PB0568 - X-Ray Structures of Thrombin Bound to Heterobifunctional Aptamer-Drug Conjugates Reveal the Mechanistic Basis for a Novel Tethered Inhibition Strategy

Background: Thrombin, a serine proteinase, is the chief effector and regulator of blood coagulation. Thrombin specificity and function is modulated by liganding at the active site or two distinct anion binding exosites (ABE1, ABE2). DNA aptamer HD22 specifically binds thrombin through ABE2 and affects functions mediated by this exosite. Dabigatran is a clinically used reversible thrombin inhibitor that binds to the active site. The combined use of aptamer and active site inhibitor has been shown to be an effective strategy to achieve potent anticoagulation in highly thrombogenic settings. This prompted the design of a heterobifunctional inhibitor by conjugating dabigatran to HD22 with a flexible oligonucleotide linker. The optimized HD22-Dabigatran conjugates (HD22-7A-DAB, and HD22-2T-DAB), showed over 100-fold higher potency in inhibiting thrombin when compared to an equimolar mixture of HD22 and dabigatran.

Aims: To resolve the X-ray structure of the drug-aptamer conjugates and obtain insights into the mechanism of inhibition.

Methods: X-ray diffraction data were collected at 2.1 Å resolution for HD22-7A-DAB, and at 2.0 Å for HD22-2T-DAB.

Results: The diffraction data allows unambiguous placement of both aptamer and dabigatran moieties onto thrombin for both HD22-7A-DAB, and HD22-2T-DAB. The first 29 nucleotides forming the core of HD22 show a duplex-quadruplex hybrid motif that presents an extended surface for molecular interaction. Linker connecting drug and aptamer is only partly resolved in both structures, implying requirement for high flexibility in the linker for unhindered presentation of the dabigatran moiety at the active site. The structural model accounts for the synergistic mechanism at play in the drug-aptamer conjugate resulting from the favorable binding of the tethered ligand at the active site upon binding of the aptamer to ABE2 of thrombin.

Conclusion(s): This readily reversible tethered inhibition approach provides a novel strategy for the potent inhibition of thrombin and other proteases in blood coagulation.