Structure-activity relationships for a series of thiobenzamide influenza fusion inhibitors derived from 1,3,3-trimethyl-5-hydroxy-cyclohexylmethylamine

The anti-influenza activity of a series of thiobenzamide fusion inhibitors derived from 1,3,3-trimethyl-5-hydroxy-cyclohexylmethylamine is profiled. Axial disposition of the thioamide moiety is essential for potent influenza inhibitory activity.     

New influenza A Virus Entry Inhibitors Derived from the Viral Fusion Peptides

Influenza A viral (IAV) fusion peptides are known for their important role in viral-cell fusion process and membrane destabilization potential which are compatible with those of antimicrobial peptides. Thus, by replacing the negatively or neutrally charged residues of FPs with positively charged lysines, we synthesized several potent antimicrobial peptides derived from the fusogenic peptides (FPs) of hemagglutinin glycoproteins (HAs) of IAV. The biological screening identified that in addition to the potent antibacterial activities, these positively charged fusion peptides (pFPs) effectively inhibited the replication of influenza A viruses including oseltamivir-resistant strain. By employing pseudovirus-based entry inhibition assays including H5N1 influenza A virus (IAV), and VSV-G, the mechanism study indicated that the antiviral activity may be associated with the interactions between the HA2 subunit and pFP, of which, the nascent pFP exerted a strong effect to interrupt the conformational changes of HA2, thereby blocking the entry of viruses into host cells. In addition to providing new peptide “entry blockers”, these data also demonstrate a useful strategy in designing potent antibacterial agents, as well as effective viral entry inhibitors. It would be meaningful in treatment of bacterial co-infection during influenza pandemic periods, as well as in our current war against those emerging pathogenic microorganisms such as IAV and HIV.     

Characterization of Potent Fusion Inhibitors of Influenza Virus

New inhibitors of influenza viruses are needed to combat the potential emergence of novel human influenza viruses. We have identified a class of small molecules that inhibit replication of influenza virus at picomolar concentrations in plaque reduction assays. The compound also inhibits replication of vesicular stomatitis virus. Time of addition and dilution experiments with influenza virus indicated that an early time point of infection was blocked and that inhibitor 136 tightly bound to virions. Using fluorescently labeled influenza virus, inhibition of viral fusion to cellular membranes by blocked lipid mixing was established as the mechanism of action for this class of inhibitors. Stabilization of the neutral pH form of hemagglutinin (HA) was ruled out by trypsin digestion studies in vitro and with conformation specific HA antibodies within cells. Direct visualization of 136 treated influenza virions at pH 7.5 or acidified to pH 5.0 showed that virions remain intact and that glycoproteins become disorganized as expected when HA undergoes a conformational change. This suggests that exposure of the fusion peptide at low pH is not inhibited but lipid mixing is inhibited, a different mechanism than previously reported fusion inhibitors. We hypothesize that this new class of inhibitors intercalate into the virus envelope altering the structure of the viral envelope required for fusion to cellular membranes.     

An Induced Pocket for the Binding of Potent Fusion Inhibitor CL-385319 with H5N1 Influenza Virus Hemagglutinin

The influenza glycoprotein hemagglutinin (HA) plays crucial roles in the early stage of virus infection, including receptor binding and membrane fusion. Therefore, HA is a potential target for developing anti-influenza drugs. Recently, we characterized a novel inhibitor of highly pathogenic H5N1 influenza virus, CL-385319, which specifically inhibits HA-mediated viral entry. Studies presented here identified the critical binding residues for CL-385319, which clustered in the stem region of the HA trimer by site-directed mutagenesis. Extensive computational simulations, including molecular docking, molecular dynamics simulations, molecular mechanics generalized Born surface area (MM_GBSA) calculations, charge density and Laplacian calculations, have been carried out to uncover the detailed molecular mechanism that underlies the binding of CL-385319 to H5N1 influenza virus HA. It was found that the recognition and binding of CL-385319 to HA proceeds by a process of "induced fit" whereby the binding pocket is formed during their interaction. Occupation of this pocket by CL-385319 stabilizes the neutral pH structure of hemagglutinin, thus inhibiting the conformational rearrangements required for membrane fusion. This "induced fit" pocket may be a target for structure-based design of more potent influenza fusion inhibitors.     

