Inhibition Of Membrane Fusion Research Articles

Antiviral Role of IFITM Proteins in African Swine Fever Virus Infection.

The interferon-induced transmembrane (IFITM) protein family is a group of antiviral restriction factors that impair flexibility and inhibit membrane fusion at the plasma or the endosomal membrane, restricting viral progression at entry. While IFITMs are widely known to inhibit several single-stranded RNA viruses, there are limited reports available regarding their effect in double-stranded DNA viruses. In this work, we have analyzed a possible antiviral function of IFITMs against a double stranded DNA virus, the African swine fever virus (ASFV). Infection with cell-adapted ASFV isolate Ba71V is IFN sensitive and it induces IFITMs expression. Interestingly, high levels of IFITMs caused a collapse of the endosomal pathway to the perinuclear area. Given that ASFV entry is strongly dependent on endocytosis, we investigated whether IFITM expression could impair viral infection. Expression of IFITM1, 2 and 3 reduced virus infectivity in Vero cells, with IFITM2 and IFITM3 having an impact on viral entry/uncoating. The role of IFITM2 in the inhibition of ASFV in Vero cells could be related to impaired endocytosis-mediated viral entry and alterations in the cholesterol efflux, suggesting that IFITM2 is acting at the late endosome, preventing the decapsidation stage of ASFV.

Line tension at lipid phase boundaries as driving force for HIV fusion peptide-mediated fusion.

Lipids and proteins are organized in cellular membranes in clusters, often called ‘lipid rafts'. Although raft-constituent ordered lipid domains are thought to be energetically unfavourable for membrane fusion, rafts have long been implicated in many biological fusion processes. For the case of HIV gp41-mediated membrane fusion, this apparent contradiction can be resolved by recognizing that the interfaces between ordered and disordered lipid domains are the predominant sites of fusion. Here we show that line tension at lipid domain boundaries contributes significant energy to drive gp41-fusion peptide-mediated fusion. This energy, which depends on the hydrophobic mismatch between ordered and disordered lipid domains, may contribute tens of kBT to fusion, that is, it is comparable to the energy required to form a lipid stalk intermediate. Line-active compounds such as vitamin E lower line tension in inhomogeneous membranes, thereby inhibit membrane fusion, and thus may be useful natural viral entry inhibitors.

Inhibition of HIV Entry by Targeting the Envelope Transmembrane Subunit gp41

The transmembrane subunit of the HIV envelope protein, gp41 is a vulnerable target to inhibit HIV entry. There is one fusion inhibitor T20 (brand name: Fuzeon, generic name: enfuvirtide) available by prescription. However, it has several drawbacks such as a high level of development of drug resistance, a short-half life in vivo, rapid renal clearance, low oral bioavailability, and it is only used as a salvage therapy. Therefore, investigators have been studying a variety of different modalities to attempt to overcome these limitations. Comprehensive literature searches were performed on HIV gp41, inhibition mechanisms, and inhibitors. The latest structural information was collected, and multiple inhibition strategies targeting gp41 were reviewed. Many of the recent advances in inhibitors were peptide-based. Several creative modification strategies have also been performed to improve inhibitory efficacy of peptides and to overcome the drawbacks of T20 treatment. Small compounds have also been an area of intense research. There is a wide variety in development from those identified by virtual screens targeting specific regions of the protein to natural products. Finally, broadly neutralizing antibodies have also been important area of research. The inaccessible nature of the target regions for antibodies is a challenge, however, extensive efforts to develop better neutralizing antibodies are ongoing. The fusogenic protein, gp41 has been extensively studied as a promising target to inhibit membrane fusion between the virus and target cells. At the same time, it is a challenging target because the vulnerable conformations of the protein are exposed only transiently. However, advances in biochemical, biophysical, structural, and immunological studies are coming together to move the field closer to an understanding of gp41 structure and function that will lead to the development of novel drugs and vaccines.

