. We confirmed that the compound produces the differentiation phenotype in part through TgBRADIN. This is the first time the Y3H system has been utilized for identification of drug targets in a pathogenic organism and demonstrates that Y3H will be a valuable tool to expand our knowledge on the edge on the targets and mechanisms of action of a number of active anti-parasitic compounds.