Design,synthesis, and biological activity of thiazole derivatives as novel influenza neuraminidase inhibitors

A series of novel influenza neuraminidase (NA) inhibitors based on thiazole core were synthesized and evaluated for their ability to inhibit NA of influenza A virus (H₃N₂). All compounds were synthesized in good yields starting from commercially available 2-amino-4-thiazole-acetic ester using a suitable synthetic strategy. These compounds showed moderate inhibitory activity against influenza A NA. The most potent compound of this series is compound 4d (IC₅₀?=?3.43 μM), which is about 20-fold less potent than oseltamivir, and could be used to design novel influenza NA inhibitors that exhibit increased activity based on thiazole ring.     

Intracytoplasmic trapping of influenza virus by a lipophilic derivative of aglycoristocetin

We report on a new anti-influenza virus agent, SA-19, a lipophilic glycopeptide derivative consisting of aglycoristocetin coupled to a phenylbenzyl-substituted cyclobutenedione. In Madin-Darby canine kidney cells infected with influenza A/H1N1, A/H3N2, or B virus, SA-19 displayed a 50% antivirally effective concentration of 0.60 μM and a selectivity index (ratio of cytotoxic versus antiviral concentration) of 112. SA-19 was 11-fold more potent than unsubstituted aglycoristocetin and was active in human and nonhuman cell lines. Virus yield at 72 h p.i. was reduced by 3.6 logs at 0.8 μM SA-19. In contrast to amantadine and oseltamivir, SA-19 did not select for resistance upon prolonged virus exposure. SA-19 was shown to inhibit an early postbinding step in virus replication. The compound had no effect on hemagglutinin (HA)-mediated membrane fusion in an HA-polykaryon assay and did not inhibit the low-pH-induced refolding of the HA in a tryptic digestion assay. However, a marked inhibitory effect on the transduction exerted by retroviral pseudoparticles carrying an HA or vesicular stomatitis virus glycoprotein (VSV-G) fusion protein was noted, suggesting that SA-19 targets a cellular factor with a role in influenza virus and VSV entry. Using confocal microscopy with antinucleoprotein staining, SA-19 was proven to completely prevent the influenza virus nuclear entry. This virus arrest was characterized by the formation of cytoplasmic aggregates. SA-19 appeared to disturb the endocytic uptake and trap the influenza virus in vesicles distinct from early, late, or recycling endosomes. The aglycoristocetin derivative SA-19 represents a new class of potent and broad-acting influenza virus inhibitors with potential clinical relevance.     

Design, synthesis and biological activity of thiazolidine-4-carboxylic acid derivatives as novel influenza neuraminidase inhibitors

A series of thiazolidine-4-carboxylic acid derivatives were synthesized and evaluated for their ability to inhibit neuraminidase (NA) of influenza A virus. All the compounds were synthesized in good yields starting from commercially available l-cysteine hydrochloride using a suitable synthetic strategy. These compounds showed moderate inhibitory activity against influenza A neuraminidase. The most potent compound of this series is compound 4f (IC(50)=0.14 μM), which is about sevenfold less potent than oseltamivir and could be used to design novel influenza NA inhibitors that exhibit increased activity based on thiazolidine ring.     

