Structural features of a duplex-quadruplex anti-thrombin aptamer: the highly effective NU172

Human α-thrombin (thrombin) is a coagulation factor that maintains blood hemostasis by balancing pro- and anti-coagulant actions. Thrombin capability to perform such different functions relies on the ability to recognize a large variety of substrates, inhibitors and cofactors. This ability is finely regulated by two distinct regions on the protein surface, known as exosites I and II, which allosterically modulate the function, thus providing specificity to the proteolytic activity of the protein [1]. Oligonucleotide aptamers, short, single-stranded DNA or RNA sequences that bind to their target with high affinity and specificity through their tridimensional structures, are extensively used to modulate the function of most of the factors involved in the coagulation pathway [2]. Particular attention has been focused on the study of anti-thrombin aptamers which adopt a G-quadruplex structure [3-5]. Nowdays, the researchers interest moves on a new class of oligonucleotides in which the addition of a duplex-forming sequences to a G-quadruplex module results in an improvement of the antithrombotic action. One of these oligonucleotides is NU172 that possesses an high anticoagulant activity with an IC50 value of 5–10 μg/mL in plasma [6] and is currently the only thrombin binding aptamer evaluated in Phase II clinical trials (ClinicalTrials.gov identifier NCT00808964) for anticoagulation in heart disease treatments by ARCA Biopharma, Inc [7]. Here we present the crystallographic structure of the thrombin-NU172 complexes in the presence of either potassium or sodium ions, which provide a reasonable interpretation of the peculiar antithrombotic properties of this oligonucleotide. Moreover, in order to investigate the role of quadruplex loops and the duplex region on the aptamer folding, conformational solution studies on NU172 variants have been performed. Finally, the anticoagulant activities of NU172 and its variants have been evaluated following the conversion of fibrinogen in fibrin promoted by thrombin by means of dynamic light scattering experiments and compared with those of TBA. Details will be discussed at the Meeting. [1] E. Di Cera J Thromb Haemost. 2007, 5 Suppl 1, 196. [2] R.S. Woodruff, B.A. Sullenger Arterioscler Thromb Vasc Biol. 2015, 35, 2083. [3] I. Russo Krauss, A. Pica, A. Merlino, L. Mazzarella, F. Sica Acta Crystallogr D Biol Crystallogr. 2013, 69, 2403. [4] I. Russo Krauss, V. Spiridonova, A. Pica, V. Napolitano, F. Sica Nucleic Acids Res. 2016, 44, 983. [5] I. Russo Krauss, V. Napolitano, L. Petraccone, R. Troisi, V. Spiridonava, C.A. Mattia, F. Sica Int J Biol Macromol. 2018, 107, 1697. [6] A.D. Keefe, S. Pai, A. Ellington Nat Rev Drug Discov. 2010, 9, 537. [7] J. Zhou, J. Rossi Nat Rev Drug Discov. 2017, 16, 181.