Recent cases of avian influenza H5N1 and the swine-origin 2009 H1N1 have caused a great concern that a global disaster like the 1918 influenza pandemic may occur again. Viral transmission begins with a critical interaction between hemagglutinin (HA) glycoprotein, which is on the viral coat of influenza, and sialic acid (SA) containing glycans, which are on the host cell surface. To elucidate the role of HA glycosylation in this important interaction, various defined HA glycoforms were prepared, and their binding affinity and specificity were studied by using a synthetic SA microarray. Truncation of the N-glycan structures on HA increased SA binding affinities while decreasing specificity toward disparate SA ligands. The contribution of each monosaccharide and sulfate group within SA ligand structures to HA binding energy was quantitatively dissected. It was found that the sulfate group adds nearly 100-fold (2.04 kcal/mol) in binding energy to fully glycosylated HA, and so does the biantennary glycan to the monoglycosylated HA glycoform. Antibodies raised against HA protein bearing only a single N-linked GlcNAc at each glycosylation site showed better binding affinity and neutralization activity against influenza subtypes than the fully glycosylated HAs elicited. Thus, removal of structurally nonessential glycans on viral surface glycoproteins may be a very effective and general approach for vaccine design against influenza and other human viruses.flu vaccine ͉ glycan binding ͉ glycosylation T he highly pathogenic H5N1 and the 2009 swine-origin influenza A (H1N1) viruses have caused global outbreaks and raised a great concern that further changes in the viruses may occur to bring about a deadly pandemic (1, 2). Important contributions to our understanding of influenza infections have come from the studies on hemagglutinin (HA), a viral coat glycoprotein that binds to specific sialylated glycan receptors in the respiratory tract, allowing the virus to enter the cell (3-6). To cross the species barrier and infect the human population, avian HA must change its receptorbinding preference from a terminally sialylated glycan that contains ␣2,3 (avian)-linked to ␣2,6 (human)-linked sialic acid motifs (7), and this switch could occur through only two mutations, as in the 1918 pandemic (8). Understanding the factors that affect influenza binding to glycan receptors is thus critical for developing methods to control any future crossover influenza strains that have pandemic potential.HA is a homotrimeric transmembrane protein with an ectodomain composed of a globular head and a stem region (3). Both regions carry N-linked oligosaccharides (9), which affect the functional properties of HA (10, 11). Among different subtypes of influenza A viruses, there is extensive variation in the glycosylation sites of the head region, whereas the stem oligosaccharides are more conserved and required for fusion activity (11). Glycans near antigenic peptide epitopes interfere with antibody recognition (12), and glycans near the proteolytic ...
As influenza viruses have developed resistance towards current drugs, new inhibitors that prevent viral replication through different inhibitory mechanisms are useful. In this study, we developed a screening procedure to search for new antiinfluenza inhibitors from 1,200,000 compounds and identified previously reported as well as new antiinfluenza compounds. Several antiinfluenza compounds were inhibitory to the influenza RNA-dependent RNA polymerase (RdRP), including nucleozin and its analogs. The most potent nucleozin analog, 3061 (FA-2), inhibited the replication of the influenza A/WSN/33 (H1N1) virus in MDCK cells at submicromolar concentrations and protected the lethal H1N1 infection of mice. Influenza variants resistant to 3061 (FA-2) were isolated and shown to have the mutation on nucleoprotein (NP) that is distinct from the recently reported resistant mutation of Y289H [Kao R, et al. (2010) Nat Biotechnol 28:600]. Recombinant influenza carrying the Y52H NP is also resistant to 3061 (FA-2), and NP aggregation induced by 3061 (FA-2) was identified as the most likely cause for inhibition. In addition, we identified another antiinfluenza RdRP inhibitor 367 which targets PB1 protein but not NP. A mutant resistant to 367 has H456P mutation at the PB1 protein and both the recombinant influenza and the RdRP expressing the PB1 H456P mutation have elevated resistance to 367. Our high-throughput screening (HTS) campaign thus resulted in the identification of antiinfluenza compounds targeting RdRP activity.high-throughput screening | antiinfluenza | influenza NP | influenza PB1 | chemical genetics
