Aptamer-Small Molecule Conjugates As Potent and Reversible Surgical Anticoagulants Targeting Thrombin

Unfractionated heparin (UFH) is the first-line anticoagulant used in millions of heart surgeries annually. The high potency of UFH is attributed to its effective activation of the antithrombin-mediated anticoagulation pathway. However, the prolonged use of heparin in heart surgeries is often associated with bleeding disorders as it depletes both patient's coagulation and anticoagulation capacity. Moreover, UFH is immunogenic, which can trigger potentially life-threatening heparin-induced thrombocytopenia (HIT) when used repeatedly. Aptamers are short oligonucleotides isolated in vitro that bind to protein or small molecule targets with high affinity and specificity. Several aptamers have been created that act as anticoagulants as they can competitively block substrate binding to anticoagulation protease exosites. Compared to UFH, aptamers have advantages, including not activating antithrombin, reversibility by administration of a well-tolerated antisense oligonucleotide, and low immunogenicity. However, the anticoagulant potency of aptamers is often limited by their inability to effectively inhibit the catalytic sites of their protease targets. Here we provide a strategy to solve this potency problem by conjugating a protease active-site inhibitor to an exosite-binding aptamer via a polynucleotide linker. The resulting bivalent conjugate binds to both the active site and exosite of the target protease to completely inhibit protease function. Meanwhile, the synergy between two binding elements significantly improves the binding affinity compared to the small molecule inhibitor or the aptamer alone. As a proof of concept, we conjugated the thrombin active site inhibitor DAB to the 5’ terminus of anti-thrombin aptamer HD22 via a poly (7) adenosine linker (HD22-7A-DAB) and characterized its thrombin inhibition activity using a fluorogenic substrate cleavage assay. The results reveal that HD22-7A-DAB has a much higher thrombin inhibition activity (IC₅₀ = 0.2 nM) than DAB (IC₅₀ = 80 nM) or the unconjugated aptamer (IC₅₀ > 1000 nM). The significantly higher inhibitory effect of HD22-7A-DAB compared to a 1:1 mixture of unconjugated DAB and HD22 also indicates that conjugation is essential to create an effective synergistic inhibitor. Notably, in the presence of 2 µM complementary DNA to HD22, the effect of HD22-7A-DAB conjugate was drastically diminished (IC₅₀ > 1000 nM), which indicates that both aptamer and small molecule in ADIC can be reversed using an antidote oligonucleotide. HD22-7A-DAB also shows a UFH-rivaling anticoagulation effect and antidote-reversible behavior in a series of in vitro clotting assays, including thrombin time (TT), prothrombin time (PT), and activated partial thromboplastin time (aPTT). Thus the development of a thrombin-targeting small molecule aptamer conjugate inhibitor yields a potent and reversible anticoagulant with clinically relevant anticoagulant activity. In addition, these studies describe a generalizable strategy for rapidly developing aptamer-small molecule active site inhibitor conjugates for generate other potent and reversible inhibitors of the other coagulation proteases. These potent and reversible anticoagulant aptamer-active site drug conjugates have the potential to become UFH substituents in surgeries to reduce the risk of UFH-associated bleeding, UFH-insensitivity and HIT.

Layzer:Basking Biosciences: Consultancy, Current holder of stock options in a privately-held company. Krishnaswamy:Bayer: Research Funding; Takeda: Consultancy. Sullenger:Basking Biosciences: Current equity holder in private company, Current holder of stock options in a privately-held company, Patents & Royalties: Duke Univeristy has licensed patents to Company on which I am inventor; Somalogic Inc: Consultancy.