Rational Design of Novel Peptiddomimetics against Influenza A Virus: Biological and Computational Studies  
[PERSON]  
1 Department of Pharmacy, University of Salerno, Via Giovanni Paolo II 132, 84084 Fisciano, Italy;  
[EMAIL_ADDRESS] (M.C.S.); pcampiglia@unisa.  
influenza strains spreading through the human population and the rise of resistance to licensed drugs have focused research into developing new treatments for influenza viruses [11; 12; 13; 14]. A viral component whose role in virus infection is crucial should be the ideal target for novel anti-influenza treatments.  
The IAV envelope contains two glycoproteins: hemagglutinin (HA) and neuraminidase (NA). Both play pivotal roles in the infection cycle: HA for binding to specific cell receptors and mediating membrane fusion, and NA for releasing viral particles from the cell surface, but they are also involved in viral attachment and entry. The majority of current treatment approaches for IAV target NA. Oseltamivir, the only oral NA inhibitor, is the most common anti-flu therapy worldwide, so its broad use led to the selection of resistant viral strains [15]. Frequently, when a tyrosine residue replaces the histidine residue 274 (position 275 of the N1 protein) on the NA gene, influenza A/H1N1 viruses develop oseltamivir resistance [16]. For example, during the 2007-2008 influenza season in Europe, many seasonal A(H1N1) viruses emerged with the H275Y NA mutation, conferring a high resistance to oseltamivir [17]. It is important to note that NA mutations responsible for oseltamivir resistance can emerge independently of the use of this drug [17]. In fact, a high proportion of oseltamivir-resistant viruses have been isolated from patients who have not received treatment, demonstrating a high rate of transmission [6]. Moreover, as these mutants are known to maintain fitness, they can easily spread globally, representing a serious public health risk. Furthermore, it is important to note that potential pandemic viruses, such as avian influenza A(H5N1) and A(H7N9), which cause severe disease in humans, are prone to the development of oseltamivir resistance in treated patients [18; 19; 20]. Finally, it has been demonstrated that mallards can transmit an avian influenza virus that is oseltamivir-resistant to chickens. As this mutant is able to cross the species barrier and also maintain fitness, it could be able to infect humans [21]. Finding alternative antiviral drugs with distinct modes of action is therefore urgently needed to fight infection by emerging viruses with pandemic potential, especially those carrying drug resistance markers. However, attempts to target HA are on the rise. The discovery of broadly neutralizing antibodies that target the HA stem has led to increased attention for HA as a target for broadly cross-protective antivirals. These antibodies are under investigation for direct use as therapeutic agents and have inspired the design of smaller cyclic peptides based on the antibody-binding interface.  
Previously, [PERSON] and coworkers demonstrated that bLf, in particular the bLf C-lobe, can bind and inhibit HA [22]. Successively, through a truncation library, we identified the tetrapeptides SLDC (**1**) and SKHS (**2**), derived from bLf C-lobe fragment 418-429, which were able to bind HA and inhibit cell infection in a concentration range of picomolar [23].  
It is well known that the direct application of peptides as medicinal entities has some severe limitations, including high degradation by proteolytic enzymes and poor cell membrane permeability. We designed new peptidomimetics derived from tetrapeptides **1** and **2** to increase metabolic stability and improve bioavailability, receptor affinity, and selectivity to overcome these drawbacks. Biological and computational assays on synthesized compounds were carried out. Direct binding assays, a bioactivity profile, and computational studies led to the identification of a very potent N-methyl peptide.