Direct Quantitative Detection of Doc2b-Induced Hemifusion in Optically Trapped Membranes

Ca2+-sensor proteins control the secretion of many neuroendocrine substances. Calcium-secretion coupling may involve several mechanisms. First, Ca2+-dependent association of their tandem C2 domains with phosphatidylserine may induce membrane curvature and thereby enhance fusion. Second, their association with SNARE complexes may inhibit membrane fusion in absence of a Ca2+ trigger. Here we present a method using two optically trapped beads coated with SNARE-free synthetic membranes to elucidate the direct role of the C2AB domain of the soluble Ca2+-sensor Doc2b. Contacting membranes are often coupled by a Doc2b-coated membrane stalk that resists forces up to 600 pN upon bead separation. Stalk formation depends strictly on Ca2+ and phosphatidylserine. Real-time fluorescence imaging shows phospholipid but not content mixing, indicating membrane hemifusion. Thus, Doc2b acts directly on membranes and stabilizes the hemifusion intermediate in this cell-free system. In living cells this mechanism may co-occur with progressive SNARE complex assembly, together defining Ca2+-secretion coupling.

Molecular mechanism of respiratory syncytial virus fusion inhibitors.

Respiratory syncytial virus (RSV) is a leading cause of pneumonia and bronchiolitis in young children and the elderly. Therapeutic small molecules have been developed that bind the RSV F glycoprotein and inhibit membrane fusion, yet their binding sites and molecular mechanisms of action remain largely unknown. Here we show that these inhibitors bind to a three-fold-symmetric pocket within the central cavity of the metastable prefusion conformation of RSV F. Inhibitor binding stabilizes this conformation by tethering two regions that must undergo a structural rearrangement to facilitate membrane fusion. Inhibitor-escape mutations occur in residues that directly contact the inhibitors or are involved in the conformational rearrangements required to accommodate inhibitor binding. Resistant viruses do not propagate as well as wild-type RSV in vitro, indicating a fitness cost for inhibitor escape. Collectively, these findings provide new insight into class I viral fusion proteins and should facilitate development of optimal RSV fusion inhibitors.

Direct quantitative detection of Doc2b-induced hemifusion in optically trapped membranes.

Ca2+-sensor proteins control the secretion of many neuroendocrine substances. Calcium-secretion coupling may involve several mechanisms. First, Ca2+-dependent association of their tandem C2 domains with phosphatidylserine may induce membrane curvature and thereby enhance fusion. Second, their association with SNARE complexes may inhibit membrane fusion in the absence of a Ca2+ trigger. Here we present a method using two optically trapped beads coated with SNARE-free synthetic membranes to elucidate the direct role of the C2AB domain of the soluble Ca2+-sensor Doc2b. Contacting membranes are often coupled by a Doc2b-coated membrane stalk that resists forces up to 600 pN upon bead separation. Stalk formation depends strictly on Ca2+ and phosphatidylserine. Real-time fluorescence imaging shows phospholipid but not content mixing, indicating membrane hemifusion. Thus, Doc2b acts directly on membranes and stabilizes the hemifusion intermediate in this cell-free system. In living cells, this mechanism may co-occur with progressive SNARE complex assembly, together defining Ca2+-secretion coupling.

Sequential conformational changes in the morbillivirus attachment protein initiate the membrane fusion process.

