Quinolinecarboxamides Inhibit the Replication of the Bovine Viral Diarrhea Virus by Targeting a Hot Spot for the Inhibition of Pestivirus Replication in the RNA-Dependent RNA Polymerase.

The bovine viral diarrhea virus (BVDV), a pestivirus from the family of Flaviviridae is ubiquitous and causes a range of clinical manifestations in livestock, mainly cattle. Two quinolinecarboxamide analogues were identified in a CPE-based screening effort, as selective inhibitors of the in vitro bovine viral diarrhea virus (BVDV) replication, i.e., TO505-6180/CSFCI (average EC50 = 0.07 µM, SD = 0.02 µM, CC50 > 100 µM) and TO502-2403/CSFCII (average EC50 = 0.2 µM, SD = 0.06 µM, CC50 > 100 µM). The initial antiviral activity observed for both hits against BVDV was corroborated by measuring the inhibitory effect on viral RNA synthesis and the production of infectious virus. Modification of the substituents on the quinolinecarboxamide scaffold resulted in analogues that proved about 7-fold more potent (average EC50 = 0.03 with a SD = 0.01 µM) and that were devoid of cellular toxicity, for the concentration range tested (SI = 3333). CSFCII resistant BVDV variants were selected and were found to carry the F224P mutation in the viral RNA-dependent RNA polymerase (RdRp), whereas CSFCI resistant BVDV carried two mutations in the same region of the RdRp, i.e., N264D and F224Y. Likewise, molecular modeling revealed that F224P/Y and N264D are located in a small cavity near the fingertip domain of the pestivirus polymerase. CSFC-resistant BVDV proved to be cross-resistant to earlier reported pestivirus inhibitors (BPIP, AG110, LZ37, and BBP) that are known to target the same region of the RdRp. CSFC analogues did not inhibit the in vitro activity of recombinant BVDV RdRp but inhibited the activity of BVDV replication complexes (RCs). CSFC analogues likely interact with the fingertip of the pestivirus RdRp at the same position as BPIP, AG110, LZ37, and BBP. This indicates that this region is a "hot spot" for the inhibition of pestivirus replication.

Computational Study Exploring the Interaction Mechanism of Benzimidazole Derivatives as Potent Cattle Bovine Viral Diarrhea Virus Inhibitors.

Bovine viral diarrhea virus (BVDV) infections are prevailing in cattle populations on a worldwide scale. The BVDV RNA-dependent RNA polymerase (RdRp), as a promising target for new anti-BVDV drug development, has attracted increasing attention. To explore the interaction mechanism of 65 benzimidazole scaffold-based derivatives as BVDV inhibitors, presently, a computational study was performed based on a combination of 3D-QSAR, molecular docking, and molecular dynamics (MD) simulations. The resultant optimum CoMFA and CoMSIA models present proper reliabilities and strong predictive abilities (with Q(2) = 0. 64, R(2)ncv = 0.93, R(2)pred = 0.80 and Q(2) = 0. 65, R(2)ncv = 0.98, R(2)pred = 0.86, respectively). In addition, there was good concordance between these models, molecular docking, and MD results. Moreover, the MM-PBSA energy analysis reveals that the major driving force for ligand binding is the polar solvation contribution term. Hopefully, these models and the obtained findings could offer better understanding of the interaction mechanism of BVDV inhibitors as well as benefit the new discovery of more potent BVDV inhibitors.

In vitro inhibition of the bovine viral diarrhoea virus by the essential oil of Ocimum basilicum (basil) and monoterpenes.

The bovine viral diarrhoea virus (BVDV) is suggested as a model for antiviral studies of the hepatitis C virus (HCV). The antiviral activity of the essential oil of Ocimum basilicum and the monoterpenes camphor, thymol and 1,8-cineole against BVDV was investigated. The cytotoxicities of the compounds were measured by the MTT (3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide) test, and the antiviral activities were tested by the plaque reduction assay. The oil or compounds were added to the assay in three different time points: a) pre-treatment of the virus (virucidal assay); b) pre-treatment of the cells; or c) post-treatment of the cells (after virus inoculation). The percentage of plaques inhibition for each compound was determined based on the number of plaques in the viral control. The results were expressed by CC50 (50% cytotoxic concentration), IC50 (inhibitory concentration for 50% of plaques) and SI (selectivity index = CC50/IC50). Camphor (CC50 = 4420.12 µg mL(-1)) and 1,8-cineole (CC50 = 2996.10 µg mL(-1)) showed the lowest cytotoxicities and the best antiviral activities (camphor SI = 13.88 and 1,8-cineol SI = 9.05) in the virucidal assay. The higher activities achieved by the monoterpenes in the virucidal assay suggest that these compounds act directly on the viral particle.

