Structural and functional properties of complexes involving G-quadruplex-based DNA aptamers

Despite significant advances in the prevention and treatment of thrombosis, this disease is still one of the leading causes of death worldwide. The anticoagulant drugs often used to control blood coagulation in many cases show adverse effects. Oligonucleotide aptamers have shown to be alternative specific anticoagulants, which are characterized by nonimmunogenicity and nontoxicity. Interestingly, a subclass of these aptamers that adopts a G-quadruplex-based structure is able to effectively modulate the activity and the generation of the human α-thrombin (thrombin). In particular, anti-thrombin aptamers that recognize either thrombin exosite I (TBA, NU172) or exosite II (HD22) were selected. Despite their excellent anticoagulant properties, new studies expand day-to-day in order to in-depth elucidate their mechanism of action and overcome some long-standing limitations, as the short circulating half-life in vivo. The research activity carried out in the frame of this PhD project has been focused on three intriguing aspects of the interaction between thrombin or its zymogen prothrombin and aptamers adopting a G-quadruplex or a mixed duplex/quadruplex structure. First, using a repertoire of different experimental and computational techniques, the impact that the binding of HD22_27mer aptamer at the exosite II has on the binding of TBA or NU172 at exosite I, and vice versa, was investigated. In particular, the crystal structure of the ternary complex formed by the thrombin with NU172 and HD22_27mer, extensive molecular dynamics simulations of different thrombin/aptamer complexes, and anticoagulant activity experiments were performed. Collectively, the findings provide a clear and detailed picture of the cooperative action that TBA or NU172 and HD22_27mer exert on thrombin inhibition. Secondly, structural studies on new analogues of thrombin binding aptamers were carried out. In particular, the crystal structures of the complexes between thrombin and three TBA variants, in which Thy3 contains functional substituents at N3 of the pyrimidine heterocycle, were solved. The results suggest an explanation for their higher binding affinity toward thrombin with respect to that of TBA. Moreover, a preliminary analysis of the crystal structures of the complexes between thrombin and two TBA variants, carrying modifications at 5ʹ and 3ʹ ends, was presented. Conversely, the structural and biochemical properties of NU172 variants incorporating hexitol nucleotides were investigated in solution, indicating the modification at the Thy9 as the most promising. Finally, a preliminary investigation of the interaction between prothrombin and some anti-thrombin aptamers recognizing exosite I was carried out. In particular, a thermodynamic analysis of the binding of TBA, RE31 and NU172 aptamers to thrombin and prothrombin was performed by means of ITC experiments. The results indicate the ability of the examined aptamers to recognize prothrombin pro-exosite I with an affinity similar to that shown for thrombin exosite I. Furthermore, the ability of prothrombin to act as molecular chaperone of aptamers was revealed by CD experiments. To increase the possibility to obtain crystallographic information on aptamer-prothrombin complexes, considering the high flexibility of the wild-type protein, the prethrombin-2 intermediate and two prothrombin mutants were recombinantly produced

Aptamers: Functional-Structural Studies and Biomedical Applications

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

Exosite Binding in Thrombin: A Global Structural/Dynamic Overview of Complexes with Aptamers and Other Ligands

: 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

Structural and functional analysis of the simultaneous binding of two duplex/quadruplex aptamers to human α-thrombin

: 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

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

Antimicrobial Applications of Green Synthesized Bimetallic Nanoparticles from Ocimum basilicum

Antibiotic resistance is an important and emerging alarm for public health that requires development of new potential antibacterial strategies. In recent years, nanoscale materials have emerged as an alternative way to fight pathogens. Many researchers have shown great interest in nanoparticles (NPs) using noble metals, such as silver, gold, and platinum, even though numerous nanomaterials have shown toxicity. To overcome the problem of toxicity, nanotechnology merged with green chemistry to synthesize nature-friendly nanoparticles from plants. Here, we describe the synthesis of NPs using silver (AgNPs) and platinum (PtNPs) alone or in combination (AgPtNPs) in the presence of Ocimum basilicum (O. basilicum) leaf extract. O. basilicum is a well-known medicinal plant with antibacterial compounds. A preliminary chemical–physical characterization of the extract was conducted. The size, shape and elemental analysis were carried out using UV–Visible spectroscopy, dynamic light scattering (DLS), and zeta potential. Transmission electron microscopy (TEM) confirmed polydisperse NPs with spherical shape. The size of the particles was approximately 59 nm, confirmed by DLS analysis, and the polydisperse index was 0.159. Fourier transform infrared (FTIR) demonstrated an effective and selective capping of the phytoconstituents on the NPs. The cytotoxic activities of AgNPs, PtNPs and AgPtNPs were assessed on different epithelial cell models, using the 3-[4.5-dimethylthiazol-2-yl]-2.5-diphenyltetrazolium bromide (MTT) cell proliferation assay, and discovered low toxicity, with a cell viability of 80%. The antibacterial potential of the NPs was evaluated against Escherichia coli (E. coli), Enterococcus faecalis (E. faecalis), Klebsiella pneumonia (K. pneumoniae), and Staphylococcus aureus (S. aureus) strains. Minimum inhibitory concentration (MIC) assays showed AgPtNP activity till the least concentration of NPs (3.15–1.56 g/mL) against ATCC, MS, and MDR E. coli, E. faecalis, and S. aureus and the Kirby–Bauer method showed that AgPtNPs gave a zone of inhibition for Gram-positive and Gram-negative bacteria in a range of 9–25 mm. In addition, we obtained AgPtNP synergistic activity in combination with vancomycin or ampicillin antibiotics. Taken together, these results indicate that bimetallic nanoparticles, synthesized from O. basilicum leaf extract, could represent a natural, ecofriendly, cheap, and safe method to produce alternative antibacterial strategies with low cytotoxicity

Protective Effects of Recombinant Human Angiogenin in Keratinocytes: New Insights on Oxidative Stress Response Mediated by RNases

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

Exosite Binding in Thrombin: A Global Structural/Dynamic Overview of Complexes with Aptamers and Other Ligands

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

Molecular dynamics simulations shed light on the cooperative mechanisms generated by the simultaneous binding of aptamers at the two exosites of thrombin”

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), 461-46