Despite large vaccination campaigns, measles virus (MeV) and canine distemper virus (CDV) cause major morbidity and mortality in humans and animals, respectively. The MeV and CDV cell entry system relies on two interacting envelope glycoproteins: the attachment protein (H), consisting of stalk and head domains, co-operates with the fusion protein (F) to mediate membrane fusion. However, how receptor-binding by the H-protein leads to F-triggering is not fully understood. Here, we report that an anti-CDV-H monoclonal antibody (mAb-1347), which targets the linear H-stalk segment 126-133, potently inhibits membrane fusion without interfering with H receptor-binding or F-interaction. Rather, mAb-1347 blocked the F-triggering function of H-proteins regardless of the presence or absence of the head domains. Remarkably, mAb-1347 binding to headless CDV H, as well as standard and engineered bioactive stalk-elongated CDV H-constructs treated with cells expressing the SLAM receptor, was enhanced. Despite proper cell surface expression, fusion promotion by most H-stalk mutants harboring alanine substitutions in the 126-138 “spacer” section was substantially impaired, consistent with deficient receptor-induced mAb-1347 binding enhancement. However, a previously reported F-triggering defective H-I98A variant still exhibited the receptor-induced “head-stalk” rearrangement. Collectively, our data spotlight a distinct mechanism for morbillivirus membrane fusion activation: prior to receptor contact, at least one of the morbillivirus H-head domains interacts with the membrane-distal “spacer” domain in the H-stalk, leaving the F-binding site located further membrane-proximal in the stalk fully accessible. This “head-to-spacer” interaction conformationally stabilizes H in an auto-repressed state, which enables intracellular H-stalk/F engagement while preventing the inherent H-stalk’s bioactivity that may prematurely activate F. Receptor-contact disrupts the “head-to-spacer” interaction, which subsequently “unlocks” the stalk, allowing it to rearrange and trigger F. Overall, our study reveals essential mechanistic requirements governing the activation of the morbillivirus membrane fusion cascade and spotlights the H-stalk “spacer” microdomain as a possible drug target for antiviral therapy.

A novel humanized antibody neutralizes H5N1 influenza virus via two different mechanisms.

Highly pathogenic avian influenza virus subtype H5N1 continues to be a severe threat to public health, as well as the poultry industry, because of its high lethality and antigenic drift rate. Neutralizing monoclonal antibodies (MAbs) can serve as a useful tool for preventing, treating, and detecting H5N1. In the present study, humanized H5 antibody 8A8 was developed from a murine H5 MAb. Both the humanized and mouse MAbs presented positive activity in hemagglutination inhibition (HI), virus neutralization, and immunofluorescence assays against a wide range of H5N1 strains. Interestingly, both human and murine 8A8 antibodies were able to detect H5 in Western blot assays under reducing conditions. Further, by sequencing of escape mutants, the conformational epitope of 8A8 was found to be located within the receptor binding domain (RBD) of H5. The linear epitope of 8A8 was identified by Western blotting of overlapping fragments and substitution mutant forms of HA1. Reverse genetic H5N1 strains with individual mutations in either the conformational or the linear epitope were generated and characterized in a series of assays, including HI, postattachment, and cell-cell fusion inhibition assays. The results indicate that for 8A8, virus neutralization mediated by RBD blocking relies on the conformational epitope while binding to the linear epitope contributes to the neutralization by inhibiting membrane fusion. Taken together, the results of this study show that a novel humanized H5 MAb binds to two types of epitopes on HA, leading to virus neutralization via two mechanisms. Recurrence of the highly pathogenic avian influenza virus subtype H5N1 in humans and poultry continues to be a serious public health concern. Preventive and therapeutic measures against influenza A viruses have received much interest in the context of global efforts to combat the current and future pandemics. Passive immune therapy is considered to be the most effective and economically prudent preventive strategy against influenza virus besides vaccination. It is important to develop a humanized neutralizing monoclonal antibody (MAb) against all of the clades of H5N1. For the first time, we report in this study that a novel humanized H5 MAb binds to two types of epitopes on HA, leading to virus neutralization via two mechanisms. These findings further deepen our understanding of influenza virus neutralization.

Structure and function of longin SNAREs.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins constitute the core membrane fusion machinery of intracellular transport and intercellular communication. A little more than ten years ago, it was proposed that the long N-terminal domain of a subset of SNAREs, henceforth called the longin domain, could be a crucial regulator with multiple functions in membrane trafficking. Structural, biochemical and cell biology studies have now produced a large set of data that support this hypothesis and indicate a role for the longin domain in regulating the sorting and activity of SNAREs. Here, we review the first decade of structure-function data on the three prototypical longin SNAREs: Ykt6, VAMP7 and Sec22b. We will, in particular, highlight the conserved molecular mechanisms that allow longin domains to fold back onto the fusion-inducing SNARE coiled-coil domain, thereby inhibiting membrane fusion, and describe the interactions of longin SNAREs with proteins that regulate their intracellular sorting. This dual function of the longin domain in regulating both the membrane localization and membrane fusion activity of SNAREs points to its role as a key regulatory module of intracellular trafficking.

