Compounds with anti-influenza activity: present and future of strategies for the optimal treatment and management of influenza. Part I: influenza life-cycle and currently available drugs

GASPARINI, R.; AMICIZIA, D.; LAI, PL.; BRAGAZZI, NL.; PANATTO, D.

HA is a homotrimeric integral type 1 membrane cylinder-like glycoprotein, approximately 13.5 nanometres long. HA monomers are synthesized as precursors (HA0) containing a hydrophobic signal sequence. After being translated, they are glycosylated and cleaved into two smaller subunits: namely, HA1 of 50 kDa and HA2 of 27 kDa, which are linked by a disulfide bridge. Each subunit is characterized by a long, central, α-helix connected to the membrane by HA2 and surmounted by HA1, a spherical head containing the sialic acid binding sites (receptor binding sites or RBSs, also known as receptor binding pocket or RBP) [57] . The apolar domain of HA2, near the cleavage site, is known as the "fusion peptide" or HAfp23 [58, 59] , since it is characterized by a domain of highly conserved N-terminal 23 residues. HAfp23 has a helical-hairpin structure consisting of two tightly-packed helices, which are fundamental to inducing the negative curvature ("fusogenic conformation" of HA) [60] . There are at least eighteen HA subtypes [61] . These are further subdivided into two groups: group 1 comprises H1, H2, H5, H6, H8, H9, H11, H12, H13, H16, H17, and H18 (from waterfowl, the last two having been recently isolated from bats in Guatemala and Peru) [61, 62] ; group 2 comprises H3, H4, H7, H10, H14, and H15 [62] . However, a recent experiment has proved that the virus can also enter into cells whose surface has been completely depleted of sialic acid-based glycoproteins and glycolipids [63] . This seems to suggest alternative entry routes. Indeed, H17 and H18 do not bind to sialic acid and their receptor is still unknown [64] . Entry is essentially through receptor-mediated endocytosis [65] , though an alternative uptake pathway, namely macropinocytosis, has quite recently been discovered [66] [67] [68] . Receptor-mediated endocytosis can be CME or mediated by lipid rafts: CDE or non-clathrin non-caveolae endocytosis [69] [70] [71] . CME is the most common pathway through which the virion is internalized. The clathrin triskelion is made up of three heavy chains, which constitute the backbone of the polyhedral structure, and of three light chains, which finely tune the assembly / disassembly of the triskelion [72] . Adaptor proteins, such as AP-2 [73] , epsin-1 [74] , epsin-15, recognize specific internalization signals located on cargo receptors, and take part in the formation of clathrin-coated pits (CCPs). Caveolae are small flask-like infoldings of the membrane, with high levels of cholesterol and glycosphingolipids and with caveolin as the integral membrane scaffolding structure. The remaining entry routes have been investigated less. In clathrin caveolae-independent endocytosis, the Ras-phosphoinositide 3-kinase (PI3K) signalling pathway may play a major role [75] . The PI3K inactive complex (PI3K p110-p85 heterodimer) moves to the plasma membrane, where the SH2 (Src Homology 2) domains of p85 engage the phosphotyrosine residues present in receptor-associated proteins, causing a molecular rearrangement of the complex, in such a way that p110 is now enzymatically active and can recruit / produce intracellular second messenger, such as PtdIns(3,4,5)P3 (phosphatidylinositol-(3,4,5)trisphosphate), from PtdIns(4,5)P2 (phosphatidylinositol-(4,5)-bisphosphate). Subsequently, several effector proteins with pleckstrin homology (PH) domains mediate an array of different signalling cascades: namely, activation of Akt, mTOR (mammalian Target Of Rapamycin), PKC (Pr