Heterobifunctional Small Molecules (hSMs) are a rapidly emerging therapeutic modality which function by hijacking the cellular Ubiquitin Proteasome System (UPS) to modulate protein levels via Targeted Protein Degradation (TPD). These molecules contain a target binding warhead covalently linked to an E3 ligase binder, inducing proximity between the therapeutic target and UPS machinery though formation of a target-hSM-ligase ternary complex. While these molecules show great promise in expanding the druggable proteome to targets previously deemed undruggable, limited solubility of these type of compounds poses a significant challenge for biophysical characterization with standard techniques such as isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) spectroscopy. Here we present a novel strategy utilizing detergent solubilization to accurately measure thermodynamic parameters of ternary complex formation. Additionally, understanding the dynamics of ternary complexes in solution along with the structural information is required for rational design of hSMs. We developed a methodology for probing the hydrodynamic properties of hSM-induced ternary complexes by utilizing pulsed field gradient (PFG) NMR with both <sup>1</sup>H and <sup>19</sup>F detection. These advances will pave the way for evaluating structure-activity relationships (SAR) of novel hSMs in search of drug-like molecules.

Characterizing thermodynamics and capturing dynamics of ternary complexes involved in targeted protein degradation

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