Vibrio effector protein VopQ inhibits fusion of V-ATPase–containing membranes

Vesicle fusion governs many important biological processes, and imbalances in the regulation of membrane fusion can lead to a variety of diseases such as diabetes and neurological disorders. Here we show that the Vibrio parahaemolyticus effector protein VopQ is a potent inhibitor of membrane fusion based on an in vitro yeast vacuole fusion model. Previously, we demonstrated that VopQ binds to the V(o) domain of the conserved V-type H(+)-ATPase (V-ATPase) found on acidic compartments such as the yeast vacuole. VopQ forms a nonspecific, voltage-gated membrane channel of 18 Å resulting in neutralization of these compartments. We now present data showing that VopQ inhibits yeast vacuole fusion. Furthermore, we identified a unique mutation in VopQ that delineates its two functions, deacidification and inhibition of membrane fusion. The use of VopQ as a membrane fusion inhibitor in this manner now provides convincing evidence that vacuole fusion occurs independently of luminal acidification in vitro.

A Novel Mode of Poxvirus Superinfection Exclusion That Prevents Fusion of the Lipid Bilayers of Viral and Cellular Membranes

Superinfection exclusion is a widespread phenomenon that prevents secondary infections by closely related viruses. The vaccinia virus A56 and K2 proteins in the cell membrane can prevent superinfection by interacting with the entry-fusion complex of subsequent viruses. Here, we described another form of exclusion that is established earlier in infection and does not require the A56 or K2 protein. Cells infected with one or more infectious virions excluded hundreds of superinfecting vaccinia virus particles. A related orthopoxvirus, but neither a flavivirus nor a rhabdovirus, was also excluded, indicating selectivity. Although superinfecting vaccinia virus bound to cells, infection was inhibited at the membrane fusion step, thereby preventing core entry into the cytoplasm and early gene expression. In contrast, A56/K2 protein-mediated exclusion occurred subsequent to membrane fusion. Induction of resistance to superinfection depended on viral RNA and protein synthesis by the primary virus but did not require DNA replication. Although superinfection resistance correlated with virus-induced changes in the cytoskeleton, studies with mutant vaccinia viruses indicated that the cytoskeletal changes were not necessary for resistance to superinfection. Interferon-inducible transmembrane proteins, which can inhibit membrane fusion in other viral systems, did not prevent vaccinia virus membrane fusion, suggesting that these interferon-inducible proteins are not involved in superinfection exclusion. While the mechanism remains to be determined, the early establishment of superinfection exclusion may provide a "winner-take-all" reward to the first poxvirus particles that successfully initiate infection and prevent the entry and genome reproduction of defective or less fit particles. The replication of a virus usually follows a defined sequence of events: attachment, entry into the cytoplasm or nucleus, gene expression, genome replication, assembly of infectious particles, and spread to other cells. Although multiple virus particles may enter a cell at the same time, mechanisms exist to prevent infection by subsequent viruses. The latter phenomenon, known as superinfection exclusion, can occur by a variety of mechanisms that are not well understood. We showed that superinfection by vaccinia virus was prevented at the membrane fusion step, which closely followed virion attachment. Thus, neither gene expression nor genome replication of the superinfecting virus occurred. Expression of early proteins by the primary virus was necessary and sufficient to induce the superinfection-resistant state. Superinfection exclusion may be beneficial to vaccinia virus by selecting particles that can infect cells rapidly, excluding defective particles and synchronizing the replication cycle.

