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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.

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.

Determination of the chemical composition of different parts of the wall paintings (pigments, mortars and binders) allows extracting information about technology of preparation of the artefact. Herein we present a multimethodological characterization of wall paintings from a Roman Archeological sites from Cuma, focusing on differences between indoor (domus) and outdoor (Temple) fabrication. Both pigments, binders and mortars were studied via a combination of destructive/microdestructutive (mass spectrometry, ionic chromatography, ICP-based techniques) and non-destructive (Raman microscopy, X-ray diffraction and SANS) methodologies. Particularly, the systematic presence of dolomite only in temple mortars suggests an intentional use of such limestone for outdoor fabrication. Differences between temple and domus extends also to binder composition: proteinaceous binders in domus (possibly egg [3] and animal glue), whereas in TCP no protein was detected but some fat acids (probably from animal fat). Ultimately, the multimethodological study allowed an overall picture of the components and technology of the paintings from fabrications with different use, proposing distinct engineering choices for indoor and outdoor walls. References [1] Cartechini L., Pucci P., Sgamellotti A., Marino G., Birolo L., “Proteomic strategies for the identification of proteinaceous binders in paintings” Analytical & Bioanalytical Chemistry 395(7), 2009, 2269-2280. [2] Gelzo M., Grimaldi M., Vergara A., Severino V., Chambery A., Dello Russo A., Piccioli C., Corso G,. Arcari P. "Comparison of binder composition in Pompeian wall painting styles from Insula Occidentalis" Chemistry Central J. 8(65), 2014, 1-10. [3] Vinciguerra R, Galano E, Vallone F, Greco G, Vergara A, Bonaduce I, Marino G, Pucci P, Amoresano A, Birolo L, “Deglycosylation Step to Improve the Identification of Egg Proteins in Art Samples” Analytical Chemistry 87 (20), 2015, 10178-10182. Acknowledgements This work was financially supported by Distretto ad Alta Tecnologia dei Beni Culturali (DATABENC scarl), progetto SNECS. “Soprintendenza Speciale per i Beni Archeologici di Napoli e Pompei” – Ufficio Archeologico di Cuma - Dr Paolo Caputo is also acknowledged.

Human angiogenin (ANG) is a 14-kDa ribonuclease involved in different pathophysiological processes including tumorigenesis, neuroprotection, inflammation, innate immunity, reproduction, the regeneration of damaged tissues and stress cell response, depending on its intracellular localization. Under physiological conditions, ANG moves to the cell nucleus where it enhances rRNA transcription; conversely, recent reports indicate that under stress conditions, ANG accumulates in the cytoplasmic compartment and modulates the production of tiRNAs, a novel class of small RNAs that contribute to the translational inhibition and recruitment of stress granules (SGs). To date, there is still limited and controversial experimental evidence relating to a hypothetical role of ANG in the epidermis, the outermost layer of human skin, which is continually exposed to external stressors. The present study collects compelling evidence that endogenous ANG is able to modify its subcellular localization on HaCaT cells, depending on different cellular stresses. Furthermore, the use of recombinant ANG allowed to determine as this special enzyme is effectively able to counter at various levels the alterations of cellular homeostasis in HaCaT cells, actually opening a new vision on the possible functions that this special enzyme can support also in the stress response of human skin.

Despite their unquestionable properties, oligonucleotide aptamers display some drawbacks that continue to hinder their applications. Several strategies have been undertaken to overcome these weaknesses, using thrombin binding aptamers as proof-of-concept. In particular, the functionalization of a thrombin exosite I binding aptamer (TBA) with aromatic moieties, e.g., naphthalene dimides (N) and dialkoxynaphthalenes (D), attached at the 5' and 3' ends, respectively, proved to be highly promising. To obtain a molecular view of the effects of these modifications on aptamers, we performed a crystallographic analysis of one of these engineered oligonucleotides (TBA-NNp/DDp) in complex with thrombin. Surprisingly, three of the four examined crystallographic structures are ternary complexes in which thrombin binds a TBA-NNp/DDp molecule at exosite II as well as at exosite I, highlighting the ability of this aptamer, differently from unmodified TBA, to also recognize a localized region of exosite II. This novel ability is strictly related to the solvophobic behavior of the terminal modifications. Studies were also performed in solution to examine the properties of TBA-NNp/DDp in a crystal-free environment. The present results throw new light on the importance of appendages inducing a pseudo-cyclic charge-transfer structure in nucleic acid-based ligands to improve the interactions with proteins, thus considerably widening their potentialities.