Recombinant pestivirus E2 glycoproteins prevent viral attachment to permissive and non permissive cells with different efficiency.

Bovine viral diarrhoea virus (BVDV) is an economically important animal pathogen, which like other pestiviruses has similar molecular biological features to hepaciviruses, including human Hepatitis C virus. The pestivirus E2 glycoproteins are the major target for virus-neutralising antibodies, as well as playing a role in receptor binding and host range restriction. In this study, recombinant E2 glycoproteins (rE2) derived from three different pestivirus species were examined for their inhibitory effects on pestivirus infectivity in cell culture. Histidine-tagged rE2 glycoproteins of BVDV type 2 strain 178003, BVDV type 1 strain Oregon C24V and CSFV strain Alfort 187 were produced in Spodoptera frugiperda insect cells and purified under native conditions. The ability of rE2 glycoprotein to inhibit the infection of permissive cells by both homologous and heterologous virus was compared, revealing that the inhibitory effects of rE2 glycoproteins correlated with the predicted similarity of the E2 structures in the recombinant protein and the test virus. This result suggests that the sequence and structure of E2 are likely to be involved in the host specificity of pestiviruses at their point of uptake into cells.

In vitro inhibition of the bovine viral diarrhoea virus by the essential oil of Ocimum basilicum (basil) and monoterpenes

The bovine viral diarrhoea virus (BVDV) is suggested as a model for antiviral studies of the hepatitis C virus (HCV). The antiviral activity of the essential oil of Ocimum basilicum and the monoterpenes camphor, thymol and 1,8-cineole against BVDV was investigated. The cytotoxicities of the compounds were measured by the MTT (3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide) test, and the antiviral activities were tested by the plaque reduction assay. The oil or compounds were added to the assay in three different time points: a) pre-treatment of the virus (virucidal assay); b) pre-treatment of the cells; or c) post-treatment of the cells (after virus inoculation). The percentage of plaques inhibition for each compound was determined based on the number of plaques in the viral control. The results were expressed by CC50 (50% cytotoxic concentration), IC50 (inhibitory concentration for 50% of plaques) and SI (selectivity index = CC50/IC50). Camphor (CC50 = 4420.12 µgmL-1) and 1,8-cineole (CC50 = 2996.10 µgmL-1) showed the lowest cytotoxicities and the best antiviral activities (camphor SI = 13.88 and 1,8-cineol SI = 9.05) in the virucidal assay. The higher activities achieved by the monoterpenes in the virucidal assay suggest that these compounds act directly on the viral particle.

Discovery of a novel, monocationic, small-molecule inhibitor of scrapie prion accumulation in cultured sheep microglia and Rov cells.

Prion diseases, including sheep scrapie, are neurodegenerative diseases with the fundamental pathogenesis involving conversion of normal cellular prion protein (PrP(C)) to disease-associated prion protein (PrP(Sc)). Chemical inhibition of prion accumulation is widely investigated, often using rodent-adapted prion cell culture models. Using a PrP(Sc)-specific ELISA we discovered a monocationic phenyl-furan-benzimidazole (DB772), which has previously demonstrated anti-pestiviral activity and represents a chemical category previously untested for anti-prion activity, that inhibited PrP(Sc) accumulation and prion infectivity in primary sheep microglial cell cultures (PRNP 136VV/154RR/171QQ) and Rov9 cultures (VRQ-ovinized RK13 cells). We investigated potential mechanisms of this anti-prion activity by evaluating PrP(C) expression with quantitative RT-PCR and PrP ELISA, comparing the concentration-dependent anti-prion and anti-pestiviral effects of DB772, and determining the selectivity index. Results demonstrate at least an approximate two-log inhibition of PrP(Sc) accumulation in the two cell systems and confirmed that the inhibition of PrP(Sc) accumulation correlates with inhibition of prion infectivity. PRNP transcripts and total PrP protein concentrations within cell lysates were not decreased; thus, decreased PrP(C) expression is not the mechanism of PrP(Sc) inhibition. PrP(Sc) accumulation was multiple logs more resistant than pestivirus to DB772, suggesting that the anti-PrP(Sc) activity was independent of anti-pestivirus activity. The anti-PrP(Sc) selectivity index in cell culture was approximately 4.6 in microglia and 5.5 in Rov9 cells. The results describe a new chemical category that inhibits ovine PrP(Sc) accumulation in primary sheep microglia and Rov9 cells, and can be used for future studies into the treatment and mechanism of prion diseases.

