Development and application of proteomics-based tools for ligand target identification
Embargo Date
2028-01-22
OA Version
Citation
Abstract
Small molecules are the foundation of modern pharmacology, yet defining their direct protein targets in the complex environment of the cell continues to challenge chemical biology. This dissertation describes the development and application of proteomics-based tools that capture ligand-protein interactions through complementary readouts of stability, conformational change, and adsorption at material interfaces. Together, these approaches extend the scope of chemoproteomics beyond conventional formats and enable the discovery of targets and mechanisms that would otherwise remain hidden. Following a general introduction in Chapter 1, the dissertation is organized into three major research efforts presented in Chapters 2 through 4.Chapter 2 establishes an approach for the discovery of ribonucleoprotein-targeted interfacial ligands. Rocaglates, acting as proximity inducers, clamp RNA to DEAD-box helicases, a mode of inhibition that depends on multicomponent assembly rather than a single binary interaction. Using proteome-wide stability assays, I demonstrate how tailoring biochemical conditions with nucleotide analogs and RNA substrates can reveal substrate-dependent interactions otherwise inaccessible, expanding the known rocaglate clamping spectrum more than two-fold.
Chapter 3 explores bioactive scaffold tunability in a disease context. Through comprehensive chemoproteomic profiling of rocaglate acyl sulfamides, I show that subtle structural changes at chemically permissive sites shift rocaglate selectivity toward DDX3X, a helicase whose engagement correlates with selective cytotoxicity in glioblastoma stem cells. Comparative proteomic assays and structural modeling converge to explain this bias, establishing these compounds as context-specific chemical probes.
Chapter 4 introduces ILIAD (Identifying Ligand Interactions through Adsorption Differences), a new derivatization-free chemoproteomic platform that reframes protein adsorption to labware surfaces as a readout of target engagement. ILIAD detects both broad remodeling with nucleotide analogs and selective engagement by tool compounds and drugs, demonstrating that adsorption can serve as an orthogonal signature of ligand binding.
Building sequentially across the three research chapters, this dissertation demonstrates how chemical context, scaffold modification, and reframing can be leveraged to expand the chemoproteomic toolkit. The approaches developed here illustrate a path toward more complete mapping of drug-protein interactions and provide conceptual and methodological advances relevant to both probe discovery and therapeutic development.
Description
2026
License
Attribution-NonCommercial-NoDerivatives 4.0 International