An approach to the identification of potent inhibitors of influenza virus fusion using parallel synthesis methodology

Structure-activity studies associated with the salicylic acid-derived inhibitor of influenza fusion, BMY-27709, were examined using a parallel synthesis approach. This SAR survey led to the discovery of potent influenza inhibitory activity in a series of aromatic amides and thioamides derived from 1,3,3-trimethyl-5-hydroxycyclohexylmethylamine. Select compounds were characterized as inhibitors of the H1 subtype of influenza A viruses that act by preventing the pH-induced fusion process, thereby blocking viral entry into host cells. In a plaque-reduction assay, the most potent inhibitors displayed EC(50) values of 0.02-0.14 microg/mL.     

Selective inhibition of cholesterol synthesis in liver versus extrahepatic tissues by HMG-CoA reductase inhibitors

Hepatic specificity of inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase may be achieved by efficient first-pass liver extraction resulting in low circulating drug levels, as with lovastatin, or by lower cellular uptake in peripheral tissues, seen with pravastatin. BMY-21950 and its lactone form BMY-22089, new synthetic inhibitors of HMG-CoA reductase, were compared with the major reference agent lovastatin and with the synthetic inhibitor fluindostatin in several in vitro and in vivo models of potency and tissue selectivity. The kinetic mechanism and the potency of BMY-21950 as a competitive inhibitor of isolated HMG-CoA reductase were comparable to the reference agents. The inhibitory potency (cholesterol synthesis assayed by 3H2O or [14C]acetate incorporation) of BMY-21950 in rat hepatocytes (IC50 = 21 nM) and dog liver slices (IC50 = 23 nM) equalled or exceeded the potencies of the reference agents. Hepatic cholesterol synthesis in vivo in rats was effectively inhibited by BMY-21950 and its lactone form BMY-22089 (ED50 = 0.1 mg/kg p.o.), but oral doses (20 mg/kg) that suppressed liver synthesis by 83-95% inhibited sterol synthesis by only 17-24% in the ileum. In contrast, equivalent doses of lovastatin markedly inhibited cholesterol synthesis in both organs. In tissue slices from rat ileum, cell dispersions from testes, adrenal, and spleen, and in bovine ocular lens epithelial cells, BMY-21950 inhibited sterol synthesis weakly in vitro with IC50 values 76- and 188-times higher than in hepatocytes; similar effects were seen for BMY-22089. However, the IC50 ratios (tissue/hepatocyte) for lovastatin and fluindostatin were near unity in these models. Thus, BMY-21950 and BMY-22089 are the first potent synthetic HMG-CoA reductase inhibitors that possess a very high degree of liver selectivity based upon differential inhibition sensitivities in tissues. This cellular uptake-based property of hepatic specificity of BMY-21950 and BMY-22089, also manifest in pravastatin, is biochemically distinct from the pharmacodynamic-based disposition of lovastatin, which along with fluindostatin exhibited potent inhibition in all tissues that were exposed to it.     

Effect of the superoxide dismutase inhibitor, diethyldithiocarbamate, on the cytotoxicity of mitomycin antibiotics