Thrombin is the key enzyme of the entire hemostatic process since it is able to exert both procoagulant and anticoagulant functions; therefore, it represents an attractive target for the developments of biomolecules with therapeutic potential. Thrombin can perform its many functional activities because of its ability to recognize a wide variety of substrates, inhibitors, and cofactors. These molecules frequently are bound to positively charged regions on the surface of protein called exosites. In this review, we carried out extensive analyses of the structural determinants of thrombin partnerships by surveying literature data as well as the structural content of the Protein Data Bank (PDB). In particular, we used the information collected on functional, natural, and synthetic molecular ligands to define the anatomy of the exosites and to quantify the interface area between thrombin and exosite ligands. In this framework, we reviewed in detail the specificity of thrombin binding to aptamers, a class of compounds with intriguing pharmaceutical properties. Although these compounds anchor to protein using conservative patterns on its surface, the present analysis highlights some interesting peculiarities. Moreover, the impact of thrombin binding aptamers in the elucidation of the cross-talk between the two distant exosites is illustrated. Collectively, the data and the work here reviewed may provide insights into the design of novel thrombin inhibitors.

Aptamers are synthetic molecules of different natures (mostly, DNA or RNA) that recognize a target molecule with high affinity and specificity [...]

The long-range communication between the two exosites of human α-thrombin (thrombin) tightly modulates the protein-effector interactions. Duplex/quadruplex aptamers represent an emerging class of very effective binders of thrombin. Among them, NU172 and HD22 aptamers are at the forefront of exosite I and II recognition, respectively. The present study investigates the simultaneous binding of these two aptamers by combining a structural and dynamics approach. The crystal structure of the ternary complex formed by the thrombin with NU172 and HD22_27mer provides a detailed view of the simultaneous binding of these aptamers to the protein, inspiring the design of novel bivalent thrombin inhibitors. The crystal structure represents the starting model for molecular dynamics studies, which point out the cooperation between the binding at the two exosites. In particular, the binding of an aptamer to its exosite reduces the intrinsic flexibility of the other exosite, that preferentially assumes conformations similar to those observed in the bound state, suggesting a predisposition to interact with the other aptamer. This behaviour is reflected in a significant increase of the anticoagulant activity of NU172 when the inactive HD22_27mer is bound to exosite II, providing a clear evidence of the synergic action of the two aptamers.

Post-SELEX modification of DNA aptamers is an established strategy to improve their affinity or inhibitory characteristics. In this study, we examined the possibility of increasing the recognition interface between the thrombin-binding aptamer HD1 (TBA) and thrombin by adding a chemically modified side chain to selected nucleotide residues. A panel of 22 TBA variants with N3-modified residues T3 and T12 was prepared by a two-step modification procedure. Aptamers were characterized by a combination of biophysical and biochemical methods. We identified mutants with enhanced affinity and improved anticoagulant activity. The crystal structures of thrombin complexes with three selected modified variants revealed that the modified pyrimidine base invariably allocates in proximity to thrombin residues Tyr76 and Ile82 due to the directing role of the unmodified TT loop. The modifications induced an increase in the contact areas between thrombin and the modified TBAs. Comparative analysis of the structural, biochemical, and biophysical data suggests that the non-equivalent binding modes of the mutants with thrombin in the T3- and T12-modified series account for the observed systematic differences in their affinity characteristics. In this study, we show that extending the recognition surface between the protein and modified aptamers is a promising approach that may improve characteristics of aptamer ligands., Graphical Abstract, Smirnov et al. describe a post-SELEX approach for the thrombin aptamer HD1 that consists in expanding the aptamer-protein interface through attachment of non-natural side chains at specific aptamer residues. The authors report synthesis and characterization of 22 aptamer mutants and present crystal structures of thrombin complexes with three selected variants.

