Microscale Thermophoresis for Thermodynamic Analysis: A Proof-of-Concept Study on LIMK Inhibitors.

LIMK1 and LIMK2, key regulators of cytoskeleton dynamics, are notoriously difficult to produce and purify in quantities sufficient for conventional biochemical studies, yet they are promising therapeutic targets due to their role in actin filament turnover and microtubule remodeling. To overcome these limitations, we developed a microscale thermophoresis (MST)-based thermodynamic assay as a proof-of-concept to study LIMK-inhibitor interactions directly in HEK293 cell lysates overexpressing fluorescent miRFP670-LIMK fusion proteins, completely bypassing protein purification. Dissociation constants (Kd) were measured across multiple temperatures and analyzed by van't Hoff plots, yielding highly linear correlations (r2 = 0.958-0.999). LIMK1 and its kinase domain (Kin1) showed comparable binding behavior to TH-257 and LX7101 (ΔG37°C = -10.3 ± 0.4 kcal mol-1), whereas LIMK2 interactions were significantly weaker (ΔG37°C lower by ∼1.5 kcal mol-1). Binding was enthalpy-driven, with entropy opposing complex formation, highlighting opportunities for rational optimization of inhibitors. This work establishes MST as a user-friendly, purification-free platform for deriving protein-ligand thermodynamics directly in cell lysates, providing a versatile proof-of-concept approach for studying challenging targets and guiding the development of therapeutically relevant inhibitors.