Discovery of a Reversible Sub-Picomolar Thrombin Inhibitor Using DCC

Dynamic combinatorial chemistry (DCC) offers a powerful yet underutilized strategy for ligand discovery, largely limited by heterogeneous kinetic constraints in exchange reactions and analytical challenges. Here we report a peptide nucleic acid (PNA)-templated trivalent DCC platform that enables rapid, target-guided exploration of 125 000 assemblies to identify ultrahigh-affinity and reversible thrombin inhibitors. Short hybridization handles allow unbiased equilibration of a three-fragment library, and size-exclusion filtration combined with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) provides a complete selection-readout cycle in under 1 h. From this library, thrombin amplifies synergistic fragment combinations engaging the active site and both exosites, yielding a trivalent inhibitor with apparent sub-picomolar affinity (K_D ≈ 84 fM) and near-stoichiometric inhibition. Despite its extreme potency, inhibition remains fully reversible: addition of a single-stranded toehold antidote rapidly disassembles the complex and restores activity in buffer and in plasma. These results establish hybridization-guided DCC as a fast, scalable route to programmable multivalent therapeutics with on-demand reversibility.