Efficacy of an antiviral compound to inhibit replication of multiple pestivirus species.

Pestiviruses are economically important pathogens of livestock. An aromatic cationic compound (DB772) has previously been shown to inhibit bovine viral diarrhea virus (BVDV) type 1 in vitro at concentrations lacking cytotoxic side effects. The aim of this study was to determine the scope of antiviral activity of DB772 among diverse pestiviruses. Isolates of BVDV 2, border disease virus (BDV), HoBi virus, pronghorn virus and Bungowannah virus were tested for in vitro susceptibility to DB772 by incubating infected cells in medium containing 0, 0.006, 0.01, 0.02, 0.05, 0.1, 0.2, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5 or 25µM DB772. The samples were assayed for the presence of virus by virus isolation and titration (BDV and BVDV 2) or PCR (HoBi, pronghorn and Bungowannah viruses). Cytotoxicity of the compound was assayed for each cell type. Complete inhibition of BVDV 2, BDV, and Pronghorn virus was detected when DB772 was included in the culture media at concentrations of 0.20µM and higher. In two of three tests, a concentration of 0.05µM DB772 was sufficient to completely inhibit HoBi virus replication. Bungowannah virus was completely inhibited at a concentration of 0.01µM DB772. Thus, DB772 effectively inhibits all pestiviruses studied at concentrations >0.20µM. As cytotoxicity is not evident at these concentrations, this antiviral compound potentially represents an effective preventative or therapeutic for diverse pestiviruses.

Synthesis of the first example of a nucleoside analogue bearing a 5'-deoxy-beta-D-allo-septanose as a seven-membered ring sugar moiety.

The first example of a nucleoside analogue bearing a 5'-deoxy-beta-D-allo-septanose as a seven-membered ring sugar moiety, namely 9-(5-deoxy-beta-D-allo-septanosyl)-adenine, is reported. This compound was synthesized in 14 steps from the commercially available D-glycero-D-gulo-1,4-lactone. When evaluated in cell culture experiments against a broad range of viruses, it did not exhibit any significant antiviral effect or cytotoxicity.

Antiviral activity of benzimidazole derivatives. I. Antiviral activity of 1-substituted-2-[(benzotriazol-1/2-yl)methyl]benzimidazoles.

Forty-three 2-[(benzotriazol-1/2-yl)methyl]benzimidazoles, bearing either linear (dialkylamino)alkyl- or bulkier (quinolizidin-1-yl)alkyl moieties at position 1, were evaluated in cell-based assays for cytotoxicity and antiviral activity against viruses representative of two of the three genera of the Flaviviridae family, i.e. Flaviviruses (Yellow Fever Virus (YFV)) and Pestiviruses (Bovine Viral Diarrhoea Virus (BVDV)), as Hepaciviruses can hardly be used in routine cell-based assays. Compounds were also tested against representatives of other virus families. Among ssRNA+ viruses were a retrovirus (Human Immunodeficiency Virus type 1 (HIV-1)), two picornaviruses (Coxsackie Virus type B2 (CVB2), and Poliovirus type-1, Sabin strain (Sb-1)); among ssRNA- viruses were a Paramyxoviridae (Respiratory Syncytial Virus (RSV)) and a Rhabdoviridae (Vesicular Stomatitis Virus (VSV)) representative. Among double-stranded RNA (dsRNA) viruses was a Reoviridae representative (Reo-1). Two representatives of DNA virus families were also included: Herpes Simplex type 1, (HSV-1; Herpesviridae) and Vaccinia Virus (VV; Poxviridae). Most compounds exhibited potent activity against RSV, with EC(50) values as low as 20 nM. Moreover, some compounds, in particular when bearing a (quinolizidin-1-yl)alkyl residue, were also moderately active against BVDV, YFV, and CVB2.

Virucidal activity of ortho-phthalaldehyde solutions against hepatitis B and C viruses.

