N-benzyl 4,4-disubstituted piperidines as a potent class of influenza H1N1 virus inhibitors showing a novel mechanism of hemagglutinin fusion peptide interaction

Abstract

The influenza virus hemagglutinin (HA) is an attractive target for antiviral therapy due to its essential role in mediating virus entry into the host cell. We here report the identification of a class of Nbenzyl-4,4,-disubstituted piperidines as influenza A virus fusion inhibitors with specific activity against the H1N1 subtype. Using the highly efficient one-step Ugi four-component reaction, a diverse library of piperidine-based analogs was synthesized and evaluated to explore the structureactivity relationships (SAR). Mechanistic studies, including resistance selection with the most active compound (2) demonstrated that it acts as an inhibitor of the low pH-induced HA-mediated membrane fusion process. Computational studies identified an as yet unrecognized fusion inhibitor binding site, which is located at the bottom of the HA 2 stem in close proximity to the fusion peptide. A direct π-stacking interaction between the N-benzylpiperidine moiety of 2 and F9 HA2 of the fusion peptide, reinforced with an additional p-stacking interaction with Y119 HA2 , and a salt bridge of the protonated piperidine nitrogen with E120 HA2 , were identified as important interactions to mediate ligand binding. This site rationalized the observed SAR and provided a structural explanation for the H1N1-specific activity of our inhibitors. Furthermore, the HA 1-S326V mutation resulting in resistance to 2 is close to the proposed new binding pocket. Our findings point to the N-benzyl-4,4,disubstituted piperidines as an interesting class of influenza virus inhibitors, representing the first example of fusion peptide binders with great potential for anti-influenza drug development. KEYWORDS Influenza virus, hemagglutinin, fusion peptide inhibitor, N-benzyl-4,4,-disubstituted piperidines 1. Introduction Each year, influenza A and B viruses are responsible for 3-5 million severe cases and 290,000-650,000 deaths worldwide [1]. The death toll rises sharply during influenza pandemics, like the 2009 pandemic that was caused by a novel swine-origin A/H1N1 virus [2]. Though widely used and recommended, influenza vaccination is limited by suboptimal effectiveness, the need for annual injection and possibility of antigen mismatch [3]. The complementary strategy, antiviral therapy, is crucial to treat and prevent complicated influenza infections, which typically develop in aged, chronically ill or hospitalized individuals [4]. Nowadays, neuraminidase inhibitors are the only effective drug class that is widely available. Hence, there is an urgent need for new influenza blockers with a distinct mode of action. Some of these are under early or advanced clinical evaluation [5,6]. The trimeric hemagglutinin (HA) glycoprotein is an attractive drug target due to its critical and dual role in virus entry [7]: i) the HA globular head recognizes sialylated cell surface glycans and mediates virus attachment [8], and ii) HA is required for membrane fusion after the virus particles have entered by endocytosis. Upon acidification of the early into late endosomes, t...

Aryl Sulfonamide Inhibits Entry and Replication of Diverse Influenza Viruses via the Hemagglutinin Protein

“…Removing or shrinking the N-ethyl group substantially diminished activity (2,3). Allyl and propargyl groups maintained activity (4,5), albeit at a decreased level, and an N-isopropyl analogue (6) had greater than 10-fold loss of activity compared to compound 1. On the aryl ring, removing the 2-methyl group cost >10× (7), but moving it to the para position was well tolerated (11).…”

“…The low endosomal pH triggers this fusion event by inducing conformation changes in HA, that expose its fusion peptide, and culminates with the release of the viral genome into the cytoplasm. This step has been the target of several HA inhibitors − that prevent fusion of HA proteins from either group 1 or group 2 but not both. Here, we identified an aryl sulfonamide as an inhibitor of influenza A virus entry that targets the HA protein, preventing viral membrane fusion.…”

Design, Synthesis, Molecular Docking Analysis and Biological Evaluations of 4-[(Quinolin-4-yl)amino]benzamide Derivatives as Novel Anti-Influenza Virus Agents

“…The target compounds were assessed for their abilities in inhibiting influenza virus replication in MDCK cells by CPE reduction assay [ 54 , 55 , 56 , 57 ]. MDCK cells were seeded into a 96-well plate and were infected with virus at an MOI of 50 CCID 50 (50% cell culture infective dose) per well.…”

Heparan Sulfate and Sialic Acid in Viral Attachment: Two Sides of the Same Coin?

“…Computational methods have contributed to optimizing the search for HA blockers, allowing for selection among thousands of compounds theoretically and the evaluation of the best candidates experimentally. Some molecules chosen by this methodology are the N-benzyl-4,4,-disubstituted piperidines [ 74 ] and the derivatives of compound NSC8556 [ 75 ]; both showed in vitro effectiveness to inhibit virus entry. So far, the only drugs used against influenza are neuraminidase inhibitors such as oseltamivir.…”