Mitomycin C (MC) and its structural analogs porfiromycin (PM), BMY-25282 and BL-6783 are toxic to EMT6 cells under aerobic and hypoxic conditions. The mitomycin antibiotics are hypothesized to exert cytotoxicity under hypoxic conditions by cross-linking DNA following reductive activation, while aerobic cytotoxicity may involve DNA cross-linking by these agents and/or damage due to the generation of oxygen radicals. Previous findings (Pritsos and Sartorelli, 1986) indicated that the rank order of cytotoxicity for a series of mitomycins was the same as the rank order for the rate of oxygen consumption induced by these agents. As an additional approach to explore the role of oxygen radicals in the aerobic cytotoxicity of the four agents studied, EMT6 cells were treated with the mitomycins in the presence of the superoxide dismutase inhibitor diethyldithiocarbamate (DETC). DETC, which decreased superoxide dismutase activity in EMT6 cells, increased the cytotoxicity of BMY-25282 and BL-6783 by half an order of magnitude, but did not affect the toxicity of PM or MC to these cells. DNA cross-links, a proposed cytotoxic lesion induced by BMY-25282, however, were not detectably increased in EMT6 cells exposed to this agent in the presence of DETC in spite of the large increase in cytotoxicity under these treatment conditions. No single strand breaks were detected in cells exposed to either BMY-25282 or BMY-25282 plus DETC. The findings support the concept that oxygen radicals may have a role in the aerobic cytotoxicity of some of the mitomycin antibiotics, and that the lesions responsible for cytotoxicity produced by oxygen radicals may not reside entirely at the level of DNA.     

BMY-14802 protects against ischemia-induced neuronal damage in the gerbil.

BMY-14802, a selective sigma ligand currently under investigation as an atypical antipsychotic agent, was tested for potential anti-ischemic activity. BMY-14802 (10, 30 and 50 mg/kg) did not produce any stereotyped behavior, ataxia or seizures. When gerbils were pretreated with 10, 30 or 50 mg/kg of BMY-14802 30 min prior to bilateral occlusion of carotid arteries for 5 min, BMY-14802 significantly protected against ischemia-induced neuronal loss in the hippocampus. Thus, BMY-14802 may also be useful as an anti-ischemic agent that does not produce psychotomimetic effects.     

Hep-G2 cells and primary rat hepatocytes differ in their response to inhibitors of HMG-CoA reductase

CI-981, a novel synthetic inhibitor of HMG-CoA reductase, was previously reported to be highly liver-selective using an ex vivo approach. In order to determine liver-selectivity at the cellular level, CI-981 was evaluated in cell culture and compared to lovastatin, pravastatin, fluvastatin and BMY-21950. Using human cell lines, none of the compounds tested showed liver-selectivity, i.e. strong inhibition of cholesterol synthesis in Hep-G2 cells (liver model) but weak inhibition in human fibroblasts (peripheral cell model). In contrast, all drugs tested produced equal and potent inhibition of sterol synthesis in primary cultures of rat hepatocytes, and CI-981, pravastatin and BMY-21950 were more than 100-fold more potent in rat hepatocytes compared to human fibroblasts. Since all compounds were also equally potent at inhibiting sterol synthesis in a rat subcellular system and in vivo, the data suggest that the use of Hep-G2 cells may not be the cell system of choice in which to study inhibition of hepatic cholesterogenesis or to demonstrate liver selectivity of inhibitors of HMG-CoA reductase.     

Different subtypes of alpha1-adrenoceptor modulate different K+ currents via different signaling pathways in canine ventricular myocytes

Multiple subtypes (alpha1A, alpha1B, and alpha1D) of alpha1-adrenoreceptors (alpha1ARs) co-exist in the heart and mediate a variety of cellular functions. We studied alphaAR modulation of inward rectifier (IK1) and transient outward (Ito) K(+) currents in canine ventricular myocytes. Phenylephrine at 10 microM depressed only Ito without affecting IK1 and at 100 microM inhibited both Ito and IK1. The effect of phenylephrine on Ito was abolished by (+)niguldipine (10 nm) to inhibit alpha1AARs but not by chloroethyclonidine (10 microM) to inactivate alpha1BARs nor by BMY-7378 to antagonize alpha1DARs. In contrast, phenylephrine inhibition of IK1 was reversed only by BMY-7378 (1 nm). PDD (100 nm, phorbol ester activator of protein kinase C (PKC)) simulates and bisindolylmaleimide (50 nm, PKC inhibitor) weakens phenylephrine modulation of Ito but not IK1. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN-93 and inhibitor peptides abolished the effects of phenylephrine on IK1. Enhancement of PKC or CaMKII activities was seen in alpha1aAR- or alpha1dAR-transfected HEK293 cells and in myocytes pretreated with 10 or 100 microM phenylephrine, respectively. Our data suggest that different subtypes of alpha1ARs selectively modulate different cardiac K(+) currents via different signal transduction mechanisms; alpha1AARs mediate Ito regulation via PKC, and alpha1DARs mediate IK1 regulation via CaMKII.     