Ortho-phthalaldehyde (OPA), a high-level disinfectant alternative to glutaraldehyde, was tested for efficacy against human hepatitis B virus (HBV) and hepatitis C virus (HCV) using surrogate animal viruses. HBV and HCV are the most prevalent human bloodborne viruses but have not yet been propagated in the laboratory. The surrogate viruses, duck hepatitis B virus (DHBV) and bovine viral diarrhea virus (BVDV), were used to assess the virucidal efficacy of OPA on HBV and HCV, respectively. After a timed exposure to the test disinfectant, the surrogate virus dried on a hard surface was neutralized and assayed to detect viable viruses using appropriate cell lines. A greater than 4-log(10) reduction in virus titer was demonstrated using dilute OPA solutions against dried DHBV and BVDV after 5 minutes of exposure at 20 degrees C. OPA was shown to be efficacious against surrogate viruses for human hepatitis B and hepatitis C virus. This is the first time that OPA efficacy has been demonstrated for HBV and HCV.

The imidazopyrrolopyridine analogue AG110 is a novel, highly selective inhibitor of pestiviruses that targets the viral RNA-dependent RNA polymerase at a hot spot for inhibition of viral replication.

Ethyl 2-methylimidazo[1,2-a]pyrrolo[2,3-c]pyridin-8-carboxylate (AG110) was identified as a potent inhibitor of pestivirus replication. The 50% effective concentration values for inhibition of bovine viral diarrhea virus (BVDV)-induced cytopathic effect, viral RNA synthesis, and production of infectious virus were 1.2 +/- 0.5 microM, 5 +/- 1 microM, and 2.3 +/- 0.3 microM, respectively. AG110 proved inactive against the hepatitis C virus and a flavivirus. AG110 inhibits BVDV replication at a time point that coincides with the onset of intracellular viral RNA synthesis. Drug-resistant mutants carry the E291G mutation in the viral RNA-dependent RNA polymerase (RdRp). AG110-resistant virus is cross-resistant to the cyclic urea compound 1453 which also selects for the E291G drug resistance mutation. Moreover, BVDV that carries the F224S mutation (because of resistance to the imidazopyridine 5-[(4-bromophenyl)methyl]-2-phenyl-5H-imidazo[4,5-c]pyridine [BPIP]and VP32947) is also resistant to AG110. AG110 did not inhibit the in vitro activity of recombinant BVDV RdRp but inhibited the activity of BVDV replication complexes (RCs). Molecular modeling revealed that E291 is located in a small cavity near the tip of the finger domain of the RdRp about 7 A away from F224. Docking of AG110 in the crystal structure of the BVDV RdRp revealed several potential contacts including with Y257. The E291G mutation might enable the free rotation of Y257, which might in turn destabilize the backbone of the loop formed by residues 223 to 226, rendering more mobility to F224 and, hence, reducing the affinity for BPIP and VP32947. It is concluded that a single drug-binding pocket exists within the finger domain region of the BVDV RdRp that consists of two separate but potentially overlapping binding sites rather than two distinct drug-binding pockets.

Antiviral 2,5-disubstituted imidazo[4,5-c]pyridines: from anti-pestivirus to anti-hepatitis C virus activity.

A novel class of inhibitors of the hepatitis C virus [substituted 2-(2-fluorophenyl)-5H-imidazo[4,5-c]pyridines] is described. Introduction of a fluorine in position 2 of the 2-phenyl substituent of the lead anti-pestivirus compound 1 (5-[(4-bromophenyl)methyl]-2-phenyl-5H-imidazo[4,5-c]pyridine) resulted in an analogue with selective activity against HCV in the subgenomic replicon system.

Brefeldin A inhibits pestivirus release from infected cells, without affecting its assembly and infectivity.

The enveloped bovine viral diarrhea virus (BVDV) is a member of the Pestivirus genus within the Flaviviridae family. While considerable information has been gathered on virus entry into the host cell, genome structure and protein function, little is known about pestivirus morphogenesis and release from cells. Here, we analyzed the intracellular localization, N-glycan processing and secretion of BVDV using brefeldin A (BFA), which blocks protein export from the endoplasmic reticulum (ER) and causes disruption of the Golgi complex with subsequent fusion of its cis and medial cisternae with the ER. BFA treatment of infected cells resulted in complete inhibition of BVDV secretion and increased co-localization of the envelope glycoproteins with the cis-Golgi marker GM 130. Processing of the N-linked glycans was affected by BFA, however, virus assembly was not perturbed and intracellular virions were fully infectious, suggesting that trafficking beyond the cis-Golgi is not a prerequisite for pestivirus infectivity.

A novel, highly selective inhibitor of pestivirus replication that targets the viral RNA-dependent RNA polymerase.