Design, synthesis, and biological activity of thiazole derivatives as novel influenza neuraminidase inhibitors

A series of novel influenza neuraminidase (NA) inhibitors based on thiazole core were synthesized and evaluated for their ability to inhibit NA of influenza A virus (H(3)N(2)). All compounds were synthesized in good yields starting from commercially available 2-amino-4-thiazole-acetic ester using a suitable synthetic strategy. These compounds showed moderate inhibitory activity against influenza A NA. The most potent compound of this series is compound 4d (IC(50)?=?3.43 μM), which is about 20-fold less potent than oseltamivir, and could be used to design novel influenza NA inhibitors that exhibit increased activity based on thiazole ring.     

Synthesis and anti-influenza activity of aminoalkyl rupestonates

A series of aminoalkyl rupestonates were designed and synthesized by reacting rupestonic acid with 1,ω-dibromoalkanes, followed by amination. All of the new compounds were bioassayed in vitro to determine their activities against influenza A (H3N2, H1N1) and B viruses. The results showed that compounds 5a-5g, which each contain a 1H-1,2,4-triazolyl moiety, were found to be the most potent set of compounds. Compound 5g was demonstrated to possess the highest inhibitory activity against influenza H3N2 and H1N1, with IC(50) values of 0.97 and 0.42 μM, respectively. Our results also indicated that compounds 2g, 3g, 4g and 5g, which contain ten-CH(2)-unit spacers between the rupestonic acid and amino functional groups, were the most potent inhibitors of influenza H1N1 among the synthesized compounds. Unfortunately, most of the synthesized compounds did not show an obvious activity against influenza B; the only exceptions were compounds 5d and 5f, which had IC(50) values of 17.3 and 3.2 μM, respectively. Compounds 4g and 5g were potent inhibitors of influenza H1N1, and they might be potentially developed as new lead anti-influenza virus compounds. Further studies of the mechanism of action are underway.     

Fusion of influenza to liposomes is not inhibited by aliphatic primary alcohols

Reports of the antiviral activity of aliphatic alcohols led us to investigate the effects of aliphatic alcohols, from 10 to 20 carbons in length, on the phase transition behaviour of model phospholipids and on the fusion of influenza to liposomes. Contrary to the effects of many other antiviral agents, we find that alcohols are potent promoters of the inverted hexagonal phase. However, we also find that aliphatic alcohols have little effect on influenza fusion to liposomes. Eicosanol is the only aliphatic alcohol tested which substantially increases in fusion of influenza virus. We also find that long chain alcohols display multi-component bilayer to hexagonal phase transitions at higher mole fractions. This suggests that eicosanol may be facilitating fusion by creating defects between alcohol-rich and alcohol-poor regions of the lipid bilayer.     

A dual reporter gene based system to quantitate the cell fusion of avian influenza virus H5N1

Membrane fusion is central to the entry of influenza virus into host cells. To quantitatively determine the fusion activity of hemagglutinin (HA) of avian influenza virus H5N1, we established a cell fusion assay based on a dual luciferase reporter gene. The HA fusion activity was assayed by measuring luciferase expression in fused cells, allowing a rapid, sensitive, and quantitative comparison of HA fusion activities at various pHs and in different cells types. The simplicity and the quantitative nature of this novel assay are ideally suited for identifying viral receptors or screening for inhibitors of viral entry in the future.     

Synthesis and evaluation of a new series of substituted acyl(thio)urea and thiadiazolo [2,3-a] pyrimidine derivatives as potent inhibitors of influenza virus neuraminidase

A series of substituted acyl(thio)urea and 2H-1,2,4-thiadiazolo [2,3-a] pyrimidine derivatives were prepared and both of their cell culture and enzymatic activity toward influenza virus were tested. Their in vitro neuraminidase inhibitory activities were in good agreement with the corresponding activities in cultured cells and they were evaluated as potent neuraminidase inhibitors. Of the analogues that demonstrated IC₅₀s < 0.1 μM, 16 and 60 were further investigated as candidates with the most potential for future development. The molecular docking work of the representative compound was described to provide more insight into their mechanism of action and further rationalize the observations of this new series herein, which represents a novel class of highly potent and selective inhibitors of influenza virus.     