Substituted Sialic Acid Prosthetic Groups as Determinants of Viral Hemagglutination

Inhibitors of hemagglutination by type A2 influenza virus and a recently isolated strain of type B influenza virus were separated by sucrose density gradient centrifugation and agarose gel filtration from horse serum. Using selected reagents, it was demonstrated that the active substituent on the horse serum inhibitor of A2 influenza virus was 4-O-acetyl-N-acetylneuraminic acid; however, the active substituent on the inhibitor of the influenza B virus was shown to be N-acetylneuraminic acid (NANA). Sodium metaperiodate treatment of a component of horse serum resulted in a 10 to 15-fold enhancement of inhibitory activity against the type B virus, whereas the A2 inhibitor was completely destroyed. Since this enhancement did not occur with influenza B viruses isolated prior to 1965, it was considered that this sensitivity to an oxidized NANA glycoside may have been a reflection of an antigenic change which occurred at that time. The use of different virus strains and selected chemical reagents to define the important sialic acid prosthetic groups active in inhibition was described.     

The effect of some anti-ulcer agents on the early vascular injury of gastric mucosa induced by ethanol in rats

The effect of some gastroprotective agents cysteamine, sodium salicylate, atropine, cimetidine, and pyrido-pyrimidine derivatives, rimazolium, Ch-127 and a mast cell stabilizer, BMY-26517-31 was studied on the enhanced vascular permeability of gastric mucosa induced by 100% ethanol, on the enhanced vascular permeability of peritoneal blood vessels due to 0.3% acetic acid and on carrageenin edema test. We found that cysteamine, sodium salicylate, rimazolium and BMY-26517-31 inhibited the alcohol-induced enhanced vascular permeability. They also decreased the carrageenin-induced edema and--with the exception of BMY-26517-31--the acetic-acid-induced enhanced vascular permeability of the peritoneal vessels. These results suggest that similar events are present in the early phase of acute inflammation and chemically induced mucosal lesions. Consequently, antiinflammatory activity might play role in the protective mechanism of some anti-ulcer agents.     

Inhibition of influenza A virus sialidase activity by sulfatide

Sulfatide, which binds to influenza A viruses and prevents the viral infection, was found to inhibit the sialidase activities of influenza A viruses in a pH-dependent manner. The kinetic parameters of the effect of sulfatide on the sialidase activities of human influenza A viruses using fluorometric assay indicated that sulfatide was a powerful and non-competitive type inhibitor in low-pH conditions.     

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.     

RWJ-270201 (BCX-1812): a novel neuraminidase inhibitor for influenza.

The influenza virus neuraminidase (NA) is important in the pathogenesis of infection and, thus, is an attractive target for agents used in the treatment and prophylaxis of influenza. This article describes preclinical and early clinical data related to RWJ-270201 (BCX-1812), a novel, orally active NA inhibitor that was rationally designed for having potent and selective activity against influenza A and B viruses. RWJ-270201 is a unique NA inhibitor with a cyclopentane ring structure and high selectivity for the influenza NA. RWJ-270201 has efficacy comparable to or better than earlier NA inhibitors against a wide range of influenza A and B isolates, including recently emerged and avian strains, both in vitro and in a lethal murine model of influenza. Based on the high selectivity and efficacy of RWJ-270201 against both type A and B influenza strains in preclinical studies as well as murine pharmacodynamic studies supporting the potential for once-daily administration, clinical trials were initiated in order to determine the tolerability and antiviral activity of RWJ-270201 in humans. To date, clinical studies have indicated that RWJ-270201 is well tolerated and has antiviral activity in human experimental influenza models when administered orally once daily.     

