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FP7

G1_G2_NAinhibit Report Summary

Project reference: 327579
Funded under: FP7-PEOPLE

Periodic Report Summary 1 - G1_G2_NAINHIBIT (Design, synthesis and evaluation of potential group-1 and group-2 neuraminidase inhibitors)

The objective of this research project is to develop new antivirals drugs with the aim of increasing the variety of drugs for influenza virus treatment. Influenza virus is an RNA virus composed of a single-stranded RNA genome enclosed within a lipoprotein envelope. This envelope contains two types of integral membrane glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Hemagglutinin is a lectin, which is a protein that binds to carbohydates, specifically to cell-surface receptors bearing terminal sialic acid residues. This recognition event mediates the viral entry in the host cells. Once the virus has been internalized and replicated by the host cell's machinery, newly formed virus particles emerge from the cell and remain bound to the membrane sialic acids by virtue of their interaction wih HA. At this point, the viral neuraminidase (NA), wich is a tetrameric hydrolytic enzyme (sialidase), cleaves the terminal sialic acid residues of the anchored glycoconjugates and allows release of new virus particles into the host organism.
Among the three types of influenza viruses, A, B and C, only Influenza A can infect humans and other animals, including birds and pigs. The nomenclature of virus subtypes designates the various identified subgroups of HA and NA proteins and new viral strains probably arise via reassortment of genes among animal and human flu viruses. Significant efforts have been dedicated to discovery of novel drugs by focussing on different molecular targets of influenza A. Flu vaccines work by raising an immune response against the surface HA and are reformulated each year to try to match antigenic HA variations, thus vaccines can be compromised by the rapidity with which influenza virus mutates. As for targeting the neuraminidase (NA) function, the most successful drugs are the structure-based drugs Oseltamivir, Zanamivir, and Peramivir, which were developed based on the structure of sialic acid bound in the active site of NA. These drugs consist of an unsaturated six-membered ring or a five-membered ring and distinctive structural features from the sialic acid-based natural substrates. These differences have ultimately led to resistant viral strains, especially in the case of Oseltamivir, underscoring the increased demand for the development of new antiviral drugs with novel structural motifs.
The design of the proposed inhibitors resulted, in the first place, from an understanding of the mechanism of action of neuraminidases (sialidases), which are retaining enzymes. The accepted mechanism for retaining sialidases involves production of a glycosylated enzyme intermediate in which both glycosylation and deglycosylation occur via short-lived transition states (TS) that have substantial oxacarbenium ion character and a distorted six-membered ring. On the basis of molecular modeling studies, it is expected that the Michaelis complex involving an influenza A N1 enzyme could be in a 4S2 or in a B2,5 conformation (Figure 1).[1]

Considering that the substrate is bound in a skew-boat conformation and the transition state for the neuraminidase-catalyzed hydrolysis of terminal sialic acid residues is presumed to be a distorted boat, which is intermediate between the structures of the Michaelis complex and the tyrosinyl–enzyme intermediate, we proposed that cyclopropane sialic acid analogues, such as 1 (Figure 1) would be a close mimic of the proposed transition state, to be our first generation target compounds. The new candidates are designed to target neuraminidase (NA) active site by mimicking the conformation of the natural substrate during the catalytic process.
Structural characterization of various influenza viral NA subtypes has recently led to the discovery of an additional cavity in the enzyme, which has been labeled as the 150-cavity, near the catalytic site of NAs. Several inhibitors have already been designed to exploit contacts in this region and increase affinity. The 150 cavity becomes available through the dynamics of the loop consisting of residues 147–152 (the 150-loop), which has been shown to have considerable flexibility, indicating that all NAs may retain the propensity for both open and closed conformations. Generally, zanamivir and oseltamivir have been shown to be good lead compounds for further optimization in the search for novel inhibitors targeting the 150 cavity. However, it is still debated whether our understanding of inhibitor selectivity between different neuraminidase groups (type 1 and 2)[2] from different strains results from inherent NA flexibility and/or from ligand induced flexibility.[3,4] Recently, we synthesized a series of triazole-containing carbocycles designed to target both the catalytic site and the 150 cavity. Similarly, in the literature several compounds have been described that appear to exploit contacts in this region by appending substituents at different positions of the central six membered ring scaffold, such as guanidinium and amino functionalities.
During the first reporting period, we successfully synthesized a novel set of possible neuraminidase inhibitors based on a fused cyclopropane scaffold 1, bearing a carboxylic acid group on the cyclopropane ring. With regards to other molecular functionalities (R substituents in Figure 1), compound 2 possesses a hydroxyl group, while compounds 3 and 4 have a basic amino group that can be further functionalized to incorporate lipophilic groups as 150-cavity binders. These constrained analogues were made via a multistep synthesis, starting from a photochemical reaction and proceeding with the introduction of the cyclopropane via a Micheal-like cyclopropanation. This reaction introduced additional structural complexity, due to the insertion of three stereocenters and led to the preparation of additional compounds. Two of the compounds synthetized, analogues of alpha and beta sialic acid, showed promising inhibitory activity against H9N2 and H5N1 strains. In particular, the kinetic analysis showed a time dependent binding, typical of neuraminidase inhibitors. Also, the synthetized amino derivatives showed low micromolar affinities for both neuraminidase subtypes. Additional experiments through a multidisciplinary approach will be performed to provide further insight into the requirements for the configuration of constrained cyclopropyl sialic acid analogues.
Influenza is a contagious infection, caused by distinct virus types and subtypes, and progress in understanding the links between viral types and infectivity is urgently required. The design of both tight and moderate-binding inhibitors provides valuable information about the mechanism of action of viral neuraminidases. Such information will help contribute to answering the intriguing questions concerning the relationships between structure and biological implications for neuraminidases; these studies involve a variety of rapidly evolving fields including NMR, biology and computational modelling. For this purpose, this project involving scientific collaborations outside Europe is critical to the reinforcement of the excellence in the European research area, with aims that include the understanding of human health and the response to global health issues.

[1] Raab M, Tvaroška I. J. Mol. Model. 2011, 17, 1445.
[2] Russell R. J. et al. Nature 2006, 443(7107), 45.
[3] a) Amaro, R. E., Swift, R. V., Votapka, L., Li, W. W., Walker, R. C., & Bush, R. M. Nat. Comm. 2011, 2, 388. b) Greenway, K. T., LeGresley, E. B., Pinto. B. M. PLOS ONE 2013, 8, e59873.
[4] Wu Y et al. Sci. Rep. 2013, 3, 2871.



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Life Sciences
Record Number: 182283 / Last updated on: 2016-05-23
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