Inhibition of viral adhesion and infection by sialic-acid-conjugated dendritic polymers

Multiple sialic acid (SA) residues conjugated to a linear polyacrylamide backbone are more effective than monomeric SA at inhibiting influenza-induced agglutination of red blood cells. However, "polymeric inhibitors" based on polyacrylamide backbones are cytotoxic. Dendritic polymers offer a nontoxic alternative to polyacrylamide and may provide a variety of potential synthetic inhibitors of influenza virus adhesion due to the wide range of available polymer structures. We evaluated several dendritic polymeric inhibitors, including spheroidal, linear, linear-dendron copolymers, comb-branched, and dendrigraft polymers, for the ability to inhibit virus hemagglutination (HA) and to block infection of mammalian cells in vitro. Four viruses were tested: influenza A H2N2 (selectively propagated two ways), X-31 influenza A H3N2, and sendai. The most potent of the linear and spheroidal inhibitors were 32-256-fold more effective than monomeric SA at inhibiting HA by the H2N2 influenza virus. Linear-dendron copolymers were 1025-8200-fold more effective against H2N2 influenza, X-31 influenza, and sendai viruses. The most effective were the comb-branched and dendrigraft inhibitors, which showed up to 50000-fold increased activity against these viruses. We were able to demonstrate significant (p < 0.001) dose-dependent reduction of influenza infection in mammalian cells by polymeric inhibitors, the first such demonstration for multivalent SA inhibitors. Effective dendrimer polymers were not cytotoxic to mammalian cells at therapeutic levels. Of additional interest, variation in the inhibitory effect was observed with different viruses, suggesting possible differences due to specific growth conditions of virus. SA-conjugated dendritic polymers may provide a new therapeutic modality for viruses that employ SA as their target receptor.     

Superior inhibition of influenza virus hemagglutinin-mediated fusion by indole-substituted spirothiazolidinones

The influenza virus hemagglutinin (HA) mediates membrane fusion after viral entry by endocytosis. The fusion process requires drastic low pH-induced HA refolding and is prevented by arbidol and tert-butylhydroquinone (TBHQ). We here report a class of superior inhibitors with indole-substituted spirothiazolidinone structure. The most active analogue 5f has an EC₅₀ value against influenza A/H3N2 virus of 1 nM and selectivity index of almost 2000. Resistance data and in silico modeling indicate that 5f combines optimized fitting in the TBHQ/arbidol HA binding pocket with a capability for endosomal accumulation. Both criteria appear relevant to achieve superior inhibitors of HA-mediated fusion.     

Studies on the effect of glycoprotein processing inhibitors on fusion of L6 myoblast cell lines

The effect of oligosaccharide processing inhibitors on the fusion of L6 myoblasts was studied. The glucosidase inhibitors, castanospermine, 1-deoxynojirimycin and N-methyl-deoxynojirimycin were potent inhibitors of myoblast fusion, as was the mannosidase II inhibitor, swainsonine. Inhibition of fusion was reversed when inhibitors were removed. However, the mannosidase I inhibitor, 1-deoxymannojirimycin did not inhibit fusion. Changes in cell membrane oligosaccharide structure were followed by monitoring the binding of concanavalin A (conA) and wheat germ agglutinin (WGA) to cell surface membranes in cells treated with processing inhibitors. All the processing inhibitors resulted in increased binding of conA and decreased binding of WGA; this is consistent with the known mechanisms of inhibition of the inhibitors used in the study. Inhibition of fusion by the processing inhibitors also resulted in reduced activities of creatine phosphokinase, an enzyme used as a marker for biochemical differentiation during fusion. Treatment of a non-differentiating conA-resistant cell line with processing inhibitors did not induce fusion, but the cells did show altered lectin-binding properties. The main conclusion drawn from these studies is that cell surface glycoproteins probably containing the mannose (Man)9 structure are important for the fusion reaction.     