Singlet oxygen effects on lipid membranes: implications for the mechanism of action of broad-spectrum viral fusion inhibitors

It was reported recently that a new aryl methyldiene rhodanine derivative, LJ001, and oxazolidine-2,4-dithione, JL103, act on the viral membrane, inhibiting its fusion with a target cell membrane. The aim of the present study was to investigate the interactions of these two active compounds and an inactive analogue used as a negative control, LJ025, with biological membrane models, in order to clarify the mechanism of action at the molecular level of these new broad-spectrum enveloped virus entry inhibitors. Fluorescence spectroscopy was used to quantify the partition and determine the location of the molecules on membranes. The ability of the compounds to produce reactive oxygen molecules in the membrane was tested using 9,10-dimethylanthracene, which reacts selectively with singlet oxygen (1O2). Changes in the lipid packing and fluidity of membranes were assessed by fluorescence anisotropy and generalized polarization measurements. Finally, the ability to inhibit membrane fusion was evaluated using FRET. Our results indicate that 1O2 production by LJ001 and JL103 is able to induce several changes on membrane properties, specially related to a decrease in its fluidity, concomitant with an increase in the order of the polar headgroup region, resulting in an inhibition of the membrane fusion necessary for cell infection.

Evidence Showing that Tetraspanins Inhibit HIV-1-Induced Cell-Cell Fusion at a Post-Hemifusion Stage

Human immunodeficiency virus type 1 (HIV-1) transmission takes place primarily through cell-cell contacts known as virological synapses. Formation of these transient adhesions between infected and uninfected cells can lead to transmission of viral particles followed by separation of the cells. Alternatively, the cells can fuse, thus forming a syncytium. Tetraspanins, small scaffolding proteins that are enriched in HIV-1 virions and actively recruited to viral assembly sites, have been found to negatively regulate HIV-1 Env-induced cell-cell fusion. How these transmembrane proteins inhibit membrane fusion, however, is currently not known. As a first step towards elucidating the mechanism of fusion repression by tetraspanins, e.g., CD9 and CD63, we sought to identify the stage of the fusion process during which they operate. Using a chemical epistasis approach, four fusion inhibitors were employed in tandem with CD9 overexpression. Cells overexpressing CD9 were found to be sensitized to inhibitors targeting the pre-hairpin and hemifusion intermediates, while they were desensitized to an inhibitor of the pore expansion stage. Together with the results of a microscopy-based dye transfer assay, which revealed CD9- and CD63-induced hemifusion arrest, our investigations strongly suggest that tetraspanins block HIV-1-induced cell-cell fusion at the transition from hemifusion to pore opening.

New Broad-Spectrum Viral Fusion Inhibitors Act by Deleterious Effect on the Viral Membrane through the Production Singlet Oxygen Molecules

Targeting membranes of enveloped viruses represents an exciting new paradigm to explore regarding the development of broad-spectrum antivirals. Recently, broad-spectrum small-molecule antiviral drugs were described, which prevent enveloped virus entry at an intermediate step, after virus binding but before virus-cell fusion [1, 2]. These compounds, an aryl methyldiene rhodanine derivate, named LJ001, and oxazolidine-2,4-dithione derivates named JL103 and JL 118, act deleteriously on the virus envelope but not at the cell membrane level. The aim of the present work was to study the interactions of active compounds (LJ001, JL103 and JL118) and LJ025 (an inactive analog used as negative control) with biological membrane models, in order to clarify the mechanism of action of these new enveloped virus entry inhibitors. Fluorescence spectroscopy was used to quantify the partition and determine the location of the molecules on membranes. The ability of the compounds to produce reactive oxygen molecules in the membrane was tested by using 9,10-dimethylanthracene, which reacts selectively with singlet oxygen. Changes on the lipid packing and fluidity of membranes were assessed by fluorescence anisotropy and generalized polarization measurements. Finally, the ability to inhibit membrane fusion was evaluated using FRET. Our results indicate that singlet oxygen production by LJ001, JL103 and JL118 is able to induce several changes on membrane properties, specially related with a decrease on its fluidity, concomitant with an increase on the order on the polar head groups region, resulting in an inhibition of the membrane fusion necessary for cell infection.1. Wolf et al. (2010) PNAS 107, 3157-62.2. Vigant et al. (2013) PLoS Pathog 9: e1003297.

