Cite this: RSC Advances, 2013, 3, 12176 Hairpin ODN-based ligands as potential inhibitors of HMGB1 cytokine activity3 Received 5th March 2013, Accepted 1st May 2013 DOI: 10.1039/c3ra41915k www.rsc.org/advances Domenica Musumeci,* a Giovanni N. Roviello, b Daniela Montesarchio, a Roberto Sapio, c Margherita Valente, c Valentina Anro ` c and Enrico M. Bucci c We here report the design, synthesis, and biological activity of a kinked hairpin-loop DNA acting at low nM concentrations as a strong inhibitor of HMGB1 (High-Mobility Group Box-1), a nuclear protein with cytokine activity in a number of inflammatory diseases. Lead compound optimization has been realized by inserting different oligo-ethylene glycol spacers at the 59-end and loop positions of the natural hairpin DNA, in order to improve its enzymatic stability and structuring capability, as well as its overall pharmacokinetic properties. Thermal stability data as well as activity assays proved that the ODN which contained two hexa-ethylene glycol spacers, one at the 59-end and the other in the loop, was the best candidate to inhibit HMGB1. Plasma stability assays and hydrodynamic volume measurements afforded further encouraging results in view of future in vivo evaluation of the optimized ligand. Introduction In previous reports 1,2 we proposed that, by screening for extracellular proteins containing nucleic acid binding domains and possibly involved in various pathologies, it should be possible to target these proteins using ad hoc designed oligonucleotide-based ligands containing the sole recognition pattern responsible for the nucleic acid-protein binding interactions, without applying SELEX (Systematic Evolution of Ligands By Exponential Enrichment) technology. In particular, by screening for proteins containing the HMG box as the DNA binding domain, a number of extracellular factors emerged, including HMGB1 (High-Mobility Group Box- 1). This is a protein involved in the architecture and reorganization of chromatin when intranuclear, 3,4 that acts as a proinflammatory mediator with immunostimulatory properties when released in serum or exposed on the cell surface. 5,6 Extracellular HMGB1 has been proposed to con- tribute to the pathogenesis of multiple chronic inflammatory and autoimmune diseases, 7–12 and high serum levels of HMGB1 have been found in several inflammatory events, including sepsis, 13 rheumatoid arthritis, 14 atherosclerosis, 15 chronic kidney disease, 16 systemic lupus erythematosus (SLE), 17,18 as well as in cancer development. 19,20 The released HMGB1 binds to the receptor for advanced glycation end products (RAGE) and activates vascular endothelial cells and macrophages/monocytes to express proinflammatory cyto- kines and chemokines. 21 Furthermore, RAGE bound by HMGB1 leads to the activation of mitogen-activated protein (MAP) kinases and to the expression of matrix metalloprotei- nases, molecules associated with tumor proliferation and invasion. 22 Therefore, inhibition of HMGB1–RAGE interaction represents a promising approach to modulate the inflamma- tory activity of the protein, as well as to block tumor growth and metastasis. This approach has been pursued through the development of molecules which interacted: 1) with RAGE or 2) with HMGB1 protein. The first strategy was realized by using recombinant box A (the truncated N-terminal domain of HMGB1) 23–25 or S100P-derived RAGE peptide, 26 which both act as competitive inhibitors of the full-length protein, interacting with RAGE without activating the receptor. Alternatively, therapies based on HMGB1-targeting involved the develop- ment of molecules which directly bind to the protein, as in the case of HMGB1-specific polyclonal and monoclonal antibo- dies, 23,24,27 or of glycyrrhizin (GR), a triterpenoid saponin glycoside of glycyrrhizic acid which bound to both HMG boxes in HMGB1 (K d 150 mM) and showed favourable effects on various HMGB1-related pathologies. 28–30 Taking into account that the interaction of HMGB1 with RAGE has been mapped to the same domain which binds the DNA (HMG box), 31 and also considering that HMGB1 is able to recognize with high affinity a range of distorted DNA structures, 32 we recently proposed that a suitable oligonucleo- tide ligand would be able to efficiently compete with the extracellular receptor of the protein. On this basis, in previous papers we reported the design, synthesis and biological activity of HMGB1 ligands based on kinked DNA duplexes. 33,34 The a Dipartimento di Scienze Chimiche, Universita ` di Napoli ‘‘Federico II’’, via Cintia 21, Complesso Universitario di Monte Sant’Angelo, I-80126, Napoli, Italy. E-mail: domymusu@alice.it; Fax: +39-081-674393; Tel: +39-081-674127 b Istituto di Biostrutture e Bioimmagini – CNR, via Mezzocannone 16, I-80134 Napoli, Italy c Bionucleon Srl, via Ribes 5, I-10010 Colleretto Giacosa (TO), Italy 3 In memory of Dr Silvano Fumero. RSC Advances PAPER 12176 | RSC Adv., 2013, 3, 12176–12184 This journal is ß The Royal Society of Chemistry 2013