During the last decades, thrombin binding aptamer (TBA) was one of the most extensively studied oligonucleotide G-quadruplex aptamers [1] and, despite the advent of new anti-thrombin aptamers [2], the search for mutations that are able to improve TBA properties is still ongoing [3]. In this context, an extensive analysis of twenty-two TBA variants with N3-modified residues Thy3 and Thy12, two key residues of the two TT loops that guide the binding with thrombin, was performed in the course of a collaborative research with Engelhardt Institute of Molecular Biology (Russian Academy of Sciences, Russia). Among them, some mutants with enhanced affinity and improved anticoagulant activity were identified (data to be published). Here, we present the crystal structures of the complexes between thrombin and three of these TBA variants, containing a modification in the Thy3 nucleobase. The data revealed that the modified pyrimidine base invariably allocates in proximity to thrombin residues Tyr76 and Ile82 due to the directing role of the unmodified TT loop. Moreover, the modifications were found to induce an increase in the contact areas between thrombin and the modified TBAs by a slight conformation adaptation of the aptamer to the thrombin binding site. Details will be presented in the poster.

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.

Molecular dynamics simulations shed light on the cooperative mechanisms generated by the simultaneous binding of aptamers at the two exosites of thrombin Human α-thrombin is a trypsin-like serine protease endowed with 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 [1]. The narrow substrate specificity of thrombin and its ability to change function are regulated by the exosite binding to different cofactors and modulators [2]. A special class of thrombin exosite synthetic ligands is represented by G-quadruplex anticoagulant aptamers, which are short single stranded DNA or RNA oligonucleotides that bind their targets with very high affinity and specificity [3]. The minimal 15mer DNA aptamer, named TBA, is the first and the most studied anti-thrombin aptamer. Based on several X-ray studies, this aptamer was established to bind the fibrinogen-binding site of thrombin (exosite I) by a pincer-like recognition mechanism involving the two TT loops [4]. The addition of a duplex motif to the G-quadruplex module has produced a new generation of aptamers with higher affinity against thrombin compared to TBA [5]. Among them, an aptamer, named HD22_27mer, recognizes exosite II with both quadruplex and duplex domains [6].In the last years, great attention has been paid to the study of the effects of the simultaneous binding of two ligands on the two exosites of thrombin. Biochemical studies have suggested that thrombin is an allosterically modulated enzyme: an interplay between its two exosites as well as between the exosites and the active site has been highlighted [7]. Furthermore, the crystallographic structures of two thrombin ternary complexes, in which exosite II is bound to HD22_27mer and exosite I interacts with TBA-like aptamers, were solved and gave structural information on the effects of the simultaneous binding of two aptamers to thrombin exosites [8]. Here we present the results of an extensive molecular dynamics study performed on free thrombin and on its binary and ternary complexes with TBA and HD22_27mer in the absence of the PPACK inhibitor that is covalently bound to the protein active site in all the crystallographic α-thrombin models. This study revealed that, in the absence of any influence of the crystal packing, an inter-exosite cross-talk and an active site-exosite communication may occur in these systems. Details will be discussed at the Meeting.[1] E. Di Cera, J Thromb Haemost., 5, (2007), 196-202.[2] S. Krishnaswamy, J Thromb Haemost., 3, (2005), 54-67.[3] A.D. Keefe, S. Pai, A. Ellington, Nat Rev Drug Discov., 9, (2010), 537-550.[4] I. Russo Krauss, A. Merlino, A. Randazzo, E. Novellino, L. Mazzarella, F. Sica, Nucleic Acids Res., 40, (2012), 8119-8128.[5] I. Russo Krauss, V. Napolitano, L. Petraccone, R. Troisi, V. Spiridonova, C.A. Mattia, F. Sica, Int J Biol Macromol., 107, (2018), 1697-1705.[6] I. Russo Krauss, A. Pica, A. Merlino, L. Mazzarella, F. Sica, Acta Cryst. D, 69, (2013), 2403-2411.[7] N.S. Petrera, A.R. Stafford, B.A. Leslie, C.A. Kretz, J.C. Fredenburgh, J.I. Weitz, J Biol Chem., 284, (2009), 25620-25629.[8] A. Pica, I. Russo Krauss, V. Parente, H. Tateishi-Karimata, S. Nagatoishi, K. Tsumoto, N. Sugimoto, F. Sica, Nucleic Acids Res., 45, (2017), 461-469

