Design and synthesis of Human Neutrophil Elastase (HNE) inhibitors

DESIGN AND SYNTHESIS OF HUMAN NEUTROPHIL ELASTASE (HNE) INHIBITORS Dottorato in area del Farmaco e Trattamenti Innovativi PhD student: Antonella Iacovone Scientific tutor: Prof.ssa Maria Paola Giovannoni Theoretical tutor: Prof.ssa Elisabetta Teodori Human Neutrophil Elastase (HNE) is an enzyme belonging to the family of serine proteases. It is a small, soluble and basic glycoprotein of approximately 30 kDa containing 218 amino acid residues that are stabilized by four disulfide bridges. HNE carries out its activity through the catalytic triad, consisting of Ser195, His57 and Asp102. It is involved in the killing of pathogens, in the regulation of inflammation and tissue homeostasis due to its proteolytic action against a variety of extracellular matrix proteins, such as elastin, collagen, fibronectin, laminin, and proteoglycans. In physiological conditions, HNE proteolytic activity is regulated by endogenous inhibitors belonging to the serpins family, including α1-antitripsin (α1-AT), α2-macroglobuline, elafin and secretory leucocyte protease inhibitor (SLPI). Alteration of the balance between HNE and serpins activity can contribute to the develop or the worsening of certain pathologies, especially affecting the respiratory system. The main pulmonary diseases involving HNE are chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF), acute respiratory distress syndrome (ARDS) and acute lung injury (ALI). Additionally, HNE is also involved in other inflammatory disorders including psoriasis, dermatitis, atherosclerosis, rheumatoid arthritis and various types of cancer. Many examples of peptide and non-peptide HNE inhibitors have been reported in the literature, however, only two drugs are currently available for clinical use: Prolastin (purified α1-AT), a peptide drug synthetized by recombinant DNA techniques and used for the treatment of α1-antitripsin deficiency (AATD) and Sivelestat (Elaspol®100), a low molecular weight non-peptide molecule. The use of Sivelestat for the treatment of acute lung injury associated with systemic inflammation is approved only in Japan and Korea. The research performed in this period as a PhD student consisted in the design and synthesis of new potential HNE inhibitors. In particular, on one side we focused on the synthesis of compounds with 7-azaindole structure, as isomers of the potent indazoles, previously investigated in our research group. On the other side, we moved our attention on the investigation of new small and flexible compounds with isoxazol-5(2H)-one scaffold; additionally we synthetized some products with benzo[c]isoxazol-3(1H)-one nucleus, as elaboration of the isoxazolone derivatives. All new products were tested as HNE inhibitors in the laboratory of Prof. Quinn, Montana University, and many of these compounds have proved to be very potent inhibitors, recording activity values in the nanomolar range. On selected isoxazolones derivatives molecular modeling studies were performed to clarify the interaction with the target enzyme, as well as studies on stability in aqueous buffer and on kinetic of HNE inhibition. References 1. Hoenderdos, K.; Condliffe, A. The neutrophil in chronic obstructive pulmonary diseases. Am. J. Respir. Cell. Mol. Biol. 2013, 48, 531-539. 2. Cools-Lartigue, J.; Spicer, J.; Najmeh, S.; Ferri, L. Neutrophil extracellular traps in cancer progression. Cell. Mol. Life Sci. 2014, 71, 4179-4194. 3. Pham, C. T. Neutrophil serine proteases: specific regulators of inflammation. Nat. Rev. Immunol. 2006, 6, 541-550. 4. Crocetti, L.; Giovannoni, M. P.; Schepetkin, I. A.; Quinn, M. T.; Khlebnikov, A. I.; Cilibrizzi, A.; Dal Piaz, V.; Graziano, A.; Vergelli, C. Design, synthesis and evaluation of N-benzoylindazole derivatives and analogues as inhibitors of human neutrophil elastase. Bioorg. Med. Chem. 2011, 19, 4460-4472. 5. Crocetti, L.; Schepetkin, I. A.; Cilibrizzi, A.; Graziano, A.; Vergelli, C.; Giomi, D.; Khlebnikov, A. I.; Quinn, M. T.; Giovannoni, M. P. Optimization of N-benzoylindazole derivatives as inhibitors of human neutrophil elastase. J. Med. Chem. 2013, 56, 6259-6272. 6. Giovannoni, M. P.; Schepetkin, I. A.; Crocetti, L.; Ciciani, G.; Cilibrizzi, A.; Guerrini, G.; Khelebnikov, A. I.; Quinn, M. I.; Vergelli, C. Cinnoline derivatives as human neutrophil elastase inhibitors. J. Enz. Inhib. Med. Chem. 2016, 31(4), 628-639. 7. Crocetti, L.; Schepetkin, I. A.; Ciciani, G.; Giovannoni, M. P.; Guerrini, G.; Iacovone, A.; Khlebnikov, A. I.; Kirpotina, L. N.; Quinn, M. T. Synthesis and farmacological evaluation of indole derivative as deaza analogues of potent human neutrophil elastase inhibitors. Drug Dev. Res. 2016, 77(6), 285-289. 8. Vergelli, C.; Schepetkin, I. A.; Crocetti, L.; Iacovone, A.; Giovannoni, M. P.; Guerrini, G.; Khlebnikov, A.I.; Ciattini, S.; Ciciani, G.; Quinn, M. T. Isoxazol-5(2H)-one: a new scaffold for potent human neutrophil elastase (HNE) inhibitors. J. Enz. Inhib. Med. Chem. 2017, 32(1), 821-831. SOLID PHASE PEPTIDE SYNTHESIS OF SMALL PEPTIDE-BASED METAL CHELATORS Supervisor: Professor Robert Hider During the last year of PhD, a visiting period at King’s College of London was spent. The host research group of Professor Robert Hider is involved for many years in the design and synthesis of Iron and Gallium chelators. In particular, as regard the Iron chelators they follow two main project. The first one concerns the synthesis of small molecules used for the treatment of the pathologies characterized by an iron overload including hemochromatosis and transfusion-dependent thalassemia; the second one concerns the synthesis of peptide as novel biosensors for the mitochondrial Labile Iron Pool (LIP) in order to better understand the role of the LIP in mitochondrial oxidative disorders. About the gallium chelators, peptide molecules have recently been developed as radiopharmaceuticals for PET imaging in particular to detect and monitor prostate cancer. In this context, the entrusted project focused on the solid-phase synthesis of tripeptide (Lys-Lys-Lys) and tetrapeptide (Lys-Lys-Lys-Lys) derivatives bearing 3,4-dihydroxy picolinic acid and the 3-(3-hydroxy-2-methyl-4-oxopyridin-1(4H)-yl)propionic acid (as chelating groups) on the amino groups of the side chains. All final compounds will be tested in the near future as iron and gallium chelators for biological and clinical applications. References 1. Hider R. C.; Zhou T. The design of orally active iron chelators. Ann N. Y. Acad. Sci. 2005, 1054, 141-154. 2. Abbate, V.; Reelfs, O.; Hider, R. C.; Pourzand, C. Design of novel fluorescent mitochondria-targeted peptides with iron selective sensing activity. Biochem. J. 2015, 469 (3), 357-366. 3. Cusnir, R.; Imberti, C.; Hider, R. C.; Blower, P. J.; Ma, M.T. Hydroxypyridinone chelators: from iron scavenging to radiopharmaceuticals for PET imaging with Gallium-68. J. Mol. Sci. 2017, 18, 116.