COMPUTATIONAL STUDY OF HIV-1 gp41 NHR TRIMER: INHIBITION MECHANISMS OF N-SUBSTITUTED PYRROLE DERIVATIVES AND FRAGMENT-BASED VIRTUAL SCREENING

Fusion of HIV-1 viral and host cellular membranes is an important step for HIV infection. The HIV-1 envelope glycoprotein mediating the membrane fusion consists of subunits gp120 and gp41 whereas gp120 recognizes the cell-surface receptors and gp41 promotes viral-cell membrane fusion. The trimeric helical complex composed of heterodimer of N-terminal and C-terminal extraviral segments has been used for the gp41 function study, and the trimeric N-terminal teptad repeat (NHR) is considered as an antiviral drug target for developing HIV-1 membrane fusion inhibitors. By using computational solvent probe mapping, we have explored druggable sites on the trimeric NHR peptides, and identified residues K574 and R579 as the hot spots for inhibitor designing. We further demonstrated that although NB-2 and NB-64 are all N-substituted Pyrrole derivatives and have very similar chemical structures, it is possible that diverse inhibitory mechanisms targeting different negative electrostatic residues (K574 and R579) exist. Results from fragment-based virtual screening identified series of potential lead compounds which could be used for further design of fusion inhibitors.

Treatment of influenza virus with Beta-propiolactone alters viral membrane fusion

Beta-propiolactone (BPL) is commonly used as an inactivating reagent to produce viral vaccines. Although BPL has been described to chemically modify nucleic acids, its effect on viral proteins, potentially affecting viral infectivity, remains poorly studied. Here, a H3N2 strain of influenza virus was submitted to treatment with various BPL concentrations (2–1000μM). Cell infectivity was progressively reduced and entirely abolished at 1mM BPL. Virus fusion with endosome being a critical step in virus infection, we analyzed its ability to fuse with lipid membrane after BPL treatment. By monitoring calcein leakage from liposomes fusing with the virus, we measured a decrease of membrane fusion in a BPL dose-dependent manner that correlates with the loss of infectivity. These data were complemented with cryo transmission electron microscopy (cryoTEM) and cryo electron tomography (cryoET) studies of native and modified viruses. In addition, a decrease of leakage irrespective of BPL concentration was measured suggesting that the insertion of HA2 fusion peptide into the target membrane was inhibited even at low BPL concentrations. Interestingly, mass spectrometry revealed that HA2 and M1 matrix proteins had been modified. Furthermore, fusion activity was partially restored by the protonophore monensin as confirmed by cryoTEM and cryoET. Moreover, exposure to amantadine, an inhibitor of M2 channel, did not alter membrane fusion activity of 1mM BPL treated virus. Taken together these results show that BPL treatment inhibits membrane fusion, likely by altering function of proteins involved in the fusion process, shedding new light on the effect of BPL on influenza virus.

Modular assembly of dimeric HIV fusion inhibitor peptides with enhanced antiviral potency

The HIV-1 envelope gp120/gp41 glycoprotein complex plays a critical role in virus-host cell membrane fusion and has been a focus for the development of HIV fusion inhibitors. In this Letter, we present the synthesis of dimers of HIV fusion inhibitor peptides C37H6 and CP32M, which target the trimeric gp41 in the pre-hairpin intermediate state to inhibit membrane fusion. Reactive peptide modules were synthesized using native chemical ligation and then assembled into dimers with varying linker lengths using Cu(I)-catalyzed azide–alkyne cycloaddition (CuAAC) ‘click’ chemistry. Cell–cell fusion inhibition assays demonstrated that dimers with a (PEG)7 linker showed enhanced antiviral potency over the corresponding monomers. Moreover, the bio-orthogonal nature of the CuAAC ‘click’ reaction provides a practical way to assemble heterodimers of HIV fusion inhibitors. Heterodimers consisting of the T20-sensitive strain inhibitor C37H6 and the T20-resistant strain inhibitor CP32M were produced that may have broader spectrum activities against both T20-sensitive and T20-resistant strains.

