SYNTHESIS AND BIOLOGICAL EVALUATION OF NEW NEURAMINIDASE INHIBITORS DERIVED FROM SIALIC ACID AS POTENTIAL ANTIVIRAL AGENTS
Creators: name: LA ROCCA, PAOLO
Publisher: name: Universit� degli Studi di Milano
Year: 2018

description: The development of new, potent and selective bacterial, viral and human sialidase (neuraminidase) inhibitors is an important issue to be pursued in order to achieve both useful therapeutical and biochemical tools. In fact, these hydrolytic enzymes can represent a good target since they play key roles in some physio-pathological processes by regulating the levels of sialic acid (such as the N-acetyl neuraminic acid; Neu5Ac) presents in glycoconjugates. In addition, in the design of inhibitors against a specific member of this class of enzymes is critical to take into account that these proteins share some common features such as the tridimensional structure of their catalytic domain, but, on the other hand, they show a very low sequence identity. Indeed, the only conserved residues are some active site amino acids essential for the catalytic mechanism. This thesis work was focused on the synthesis of hemagglutinin-neuraminidase (HN) inhibitors against the Newcastle virus (NDV), a member of the Paramyxoviridae family and strictly related to human parainfluenza viruses (hPIVs). NDV is a single-stranded RNA virus which could affect most species of both domestic and wild birds, causing significant and substantial economic losses in the poultry industry. To date, vaccination is the preferential instrument to border the infection, but when this procedure is not applicable, an efficient antiviral therapy could be the only useful way to control NDV outbreaks. At this purpose, the HN glycoproteins of paramyxoviruses represent an excellent target to be hit because they have some key roles in viral lifecycle: a) allowing viral attachment to the target cell; b) promoting the fusion process and, finally c) ensuring the release of the neo-synthesized virions. Over the past years, while some 2,3-unsaturated Neu5Ac derivatives (DANA derivatives) have been marketed as inhibitors against influenza virus neuraminidases (belonging to Orthomyxoviridae family), no compounds reach the clinical phase for paramyxoviruses treatment. In particular, few molecules have been developed for NDV-HN, and the N-trifluoroacetyl derivative of DANA (FANA) was still the best inhibitor until my thesis work. So, the necessity to find new, potent and possibly selective inhibitors against paramyxoviruses-HNs remain a key issue. At this purpose, the successful strategy, resulted fundamental to develop new NDV-HN inhibitors, was based on a multidisciplinary approach that combined the use of a) the chemical synthetic procedures, b) the computational docking studies and c) some biochemical activity assays. More in detail, the attention was directed to the study of two classes of inhibitors: ? Some C5 or C4/C5 modified 2,3-unsaturated DANA derivatives, as reversible inhibitors. ? Some scarcely investigated C2 modified 3,4-unsaturated Neu5Ac analogues, as irreversible ones. We finally reached satisfying results, regarding both classes of inhibitors: a) The understanding of the influence of the C5 N-perfluorinated substituents on the inhibitory activity of some 2,3-unsaturated DANA analogues, as potent and reversible NDV-HN inhibitors. b) The discovery of a new C5 N-perfluorinated inhibitor against NDV-HN as potent as FANA (the best NDV-HN inhibitor previously published) but more selective for NDV-HN towards human NEU3. c) The significant achievements of five new, potent and selective C4 and C5 modified 2,3-unsaturated DANA derivatives. All these compounds, combining the C4 azido or C4 p-toluensolfonamido group with the C5 N-perfluorinated chains, showed IC50 values in the nanomolar range; thus, they are up to 15-fold more potent than FANA. d) The set-up of more efficient synthetic procedures to achieve the 3,4-unsaturated Neu5Ac derivatives in high yields and ?-anomeric stereoselectivity. e) The set-up of a smart and rapid method to unequivocally attribute the C2 configuration of the 3,4-unsaturated Neu5Ac inhibitors, via a 1,7-lactonization reaction. f) The mechanism elucidation of an unreported and unexpected chemical scrambling between the C4 and the C5 position of Neu5Ac derivatives (through a previously uncharacterized reaction intermediate). In addition, the rigid and induced fit docking simulation results permitted me to speculate on the interactions of the synthesized inhibitors with some active site amino acids, such as Lys236, a well know key residue involved in NDV-NH catalytic site activation mechanism and in fusion promotion activity. The comprehension of ligand/receptor interactions could lead to the development of molecules able to block, not only the neuraminidase activity of NDV-HN or other paramyxoviruses-HN, but also other viral functions mediated by these enzymes. Some of the obtained results allowed the publication of two scientific articles: ? Rota, P., La Rocca, P., Piccoli, M., Montefiori, M., Cirillo, F., Olsen, L., Orioli, M., Allevi, P., and Anastasia, L. (2018) Potent Inhibitors against Newcastle Disease Virus Hemagglutinin-Neuraminidase, ChemMedChem 13, 236-240. ? Rota, P., Papini, N., La Rocca, P., Montefiori, M., Cirillo, F., Piccoli, M., Scurati, R., Olsen, L., Allevi, P., and Anastasia, L. (2017) Synthesis and chemical characterization of several perfluorinated sialic acid glycals and evaluation of their in vitro antiviral activity against Newcastle disease virus, MedChemComm 8, 1505-1513.