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G1_G2_NAINHIBIT Report Summary

Project ID: 327579
Funded under: FP7-PEOPLE
Country: Italy

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

The main objective of this project was the development of novel influenza A neuraminidase inhibitors aiming at possibly increasing the number and variety of drugs for influenza A virus treatment.

Influenza A is a contagious respiratory disease that causes severe illness and death in high-risk populations on a global scale. Structurally, Influenza A virus is an RNA virus enclosed within a lipoprotein envelope. This envelope contains two critical membrane glycoproteins, hemagglutinin (HA) and neuraminidase (NA). HA is a lectin which binds 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 virions emerge from the cell and remain bound to the membrane sialic acids by means of their interaction with HA. At this stage, the viral neuraminidase (NA), which is a tetrameric hydrolytic enzyme (sialidase), cleaves the terminal sialic acid residues of the anchored glycoconjugates and allows release of the newly budded virus particles into the host organism, spreading the infection. 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 arise via reassortment of genes among animal and human flu viruses. Significant effort has been dedicated to the discovery of novel drugs targeting HA (vaccines) and NA (antivirals). Influenza A vaccines work by raising an immune response against the surface HA and are reformulated each year, trying to match that year's HA variations. Thus, vaccines can be compromised by the rapidity with which influenza virus mutates. As for influenza A antivirals, the most successful drugs are Oseltamivir, Zanamivir, and Peramivir. These molecules consist of functionalized unsaturated six- or five-membered rings mimicking the sialic acid natural substrate bound in the active site of NA. However, with the emergence of oseltamivir-resistant influenza virus strains, it is important to develop new antiviral drugs, with novel structural motifs and substitution patterns.

In particular, we explored the use of a bicyclic (bicyclo[3.1.0]hexane) analogue of sialic acid that has been designed to mimic the conformation adopted during the enzymatic cleavage within the neuraminidase binding site (Figure 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, we proposed that the bicyclo[3.1.0]hexane scaffold, appropriately functionalized, would be a close mimic of the proposed transition state. Additional functionalization was also proposed as a way to increase the affinity by reaching an additional binding region, the so-called neuraminidase 150-cavity. The 150 cavity becomes available through the dynamics of a loop in NA consisting of residues 147–152 (the 150-loop), which has been shown to have considerable flexibility, indicating that all NAs may retain the propensity of loop opening for additional interactions within the cavity.

The scientific goals of the project have been pursued with a multidisciplinary approach, including:
- the development and optimization of synthetic routes to build the general bicyclo[3.1.0]hexane scaffold;
- Functionalization of the scaffold with different groups, to target more efficiently the enzyme pocket (catalytic site) and the additional 150-cavity;
- Inhibitors evaluation with fluorometric enzymatic assays;
- Inhibitors evaluation with influenza replication inhibition test with MDCK cells;
- Further inhibitors design with molecular modelling.

To reach these goals we proposed a research program divided in two phases, overlapping with the outgoing and return phases.
1) During the outgoing phase of two years at Simon Fraser University, we successfully synthesized a first series of compounds based on the proposed bicyclo[3.1.0]hexane scaffold, bearing a carboxylic acid group on the cyclopropane ring and additional functionalities on the five member ring, comprising the 3-pentyl side chain, which is present in Oseltamivir, and lipophilic groups as 150-cavity binders. These constrained molecules were made via a multistep synthesis, starting from a photochemical reaction and proceeding with the introduction of the cyclopropane via a Micheal-like cyclopropanation reaction. We generate, in the synthetic process, six stereocenters for a total of six skeleton carbons. Given that this first series of bicyclo[3.1.0]hexane inhibitors were at least four orders of magnitude less active than available drugs, we hypothesized that the new carbon skeleton didn’t produce the same set of interactions as the cyclohexene frameworks used in previous discovery efforts. We tried to address this critical point with the aid of molecular modelling and we proposed new structures with different functionalization, such as the introduction of amino and guanidinium groups and different ether side chain other than the 3-pentyl side chain, the characteristic side chain in Oseltamivir.
2) These derivatives were successfully synthetized during the return phase of one year at Università degli Studi di Milano. A highly simplified synthetic route was developed, starting from the cyclopropanation of cyclopentenone and followed by an aziridination and further functionalization of the five-member ring. This allowed us to efficiently prepare a small library of bicyclic ligands that, unfortunately, did not result in improved activity against various influenza A neuraminidases. Overall, the information collected in this project allowed us to speculate that the 3-pentyl side chain could be of crucial importance for productive interactions of sialic acid mimics with influenza A neuraminidases. The functionalizations proposed and installed on the bicyclo[3.1.0]hexane scaffold in the second phase of the project did not result in productive interactions with neuraminidases. We conclude that the choice and the positioning of functional groups on the bicyclo[3.1.0]hexyl system still need to be properly tuned for producing complementary interactions within the catalytic site.

Overall, the research project has yielded the following scientific achievements:
- The successful synthesis of bicyclo[3.1.0]hexane derivatives containing a variety of functional groups. The synthesis of these highly functionalized and structurally complex molecules that incorporate six stereocenters on such a small framework is a stand-alone achievement.
- The screening of the potential inhibitors against NAs from group 1 and group 2;
- Elaboration of the results with computational techniques and design of new potential drugs;
- The simplification of the synthetic route for the generation and evaluation of new structurally variant of the scaffold with the amino and guanidinium functionalities.

In addition to these scientific achievements, Dr. C. Colombo, during this fellowship, acquired the ability to conceive and implement a substantial program of innovative research, and to adapt the program as it evolves in complexity. The progresses in the research project have been presented at high level scientific conferences and one manuscript has been published, ensuring exposure to the peer review process. Two manuscripts are currently under preparation. The scientific contacts and training that Dr. C. Colombo has acquired during the outgoing phase will be essential for future collaborative research projects. The intention is to continue the collaboration between the host laboratories at Simon Fraser University and Università degli Studi di Milano. This project represented the first opportunity for this collaboration to develop and undoubtedly was the seed to crystallize a broader program of future collaboration in fields of common interest, such as the use of glycochemistry to fight infectious diseases.

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