Small Molecule Target Identification using Drug Affinity Responsive Target Stability (DARTS)

Small molecule target identification is a monumental task. Knowledge of the binding interactions between small molecule compounds and cellular macromolecules such as proteins is crucial for understanding their biological properties and mechanisms of action, yet discovering these interactions remains a considerable challenge. To overcome the limitations of current target ID methods, we developed a novel technique that can identify direct binding interactions using the native, unmodified compounds. This method, called drug affinity responsive target stability (DARTS), takes advantage of the stabilization of protein structure upon drug binding that provides the target protein with resistance to protease degradation. When a small molecule is incubated with protein lysates, it binds and protects target proteins from proteolysis while degradation of non-target proteins is unaffected. Using proteomics, target proteins are then identified based on their enrichment in the compound-treated sample versus a control sample post-proteolysis. After demonstration of DARTS using a variety of known drug-protein pairs, DARTS was used to discover that the protein translation initiation machinery is a novel target of resveratrol. Then, to improve the sensitivity of DARTS, it was combined with a powerful gel-free fractionation technique and mass spectrometry analysis, which allowed for the identification of several potential new targets for the anti-cancer marine natural product didemnin B (DB). The TCP1 chaperonin complex (TRiC) was validated to be a target of DB using both biochemical and cell-based assays. Importantly, this is the first non ATP-mimetic compound reported to bind and inhibit TRiC, and could help design more specific and potent inhibitors of this complex. Finally, we demonstrated that DARTS can also be used for mapping the binding domains of small molecules within their target proteins. DARTS provided the first experimental evidence that DB binds both domains 1 and 2 of the eukaryotic elongation factor-1 alpha and verified the binding of pateamine A and hippuristanol to the N-terminal and C-terminal domains of the eukaryotic translation initiation factor 4a, respectively. Collectively, the results presented in this dissertation establish DARTS as a powerful technique for identifying and studying protein-small molecule interactions that will have a profound impact on the chemical biology field.