The nucleoprotein (NP) of the influenza virus exists as trimers, and its tail-loop binding pocket has been suggested as a potential target for antiinfluenza therapeutics. The possibility of NP as a drug target was validated by the recent reports that nucleozin and its analogs can inhibit viral replication by inducing aggregation of NP trimers. However, these inhibitors were identified by random screening, and the binding site and inhibition mechanism are unclear. We report a rational approach to target influenza virus with a new mechanism-disruption of NP-NP interaction. Consistent with recent work, E339A, R416A, and deletion mutant Δ402-428 were unable to support viral replication in the absence of WT NP. However, only E339A and R416A could form hetero complex with WT NP, but the complex was unable to bind the RNA polymerase, leading to inhibition of viral replication. These results demonstrate the importance of the E339…R416 salt bridge in viral survival and establish the salt bridge as a sensitive antiinfluenza target. To provide further support, we showed that peptides encompassing R416 can disrupt NP-NP interaction and inhibit viral replication. Finally we performed virtual screening to target E339…R416, and some small molecules identified were shown to disrupt the formation of NP trimers and inhibit replication of WT and nucleozinresistant strains. This work provides a new approach to design antiinfluenza drugs.T he RNA-dependent RNA polymerase (RDRP) of the influenza A virus is composed of polymerase basic protein 1 (PB1), basic protein 2 (PB2), and acidic protein (PA) (1). The function of RDRP for viral replication requires association with the nucleoprotein (NP) (2) to form the ribonucleoprotein (RNP) complex. Only low resolution structures of the RNP complex are available from cryo-EM studies (2-9), whereas high resolution structures have been reported for some individual components or fragments (10-12). Crystal structures of NP indicate that it exists in trimers (13,14), with the tail-loop (residues 402-428) region playing an important role in the trimerization (Fig. 1A). Based on the structural information, it was suggested that the tail-loop binding pocket could be a target for antiinfluenza therapeutics (13,14).Disruption of the NP-NP interaction as a strategy for designing antiinfluenza drugs has been further reported. Many mutants of NP, including some tail-loop mutants, lose the ability to support the RDRP activity in reconstitution experiments (2,(15)(16)(17)(18). In addition, some of the mutants are shown to exist in monomers instead of trimers. These results support the importance of NP in the RDRP activity and viral replication, and the possibility of NP as a drug target. However, it remains to be shown that molecules capable of disrupting the NP-NP interaction would inhibit viral replication.Recently Kao et al. (19) and our group (20) reported the use of high throughput screening to identify nucleozin and its analogs as inhibitors that halt viral replication by binding to NP and causing it...
The proteolytic processing of polyproteins by the 3CL protease of severe acute respiratory syndrome coronavirus is essential for the viral propagation. A series of tripeptide alpha,beta-unsaturated esters and ketomethylene isosteres, including AG7088, are synthesized and assayed to target the 3CL protease. Though AG7088 is inactive (IC50 > 100 microM), the ketomethylene isosteres and tripeptide alpha,beta-unsaturated esters containing both P1 and P2 phenylalanine residues show modest inhibitory activity (IC50 = 11-39 microM). The Phe-Phe dipeptide inhibitors 18a-e are designed on the basis of computer modeling of the enzyme-inhibitor complex. The most potent inhibitor 18c with an inhibition constant of 0.52 microM is obtained by condensation of the Phe-Phe dipeptide alpha,beta-unsaturated ester with 4-(dimethylamino)cinnamic acid. The cell-based assays also indicate that 18c is a nontoxic anti-SARS agent with an EC50 value of 0.18 microM.
Using d-xylose as an appropriate chiral precursor, we have synthesized active neuraminidase inhibitor oseltamivir, antiflu drug Tamiflu, and novel phosphonate congeners that exhibit even stronger antiflu activities by inhibiting the neuraminidases of the wild-type and H274Y mutant of H1N1 and H5N1 viruses. Molecular modeling of the neuraminidase−phosphonate complex indicates a pertinent binding mode of the phosphonate with three arginine residues in the active site. Discovery of such potent neuraminidase inhibitors will offer an opportunity to the development of new anti-influenza drugs.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.