Induction of an Inhibitor of Influenza Virus Hemagglutination by Treatment of Serum with Periodate

Serum is commonly treated with potassium periodate to destroy nonspecific inhibitors of influenza virus hemagglutination. We have observed, however, that such treatment of serum without pre-existing inhibitor produced high titers of inhibitor against certain strains of influenza virus. Inhibitor was induced in the serum from several different animal species but not in hamster or mouse serum. Periodate treatment of serum albumin, fraction V, from several animals, including man, creates this inhibitor. Our data indicate that the inhibitor induced in the serum of various animal species differs in its mechanism of induction and in its resistance to receptor-destroying enzyme and trypsin. Hemagglutination by B/Singapore/3/64, B/Colorado/2/65, B/Georgia/1/65, and B/Massachusetts/3/66 strains of influenza virus is inhibited by periodate-treated human serum at dilutions as high as 1:5,120. The routine use of periodate treatment of serum in diagnostic and surveillance studies of influenza virus infections is not recommended.     

Trophic effects induced by alpha1D-adrenoceptors on endothelial cells are potentiated by hypoxia

Catecholamines have been shown to be involved in vascular remodeling through the stimulation of alpha(1)-adrenoceptors (alpha(1)-ARs). Recently, it has been demonstrated that catecholamines can stimulate angiogenesis in pathological conditions, even if the mechanisms and the AR subtypes involved still remain unclear. We investigated the influence of hypoxia (3% O(2)) on the ability of picomolar concentrations of phenylephrine (PHE), which are unable to induce any vascular contraction, to induce a trophic effect in human endothelial cells through stimulation of the alpha(1D)-subtype ARs. PHE, at picomolar concentrations, significantly promoted pseudocapillary formation from fragments of human mature vessels in vitro. Exposure to hypoxia significantly potentiated this effect, which was inhibited by the selective alpha(1D)-AR antagonist BMY-7378 and by the nitric oxide synthase inhibitor L-NAME, suggesting that alpha(1D)-ARs were involved in this effect through activation of the nitric oxide pathway. Proliferation and migration of HUVEC were also affected by picomolar PHE concentrations. Again, these effects were significantly potentiated in cells exposed to hypoxia and were inhibited by BMY-7378 and by N(G)-nitro-L-arginine methyl ester. Conversely, the alpha(1A)-AR-selective antagonist (S)-(+)-niguldipine hydrochloride and the alpha(1B)-AR antagonist chloroethylclonidine dihydrochloride did not modify endothelial cell migration and proliferation in response to PHE. These results demonstrate that the stimulation of alpha(1D)-ARs, triggered by picomolar PHE concentrations devoid of any contractile vascular effects, induces a proangiogenic phenotype in human endothelial cells that is enhanced in a hypoxic environment. The role of alpha(1D)-ARs may become more prominent in the adaptive responses to hypoxic vasculature injury.     

Virus-inhibiting properties of the carbonyl-conjugated pentaene roseofungin

The antiinfluenza activity of roseofungin, a polyenic macrolide antibiotic was studied in vitro on surviving fragments of the chick embryo chorionallantoic membranes and in ovo on growing chick embryos. It was shown that the antibiotic activity against influenza A and B viruses was sufficiently high. The activity of roseofungin against influenza A virus did not differ from that of remantadin, the most active inhibitor of influenza virus reproduction. However, the activity of roseofungin against influenza B virus was an advantage of this antibiotic over remantadin, which had practically no effect on this virus type. A statistically significant protective effect of roseofungin (p less than 0.05) was shown on the animals with experimental influenza. The study on the antiviral activity of roseofungin against the DNA-containing variolovaccine virus revealed that it markedly inhibited the plague reduction. Roseofungin had a pronounced inhibitory effect on cell neoplastic transformation induced by the RNA-containing oncogenic virus of Rous sarcoma.     

