Multidomain Targeting Generates a High‐Affinity Thrombin‐Inhibiting Bivalent Aptamer

Thrombin is a multifunctional serine protease and the key enzyme of the hemostatic network. It initiates the formation of a hemostatic plug through the activation of platelets and induction of fibrin formation through proteolytic cleavage of fibrinogen. Regulation of thrombin activity by thrombin-specific inhibitors is a promising therapeutic measure in the treatment of patients with arterial and venous thromboembolism. Inhibition of the enzymatic activity of thrombin can be achieved by blocking the catalytic triad or by blocking the anion-binding exosites I and II, which are located near the active site and are involved in substrate recognition and binding (Figure 1A). Hirudin, a 7 kDa protein, is the most potent thrombin inhibitor, with an inhibition constant of 2310 m. It interacts with exosite I and the active site to result in inhibition of all functions of thrombin. Recombinant versions of hirudin have been shown to be superior to established anticoagulants, such as heparins, in various clinical settings such as the treatment of coronary artery disease or prevention of deep-vein thrombosis. However, hirudin-treated patients are at an increased risk of bleeding owing to its narrow therapeutic window. The safety profile is somewhat better with bivalirudin, a reversible thrombin inhibitor. Bivalirudin is a bifunctional 20-amino-acid peptide, whose structure is analogous to that of the COOH terminus of hirudin but with an NH2-terminal that binds to the active site of thrombin. After binding, the NH2-terminal segment is hydrolyzed by thrombin to result in the regeneration of free thrombin and the inactive proteolytic product. Bivalirudin has been shown to be as effective in patients undergoing percutaneous coronary intervention as heparin plus glycoprotein IIb/IIIa inhibition. Thrombin-inhibiting aptamers are alternatives to proteinbased thrombin inhibitors. Aptamers are short single-stranded nucleic acids that fold into well-defined three-dimensional shapes. They bind with high affinity and specificity to their target molecules and possess great inhibitory potential. 5] These characteristics make them very interesting tools for molecular biology as well as for diagnostic and therapeutic applications. Compared to conventional drugs, aptamers cause almost no immune response and are nontoxic. Additionally, the anticoagulant activity of aptamers has been shown to be easily controlled by the addition of complementary strands or small molecules. One of the most intensively studied aptamers is the 15-mer ssDNA molecule HD1, which binds to and inactivates human a-thrombin (Figure 1C). The aptamer folds into a stable G-quadruplex structure that consists of six thymidine and nine guanosine nucleotides. Structural analysis revealed that the anticoagulant activity of the HD1 aptamer is mediated by binding to the exosite I of thrombin. However, clinical trials evaluating HD1 (ARC183) as an anticoagulant during coronary artery bypass graft surgery (CABG) were halted after phase I due to suboptimal dosing profiles, primarily caused by the restricted binding affinity of the aptamer. Generally, enhancing the activity of aptamers might be possible by mutagenesis, reselection, or site-directed modification. However, these approaches are unpredictable, time-consuming, and limited. They also strongly depend on the nature of the target molecule and the chemistry at the interface of the receptor–ligand interaction. Here we report an alternative strategy to enhance and modify the activity of aptamers. Our approach makes use of two functionally distinct aptamers that target the same protein, while the interaction of each aptamer is mediated by different protein subdomains. Approaches relying on the assembly of aptamers that target distinct protein subdomains have been reported previously, but only for diagnostic purposes. Here we have constructed fusion aptamers that combine two aptamer functionalities in one molecule. We hypothesized that the resultant fusion aptamer would not only possess the functionalities of its precursor molecules but would also reveal improved activities due to increased overall affinity. In a first attempt to validate this hypothesis, we constructed a fusion aptamer that was assembled by two distinct aptamers that target thrombin—one via exosite I, the other via exosite II. For this purpose, we attached the exosite I-binding aptamer HD1 through a nucleotide linker region to the exosite II-binding aptamer, termed 60.29. At an appropriate length of the linker region, the resultant fusion aptamer should simultaneously interact with both exosites, thereby combining the features of the individual aptamers in one molecule: high affinity binding of 60.29 coupled with the anticoagulant activity of HD1. We decided to use a poly-dA linker to connect the aptamers. This has the advantage that positively charged residues of the thrombin molecule, residing in close proximity to exosites I and II, might serve as further anchor regions, thus resulting in an enhanced affinity due to additional electrostatic interactions with the linker region of the fusion aptamer. Poly-dA also has the advantage of not interfering with the G-quadruplex formation of the individual aptamers. Based on the crystal structure of the HD1–thrombin complex, the distance between exosites I and II was determined to be around 26 D. Thus, we rea[a] Dr. J. M ller, Prof. Dr. B. Pçtzsch Institute of Experimental Haematology and Transfusion Medicine Universit$t Bonn Sigmund-Freud-Strasse 25, 53127 Bonn (Germany) [b] B. Wulffen, Dr. G. Mayer Life and Medical Sciences (LIMES) Program Unit Chemical Biology and Medicinal Chemistry c/o Kekul6-Institut f r Organische Chemie und Biochemie Universit$t Bonn Gerhard-Domagk-Strasse 1, 53121 Bonn (Germany) Fax: (+49)228-734809 E-mail : gmayer@uni-bonn.de Supporting information for this article is available on the WWW under http://www.chembiochem. org or from the author.