Human α-thrombin is a trypsin-like serine protease playing a pivotal role in the haemostatic pathway. Apart from the active site, thrombin has two electropositive patches on its surface, named exosites I and II, which determine the biological properties of the enzyme through the binding of substrates and cofactors [1,2]. The two exosites also represent the anchoring sites for 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 3D structures [3,4]. New generation anti-thrombin aptamers, which adopt a mixed duplex/quadruplex structure and possess improved binding or anticoagulant properties, are currently under study [5]. Among them, NU172, the only thrombin binding aptamer in advanced clinical trial, recognizes exosite I with its quadruplex domain [6]. On the other hand, HD22_27mer oligonucleotide binds exosite II with both quadruplex and duplex domains [7]. Both these bimodular aptamers show high affinity toward their target. Recently, great attention has been paid to the study of the effects of the simultaneous binding of two ligands on both thrombin exosites [8-11]. In particular, an efficient modulation of thrombin activity could be obtained by the combined use of NU172 and HD22_27mer. Here we present the crystal structure of the ternary complex, in which thrombin is sandwiched between NU172 at exosite I and HD22_27mer at exosite II. Even though the crystalline packing reduces the conformational mobility of the two aptamers, the comparison of the present model with those of the relative binary complexes has revealed small but significant clues of an inter-exosites cross-talk. To evaluate the actual influence of the crystal packing on the long-range communication between the two exosites, a molecular dynamics study of the ternary complex is in progress. Details will be discussed at the Meeting. [1] E. Di Cera J Thromb Haemost. 2007, 5, 196. [2] S. Krishnaswamy J Thromb Haemost. 2005, 3, 54. [3] A.D. Keefe, S. Pai, A. Ellington Nat Rev Drug Discov. 2010, 9, 537. [4] I. Russo Krauss, A. Merlino, A. Randazzo, E. Novellino, L. Mazzarella, F. Sica Nucleic Acids Res. 2012, 40, 8119. [5] I. Russo Krauss, V. Napolitano, L. Petraccone, R. Troisi, V. Spiridonova, C.A. Mattia, F. Sica Int J Biol Macromol. 2018, 107, 1697. [6] R. Troisi, V. Napolitano, V. Spiridonova, I. Russo Krauss, F. Sica Nucleic Acids Res. 2018, 46, 12177. [7] I. Russo Krauss, A. Pica, A. Merlino, L. Mazzarella, F. Sica Acta Cryst. D 2013, 69, 2403. [8] N.S. Petrera, A.R. Stafford, B.A. Leslie, C.A. Kretz, J.C. Fredenburgh, J.I. Weitz J Biol Chem. 2009, 284, 25620. [9] X. Feng, C. Yu, F. Feng, P. Lu, Y. Chai, Q. Li, D. Zhang, X. Wang, L. Yao Chemistry 2019, 25, 2978. [10] K. Derszniak, K. Przyborowski, K. Matyjaszczyk, M. Moorlag, B. de Laat, M. Nowakowska, S. Chlopicki Front Pharmacol. 2019, 10, 68. [11] A. Pica, I. Russo Krauss, V. Parente, H. Tateishi-Karimata, S. Nagatoishi, K. Tsumoto, N. Sugimoto, F. Sica Nucleic Acids Res., 2017, 45, 461.