Structurally Conserved Binding Sites of Hemagglutinin as Targets for Influenza Drug and Vaccine Development

ProBiS is a new method to identify the binding site of protein through local structural alignment against the nonredundant Protein Data Bank (PDB), which may result in unique findings compared to the energy-based, geometry-based, and sequence-based predictors. In this work, binding sites of Hemagglutinin (HA), which is an important target for drugs and vaccines in influenza treatment, have been revisited by ProBiS. For the first time, the identification of conserved binding sites by local structural alignment across all subtypes and strains of HA available in PDB is presented. ProBiS finds three distinctive conserved sites on HA's structure (named Site 1, Site 2, and Site 3). Compared to other predictors, ProBiS is the only one that accurately defines the receptor binding site (Site 1). Apart from that, Site 2, which is located slightly above the TBHQ binding site, is proposed as a potential novel conserved target for membrane fusion inhibitor. Lastly, Site 3, located around Helix A at the stem domain and recently targeted by cross-reactive antibodies, is predicted to be conserved in the latest H7N9 China 2013 strain as well. The further exploration of these three sites provides valuable insight in optimizing the influenza drug and vaccine development.

Computational Identification of a New Binding Site in Influenza Virus Hemagglutinin for Membrane Fusion Inhibitors

The influenza virus hemagglutinin is a potential drug target for antivirus treatment. A variety of membrane fusion inhibitors targeting hemagglutinin have been discovered, but the binding sites and modes, important for understanding membrane fusion and rational drug design, have not yet been elucidated. In this article, we investigated the possible hemagglutinin binding sites for the current membrane fusion inhibitors. Four possible binding pockets (Pocket A, B, C, and D) at the stalk region of hemagglutinin were detected and defined using the CAVITY program. Most of the current membrane fusion inhibitors were reported to bind to Pocket C by amino acid mutation experiments and molecular modeling simulation. However, our binding site prediction suggested that Pocket A is the best ligand binding site other than Pocket C. Using a specific computational protocol combining molecular docking, three-dimensional QSAR, and receptor mimicking, we further found that Pocket A is the putative binding site for a series of membrane fusion inhibitors (1-phenyl-cycloalkane carbamides). This is further proven by the antiviral spectrum of the inhibitors. This protocol for the identification of ligand binding sites in influenza hemagglutinin is also applicable for the analysis of other protein targets with no explicit binding information.

Design of inhibitors of influenza virus membrane fusion: Synthesis, structure–activity relationship and in vitro antiviral activity of a novel indole series

The fusion of virus and endosome membranes is an essential early stage in influenza virus infection. The low pH-induced conformational change which promotes the fusogenic activity of the haemagglutinin (HA) is thus an attractive target as an antiviral strategy. The anti-influenza drug Arbidol is representative of a class of antivirals which inhibits HA-mediated membrane fusion by increasing the acid stability of the HA. In this study two series of indole derivatives structurally related to Arbidol were designed and synthesized to further probe the foundation of its antiviral activity and develop the basis for a structure–activity relationship (SAR). Ethyl 5-(hydroxymethyl)-1-methyl-2-(phenysulphanylmethyl)-1H-indole-3-carboxylate (15) was identified as one of the most potent inhibitors and more potent than Arbidol against certain subtypes of influenza A viruses. In particular, 15 exhibited a much greater affinity and preference for binding group 2 than group 1 HAs, and exerted a greater stabilising effect, in contrast to Arbidol. The results provide the basis for more detailed SAR studies of Arbidol binding to HA; however, the greater affinity for binding HA was not reflected in a comparable increase in antiviral activity of 15, apparently reflecting the complex nature of the antiviral activity of Arbidol and its derivatives.