We report on the highly potent and selective antipestivirus activity of 5-[(4-bromophenyl)methyl]-2-phenyl-5H-imidazo[4,5-c]pyridine (BPIP). The 50% effective concentration (EC50) for inhibition of bovine viral diarrhea virus (BVDV)-induced cytopathic effect formation was 0.04 +/- 0.01 microM. Comparable reduction of viral RNA synthesis (EC50 = 0.12 +/- 0.02 microM) and production of infectious virus (EC50= 0.074 +/- 0.003 microM) were observed. The selectivity index (ratio of 50% cytostatic concentration/EC50) of BPIP was approximately 2,000. BPIP was inactive against the hepatitis C virus subgenomic replicon and yellow fever virus but demonstrated weak activity against GB virus. Drug-resistant mutants were at least 300-fold less susceptible to BPIP than wild-type virus; showed cross-resistance to N-propyl-N-[2-(2H-1,2,4-triazino[5,6-b]indol-3-ylthio)ethyl]-1-propanamine (VP32947), and carried the F224S mutation in the viral RNA-dependent RNA polymerase (RdRp). When the F224S mutation was introduced into an infectious clone, the drug-resistant phenotype was obtained. BPIP did not inhibit the in vitro activity of recombinant BVDV RdRp, but did inhibit the activity of replication complexes (RCs). Computational docking revealed that F224 is located at the top of the finger domain of the polymerase. Docking of BPIP in the crystal structure of the BVDV RdRp revealed aromatic ring stacking, some hydrophobic contacts, and a hydrogen bond. Since two structurally unrelated compounds, i.e., BPIP and VP32947, target the same region of the BVDV RdRp, this position may be expected to be critical in the functioning of the polymerase or assembly of the RC. The potential of BPIP for the treatment of pestivirus and hepacivirus infections is discussed.

Mechanism of action of a pestivirus antiviral compound.

We report here the discovery of a small molecule inhibitor of pestivirus replication. The compound, designated VP32947, inhibits the replication of bovine viral diarrhea virus (BVDV) in cell culture at a 50% inhibitory concentration of approximately 20 nM. VP32947 inhibits both cytopathic and noncytopathic pestiviruses, including isolates of BVDV-1, BVDV-2, border disease virus, and classical swine fever virus. However, the compound shows no activity against viruses from unrelated virus groups. Time of drug addition studies indicated that VP32947 acts after virus adsorption and penetration and before virus assembly and release. Analysis of viral macromolecular synthesis showed VP32947 had no effect on viral protein synthesis or polyprotein processing but did inhibit viral RNA synthesis. To identify the molecular target of VP32947, we isolated drug-resistant (DR) variants of BVDV-1 in cell culture. Sequence analysis of the complete genomic RNA of two DR variants revealed a single common amino acid change located within the coding region of the NS5B protein, the viral RNA-dependent RNA polymerase. When this single amino acid change was introduced into an infectious clone of drug-sensitive wild-type (WT) BVDV-1, replication of the resulting virus was resistant to VP32947. The RNA-dependent RNA polymerase activity of the NS5B proteins derived from WT and DR viruses expressed and purified from recombinant baculovirus-infected insect cells confirmed the drug sensitivity of the WT enzyme and the drug resistance of the DR enzyme. This work formally validates NS5B as a target for antiviral drug discovery and development. The utility of VP32947 and similar compounds for the control of pestivirus diseases, and for hepatitis C virus drug discovery efforts, is discussed.

A gloverin-like antibacterial protein is synthesized in Helicoverpa armigera following bacterial challenge.

A bacteria inducible antibacterial protein, P2, was isolated from the old world bollworm Helicoverpa armigera. Fifth-instar larvae were injected with live Escherichia coli NCTC 8196. P2 was isolated by HPLC using reversed-phase and size-exclusion columns. In addition, P2 was isolated by an alternative method of sequential cation-exchange and reversed-phase HPLC. The structure of P2 was determined by N-terminal Edman degradation and mass spectrometry. P2 had similar mass (14.1 kDa) structure and activity to gloverin, an inducible glycine-rich antibacterial protein isolated from Hyalophora gloveri [Axén, A.; Carlsson, A.; Engström, A.; Bennich, H. Eur. J. Biochem. 247:614-619; 1997]. At the N-terminus P2 had approximately 60% identity with gloverin. P2 is basic, heat stable, and displayed rapid antibacterial action. P2 was active against the Gram-negative bacteria tested and was inactive against the Gram-positive bacteria, Candida albicans, a bovine turbinate cell line, and pestivirus.