Inhibitor fluorination pattern tunes chemically induced protein dimerization.

Chemically induced dimerization of proteins is a powerful approach to regulate biomolecular functions through small molecule ligands acting as "molecular glues". Here, we demonstrate that simple, thienopyrimidinone scaffold-based inhibitors efficiently promote homodimerization of an essential oxidoreductase from the human pathogenic parasite through selective covalent attachment and self-assembly. A fluorine walk strategy, commonly used to optimize small molecule properties, resulted in tuning induced dimer affinity across two orders of magnitude. NMR spectroscopy, MD simulations, chromatography, multi-angle light scattering, mass spectrometry, calorimetry, and functional assays reveal how the inhibitor fluorination pattern alters the dynamics and interactions of the enzyme-bound inhibitor and surface-exposed aromatic protein side chains, affecting both enzyme inhibition kinetics and induced dimerization. This work highlights how site-specific fluorination can modulate protein interactions and offers a framework for the design of novel molecular glues with broad applications in chemical biology and drug development.