Discovery of a Reversible Sub-Picomolar Thrombin Inhibitor Using DCC

Area of Science:

• Molecular Biology
• Medicinal Chemistry
• Biochemistry

Background:

• Dynamic combinatorial chemistry (DCC) is a powerful strategy for ligand discovery.
• Current DCC methods face limitations due to kinetic constraints and analytical challenges.

Purpose of the Study:

• To develop a peptide nucleic acid (PNA)-templated trivalent DCC platform for rapid identification of ultrahigh-affinity and reversible thrombin inhibitors.

Main Methods:

• Utilized a PNA-templated trivalent DCC platform for exploring 125,000 assemblies.
• Employed short hybridization handles for unbiased fragment library equilibration.
• Integrated size-exclusion filtration and MALDI-MS for rapid selection and readout.

Main Results:

• Identified ultrahigh-affinity and reversible thrombin inhibitors with apparent sub-picomolar affinity (K_D ≈ 84 fM).
• Achieved near-stoichiometric inhibition due to synergistic fragment combinations engaging multiple thrombin sites.
• Demonstrated on-demand reversibility of inhibition via a toehold antidote.

Conclusions:

• Established hybridization-guided DCC as a fast, scalable method for programmable multivalent therapeutics.
• Highlighted the potential for designing reversible inhibitors with precise control over activity.