A novel class of potent influenza virus inhibitors: polysubstituted acylthiourea and its fused heterocycle derivatives

A series of polysubstituted and fused heterocycle derivatives of acylthiourea was prepared and the biological activity against influenza virus was evaluated. Of the analogues that demonstrated IC(50)s<0.1 microM, acylthiourea derivatives 16 and 50 were further investigated as candidates with the most potential for future development. The SAR of these compounds are discussed and they represent a novel class of highly potent and selective inhibitors of influenza virus.     

Capturing a fusion intermediate of influenza hemagglutinin with a cholesterol-conjugated peptide, a new antiviral strategy for influenza virus

We previously described fusion-inhibitory peptides that are targeted to the cell membrane by cholesterol conjugation and potently inhibit enveloped viruses that fuse at the cell surface, including HIV, parainfluenza, and henipaviruses. However, for viruses that fuse inside of intracellular compartments, fusion-inhibitory peptides have exhibited very low antiviral activity. We propose that for these viruses, too, membrane targeting via cholesterol conjugation may yield potent compounds. Here we compare the activity of fusion-inhibitory peptides derived from the influenza hemagglutinin (HA) and show that although the unconjugated peptides are inactive, the cholesterol-conjugated compounds are effective inhibitors of infectivity and membrane fusion. We hypothesize that the cholesterol moiety, by localizing the peptides to the target cell membrane, allows the peptides to follow the virus to the intracellular site of fusion. The cholesterol-conjugated peptides trap HA in a transient intermediate state after fusion is triggered but before completion of the refolding steps that drive the merging of the viral and cellular membranes. These results provide proof of concept for an antiviral strategy that is applicable to intracellularly fusing viruses, including known and emerging viral pathogens.     

Salicylamide inhibitors of influenza virus fusion

Structural variation of the quinolizidine heterocycle of the influenza fusion inhibitor BMY-27709 was examined by several topological dissections in order to illuminate the critical features of the ring system. This exercise resulted in the identification of a series of synthetically more accessible decahydroquinolines that retained the structural elements of BMY-27709 important for antiviral activity. The 2-methyl-cis-decahydroquinoline 6f was the most potent influenza inhibitor identified that demonstrated an EC50 of 90 ng/mL in a plaque reduction assay.     

Pyrrolidinobenzoic acid inhibitors of influenza virus neuraminidase: modifications of essential pyrrolidinone ring substituents

We recently reported the first benzoic acid, 1-[4-carboxy-2-(3-pentylamino)phenyl]-5,5-bis(hydroxymethyl)pyrrolidin-2-one (8), that is a potent inhibitor of avian influenza A neuraminidase (N9) and, unlike other reported potent neuraminidase inhibitors, does not contain a basic aliphatic amine or guanidine nor a simple N-acetyl grouping. However, 8 was a poor inhibitor of influenza B neuraminidase. In the present study we further evaluated 8 as an inhibitor of human influenza A NA isolates, and it was effective against N2NA but found to be 160-fold less active against N1NA. We also synthesized analogues of 8 involving moderate modifications of essential substituents on the pyrrolidinone ring. Specifically, the aminomethyl (9), hydroxyethyl (10), and aminoethyl (11) analogues were prepared. Only the most conservative change (compound 9) resulted in continued effective inhibition of influenza A, in addition to a noteworthy increase in the activity of 9 for N1NA. The effectiveness of 9 against influenza B neuraminidase was furthermore improved 10-fold relative to 8, but this activity remained 50-fold poorer than for type A NA.     

Design of benzoic acid inhibitors of influenza neuraminidase containing a cyclic substitution for the N-acetyl grouping.

A 2-pyrrolidinone ring containing a single hydroxymethyl side chain effectively replaces the N-acetylamino group of 4-(N-acetylamino)-3-guanidinobenzoic acid, a low micromolar inhibitor of influenza neuraminidase. This novel structural template affords new opportunities to evolve more potent benzoic acid inhibitors.