Microwave assisted synthesis and anti-influenza virus activity of 1-adamantyl substituted N-(1-thia-4-azaspiro[4.5]decan-4-yl)carboxamide derivatives

A microwave-assisted three-component one-pot cyclocondensation method was applied for the synthesis of novel N-(1-thia-4-azaspiro[4.5]decan-4-yl)carboxamide compounds carrying an adamantyl moiety. The structures of the compounds were confirmed by spectral and elemental analysis. All compounds were evaluated for antiviral activity against influenza A (H1N1 and H3N2) and influenza B virus in MDCK cell cultures. The compounds displayed a confined structure-activity relationship. The N-(2,8-dimethyl-3-oxo-1-thia-4-azaspiro[4.5]dec-4-yl)adamantane-1-carboxamide 3b was the most potent inhibitor [antiviral EC₅₀: 1.4 μM against influenza A/H3N2 virus]. Its strong inhibitory effect in a virus hemolysis assay supports that 3b acts as an influenza virus fusion inhibitor by preventing the conformational change of the influenza virus hemagglutinin at low pH.     

GBR-12909 and fluspirilene potently inhibited binding of [3H] (+)3-PPP to sigma receptors in rat brain

Fluspirilene and GBR-12909, two compounds structurally similar to BMY-14802 and haloperidol, were assessed for their ability to interact with sigma receptors. Fluspirilene, an antipsychotic agent that interacts potently with dopamine receptors, inhibited the binding of [3H]-(+) 3-PPP (IC50 = 380 nM) more potently than rimcazole, a putative sigma antagonist that was tested clinically for antipsychotic activity. GBR-12909, a potent dopamine uptake blocker, also inhibited the binding of [3H]-(+) 3-PPP with an IC50 of 48 nM. However, other compounds that block the re-uptake of catecholamines, such as nomifensine, desipramine, imipramine, xylamine, benztropine and cocaine, were much weaker than GBR-12909 as sigma ligands. Thus, GBR-12909 and fluspirilene, compounds structurally similar to BMY-14802, are potent sigma ligands.     

Efficacy of the New Neuraminidase Inhibitor CS-8958 against H5N1 Influenza Viruses

Currently, two neuraminidase (NA) inhibitors, oseltamivir and zanamivir, which must be administrated twice daily for 5 days for maximum therapeutic effect, are licensed for the treatment of influenza. However, oseltamivir-resistant mutants of seasonal H1N1 and highly pathogenic H5N1 avian influenza A viruses have emerged. Therefore, alternative antiviral agents are needed. Recently, a new neuraminidase inhibitor, R-125489, and its prodrug, CS-8958, have been developed. CS-8958 functions as a long-acting NA inhibitor in vivo (mice) and is efficacious against seasonal influenza strains following a single intranasal dose. Here, we tested the efficacy of this compound against H5N1 influenza viruses, which have spread across several continents and caused epidemics with high morbidity and mortality. We demonstrated that R-125489 interferes with the NA activity of H5N1 viruses, including oseltamivir-resistant and different clade strains. A single dose of CS-8958 (1,500 µg/kg) given to mice 2 h post-infection with H5N1 influenza viruses produced a higher survival rate than did continuous five-day administration of oseltamivir (50 mg/kg twice daily). Virus titers in lungs and brain were substantially lower in infected mice treated with a single dose of CS-8958 than in those treated with the five-day course of oseltamivir. CS-8958 was also highly efficacious against highly pathogenic H5N1 influenza virus and oseltamivir-resistant variants. A single dose of CS-8958 given seven days prior to virus infection also protected mice against H5N1 virus lethal infection. To evaluate the improved efficacy of CS-8958 over oseltamivir, the binding stability of R-125489 to various subtypes of influenza virus was assessed and compared with that of other NA inhibitors. We found that R-125489 bound to NA more tightly than did any other NA inhibitor tested. Our results indicate that CS-8958 is highly effective for the treatment and prophylaxis of infection with H5N1 influenza viruses, including oseltamivir-resistant mutants.     

