PNA-templated trivalent inhibitor achieves sub-picomolar thrombin affinity with on-demand reversibility

The discovery of high-affinity ligands is crucial for developing effective therapeutics, yet traditional dynamic combinatorial chemistry (DCC) methods are often hampered by slow kinetics and analytical challenges. This limits their utility in rapidly exploring vast chemical spaces. For thrombin, a key enzyme in the coagulation cascade, potent and reversible inhibitors are highly sought after to manage thrombotic disorders. Current anticoagulants often carry bleeding risks, highlighting the need for agents with precise control over their activity. This research addresses the gap by developing a rapid, scalable DCC platform to identify ultrahigh-affinity, programmable, and reversible thrombin inhibitors.

Researchers developed a peptide nucleic acid (PNA)-templated trivalent DCC platform to screen 125,000 potential assemblies. Short hybridization handles on the PNA template allowed for unbiased equilibration of a three-fragment library. The selection-readout cycle was expedited using size-exclusion filtration combined with MALDI-MS (matrix-assisted laser desorption/ionization mass spectrometry), completing the process in under 1 hour. The platform was used to identify a trivalent inhibitor targeting thrombin. Reversibility was assessed by adding a single-stranded toehold antidote to disassemble the inhibitor-thrombin complex, and activity restoration was measured in both buffer and plasma.

The PNA-templated DCC platform successfully identified a trivalent inhibitor with an apparent sub-picomolar affinity for thrombin, specifically a KD of approximately 84 fM. This inhibitor demonstrated near-stoichiometric inhibition, indicating highly efficient binding. The design allowed for synergistic fragment combinations that simultaneously engaged thrombin's active site and both exosites, contributing to its extreme potency. Importantly, the inhibition proved to be fully reversible. > Addition of a single-stranded toehold antidote rapidly disassembled the inhibitor-thrombin complex, restoring thrombin activity completely in both buffer and human plasma. This on-demand reversibility was a key functional outcome, demonstrating precise control over the inhibitor's action. The rapid MALDI-MS readout enabled high-throughput screening, accelerating the discovery process significantly.