Several structural motifs cooperate in determining the highly effective anti-thrombin activity of NU172 aptamer

Despite aptamers are very promising alternative to antibodies, very few of them are under clinical trials or are used as drugs. Among them, NU172 is currently in Phase II as anticoagulant in heart disease treatments. It inhibits thrombin activity much more effectively than TBA, the best-known thrombin binding aptamer. The crystal structure of thrombin-NU172 complex reveals a bimodular duplex/quadruplex architecture for the aptamer, which binds thrombin exosite I through a highly complementary surface involving all three loops of the G-quadruplex module. Although the duplex domain does not interact directly with thrombin, the features of the duplex/quadruplex junction and the solution data on two newly designed NU172 mutants indicate that the duplex moiety is important for the optimization of the protein-ligand interaction and for the inhibition of the enzyme activity. Our work discloses the structural features determining the inhibition of thrombin by NU172 and put the basis for the design of mutants with improved properties

Structural motifs determining the high anti-thrombin activity of NU172, an aptamer currently in Phase II clinical trials

Human α-thrombin is a trypsin-like serine protease that has the unique ability to convert soluble fibrinogen in insoluble fibrin clot. In addition to the active site, this enzyme owns two electropositive regions, (exosite I and II), located at opposite sides of its globular shape and playing an essential role in the recognition of many cofactors and substrates.1 A special class of thrombin synthetic ligands is represented by aptamers, which are short single stranded DNA or RNA oligonucleotides that bind their targets with very high affinity and specificity by adopting defined and stable three dimensional structural motifs.2 In particular, anti-thrombin aptamers adopt a G-quadruplex structure.3-5 Recently, the researchers interest has moved 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 plasma6 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 The crystallographic characterization of thrombin-NU172 complex in the presence of either potassium or sodium ions provides together with conformational solution studies on NU172 variants and anticoagulant assays have provided a reasonable interpretation of the peculiar antithrombotic properties of NU172. Details on our results will be discussed at the Meeting. References: 1. Di Cera, E. J Thromb Haemost. 2007, 5 Suppl 1, 196 – 202. 2. Woodruff, R.S.; Sullenger, B.A. Arterioscler Thromb Vasc Biol. 2015, 35, 2083 – 2091. 3. Russo Krauss, I; Pica, A.; Merlino, A.; Mazzarella, L.; Sica, F. Acta Crystallogr D Biol Crystallogr. 2013, 69, 2403 – 2411. 4. Russo Krauss, I.; Spiridonova, V.; Pica, A.; Napolitano, V.; Sica, F. Nucleic Acids Res. 2016, 44, 983 – 991. 5. Russo Krauss, I.; Napolitano, V.; Petraccone, L.; Troisi, R.; Spiridonava, V.; Mattia, C.A.; Sica, F. Int J Biol Macromol. 2018, 107, 1697 – 1705. 6. Keefe, A.D.; Pai, S.; Ellington, A. Nat Rev Drug Discov. 2010, 9, 537 – 550. 7. Zhou, J.; Rossi, J. Nat Rev Drug Discov. 2017, 16, 181 – 202

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

Duplex/quadruplex oligonucleotides: Role of the duplex domain in the stabilization of a new generation of highly effective anti-thrombin aptamers

Recently, mixed duplex/quadruplex oligonucleotides have attracted great interest for use as biomedical aptamers. In the case of anti-thrombin aptamers, the addition of duplex-forming sequences to a G-quadruplex module identical or very similar to the best-known G-quadruplex of the Thrombin Binding Aptamer (HD1) results in new or improved biological properties, such as higher activity or different recognition properties with respect to HD1. Remarkably, this bimodular fold was hypothesized, based on its sequence, for the only anti-thrombin aptamer in advanced clinical trial, NU172. Whereas cation modulation of G-quadruplex conformation and stability is well characterized, only few data from similar analysis on duplex/quadruplex oligonucleotides exist. Here we have performed a characterization of structure and stability of four different duplex/quadruplex anti-thrombin aptamers, including NU172, in the presence of different cations and in physiological-mimicking conditions in comparison to HD1, by means of spectroscopic techniques (UV and circular dichroism) and differential scanning calorimetry. Our data show a strong reciprocal influence of each domain on the stability of the other and in particular suggest a stabilizing effect of the duplex region in the presence of solutions mimicking the physiological conditions, strengthening the idea that bimodular aptamers present better therapeutic potentialities than those containing a single G-quadruplex domain

Anti-TNF treatment response in rheumatoid arthritis patients with moderate disease activity: a prospective observational multicentre study (MODERATE)

Rheumatoid arthritis (RA) patients with moderate disease activity show progression of joint damage and have impaired quality of life, physical function, work and daily activities. Little is known about management of patients with moderate RA. The aim of the study was to assess the 1-year response to anti-TNF in biologic-na\uefve RA patients with moderate (3.2 <DAS28 645.1) disease activity despite DMARD treatment, in the Italian clinical practice