Date of Award
Bioactive small molecules are valuable tools to understand and manipulate biological pathways. In order to be effective as either probes for understanding cell biology or as clinical drugs, the small moleculeâ€™s mechanism of action must be characterized. However, identifying a small moleculeâ€™s target and characterizing its interaction with that target remain major challenges in the chemical biology field. In this thesis, I describe methods to improve the process of drug target identification and binding site characterization. In order to identify the target of a small molecule, I have developed an approach in which multiple drug resistant clones are isolated and transcriptome sequencing is used to find mutations in each clone. Further analysis of mutations common to more than one drug-resistant clone can identify a drugâ€™s physiological target and indirect resistance mechanisms. In proof-of-concept studies I analyze clones resistant to two cytotoxic anti-cancer drugs, BI 2536 and bortezomib. For both compounds I detect mutations in the known target that confer resistance to the drug. Unlike other target identification methods, this approach can establish a genetic proof of the target in human cells. I have also developed a method to characterize a small moleculeâ€™s binding site after its target is known. In this method, called Stable Isotope Labeled Inhibitors for Crosslinking (SILIC), structure-activity relationship data is used to design inhibitor analogs that incorporate a photo-crosslinking group along with either natural or heavy stable isotopes. An equimolar mixture of these inhibitor analogs is crosslinked to the target protein to yield a robust signature for identifying inhibitor-modified peptide fragments in complex mass spectrometry data. I applied this approach to an ATP-competitive inhibitor of kinesin-5, a widely conserved motor protein required for cell division. This analysis, along with mutagenesis studies, suggests that the inhibitor binds at an allosteric site in the motor protein.
Wacker, Sarah Anne, "Approaches to Study Small Molecule Inhibitors and Their Targets" (2012). Student Theses and Dissertations. 174.