Requirement for vacuolar proton-ATPase activity during entry of influenza virus into cells.

The role that endosomal acidification plays during influenza virus entry into MDCK cells has been analyzed by using the macrolide antibiotics bafilomycin A1 and concanamycin A as selective inhibitors of vacuolar proton-ATPase (v-[H+]ATPase), the enzyme responsible for the acidification of endosomes. Bafilomycin A1 and concanamycin A, present at the low concentrations of 5 x 10(-7) and 5 x 10(-9) M, respectively, prevented the entry of influenza virus into cells when added during the first minutes of infection. Attachment of virion particles to the cell surface was not the target for the action of bafilomycin A1. N,N'-Dicyclohexylcarbodiimide, a nonspecific inhibitor of proton-ATPases, also blocked virus entry, whereas elaiophylin, an inhibitor of the plasma-proton ATPase, had no effect. The inhibitory actions of bafilomycin A1 and concanamycin A were tested in culture medium at different pHs. Both antibiotics powerfully prevented influenza virus infection when the virus was added under low-pH conditions. This inhibition was reduced if the virus was bound to cells at 4 degrees C prior to the addition of warm low-pH medium. Moreover, incubation of cells at acidic pH potently blocked influenza virus infection, even in the absence of antibiotics. These results indicate that a pH gradient, rather than low pH, is necessary for efficient entry of influenza virus into cells.     

Pharmacologic Inhibition of COX-1 and COX-2 in Influenza A Viral Infection in Mice

Background

We previously demonstrated that cyclooxygenase (COX)-1 deficiency results in greater morbidity and inflammation, whereas COX-2 deficiency leads to reduced morbidity, inflammation and mortality in influenza infected mice.

Methodology/Principal Findings

We investigated the effects of COX-1 and COX-2 inhibitors in influenza A viral infection. Mice were given a COX-1 inhibitor (SC-560), a COX-2 inhibitor (celecoxib) or no inhibitor beginning 2 weeks prior to influenza A viral infection (200 PFU) and throughout the course of the experiment. Body weight and temperature were measured daily as indicators of morbidity. Animals were sacrificed on days 1 and 4 post-infection and bronchoalveolar lavage (BAL) fluid was collected or daily mortality was recorded up to 2 weeks post-infection. Treatment with SC-560 significantly increased mortality and was associated with profound hypothermia and greater weight loss compared to celecoxib or control groups. On day 4 of infection, BAL fluid cells were modestly elevated in celecoxib treated mice compared to SC-560 or control groups. Viral titres were similar between treatment groups. Levels of TNF-α and G-CSF were significantly attenuated in the SC-560 and celecoxib groups versus control and IL-6 levels were significantly lower in BAL fluid of celecoxib treated mice versus control and versus the SC-560 group. The chemokine KC was significantly lower in SC-560 group versus control.

Conclusions/Significance

Treatment with a COX-1 inhibitor during influenza A viral infection is detrimental to the host whereas inhibition of COX-2 does not significantly modulate disease severity. COX-1 plays a critical role in controlling the thermoregulatory response to influenza A viral infection in mice.     

A reverse genetics approach for recovery of recombinant influenza B viruses entirely from cDNA

The recovery of recombinant influenza A virus entirely from cDNA was recently described (9, 19). We adapted the technique for engineering influenza B virus and generated a mutant bearing an amino acid change E116G in the viral neuraminidase which was resistant in vitro to the neuraminidase inhibitor zanamivir. The method also facilitates rapid isolation of single-gene reassortants suitable as vaccine seeds and will aid further investigations of unique features of influenza B virus.