9-Aminoacridine-based agents impair the bovine viral diarrhea virus (BVDV) replication targeting the RNA-dependent RNA polymerase (RdRp).

Bovine viral diarrhea virus (BVDV) infection is still a plague that causes important livestock pandemics. Despite the availability of vaccines against BVDV, and the implementation of massive eradication or control programs, this virus still constitutes a serious agronomic burden. Therefore, the alternative approach to combat Pestivirus infections, based on the development of antiviral agents that specifically inhibit the replication of these viruses, is of preeminent actuality and importance. Capitalizing from a long-standing experience in antiviral drug design and development, in this work we present and characterize a series of small molecules based on the 9-aminoacridine scaffold that exhibit potent anti-BVDV activity coupled with low cytotoxicity. The relevant viral protein target - the RNA-dependent RNA polymerase - the binding mode, and the mechanism of action of these new antivirals have been determined by a combination of in vitro (i.e., enzymatic inhibition, isothermal titration calorimetry and site-directed mutagenesis assays) and computational experiments. The overall results obtained confirm that these acridine-based derivatives are promising compounds in the treatment of BVDV infections and, based on the reported structure-activity relationship, can be selected as a starting point for the design of a new generation of improved, safe and selective anti-BVDV agents.

Molecular mechanism of viral resistance to a potent non-nucleoside inhibitor unveiled by molecular simulations.

Recently, we reported on a potent benzimidazole derivative (227G) that inhibits the growth of the bovine viral diarrhea virus (BVDV) in cell-based and enzyme assays at nanomolar concentrations. The target of 227G is the viral RNA-dependent RNA polymerase (RdRp), and the I261M mutation located in motif I of the RdRp finger domain was found to induce drug resistance. Here we propose a molecular mechanism for the retained functionality of the enzyme in the presence of the inhibitor, on the basis of a thorough computational study of the apo and holo forms of the BVDV RdRp either in the wild type (wt) or in the form carrying the I261M mutation. Our study shows that although the mutation affects to some extent the structure of the apoenzyme, the functional dynamics of the protein appear to be largely maintained, which is consistent with the retained functionality of this natural mutant. Despite the binding site of 227G not collapsing or undergoing drastic structural changes upon introduction of the I261M substitution, these alterations reflect crucially on the binding mode of 227G, which is significantly different from that found in wt RdRp. In particular, while in the wt system the four loops lining the template entrance site embrace 227G and close the template passageway, in the I261M variant the template entrance is only marginally occluded, allowing in principle the translocation of the template to the interior of the enzyme. In addition, the mutated enzyme in the presence of 227G retains several characteristics of the wt apoprotein. Our work provides an original molecular picture of a resistance mechanism that is consistent with published experimental data.

Residues Arg283, Arg285, and Ile287 in the nucleotide binding pocket of bovine viral diarrhea virus NS5B RNA polymerase affect catalysis and fidelity.

Residues Arg283, Arg285, and Ile287 are highly conserved amino acids in bovine viral diarrhea virus RNA polymerase (BVDV RdRp) and RdRps from related positive-strand RNA viruses. This motif is an important part of the binding pocket for the nascent RNA base pair during initiation and elongation. We found that replacement of the arginines with alanines or more conserved lysines or replacement of isoleucine with alanine or valine alters the ability of the mutant RdRps to incorporate ribonucleotides efficiently. The reduced RdRp activity stems from both decreased ribonucleotide binding and decreased catalytic efficiency in both primer-dependent and de novo initiation, as shown by kinetic studies. In line with other studies on flaviviral RdRps, our data suggest that Arg283 and Ile287 may be implicated in ribonucleotide binding and positioning of the template base in the active site. Arg285 appears to be involved directly in the selection of cognate nucleotide. The findings for Arg285 and Ile287 mutants also agree with similar data from picornavirus RdRps.

Different molecular mechanisms of inhibition of bovine viral diarrhea virus and hepatitis C virus RNA-dependent RNA polymerases by a novel benzimidazole.

The virus-encoded RNA-dependent RNA polymerase (RdRp) has emerged as a primary target in the search for selective inhibitors of Flaviviridae. Recently, we reported on the selective inhibition, in cell-based assays, of both BVDV (EC50 = 0.80 ± 0.06 µM) and HCV (EC50 = 1.11 ± 0.15 µM) by 2-{1-[2-(2,4-dimethoxyphenyl)-1H-benzimidazol-5-yl]ethylidene}hydrazinecarbothioamide (227G). Here we show that, in enzyme assays with recombinant enzymes, 227G inhibits, in a dose-dependent manner, the RdRp of both BVDV (IC50 = 0.0020 ± 0.0004 µM) and HCV (IC50 = 0.40 ± 0.04 µM). Furthermore, we report on the selection and molecular analysis of a BVDV-resistant mutant, characterized by the presence of the I261M mutation. By applying a multilevel computational approach, we identified different 227G binding sites on the two RdRps. They were further validated by the good agreement between the calculated affinities and those extrapolated from IC50 values. Our findings suggest different molecular mechanisms of inhibition of the HCV and BVDV RdRps by 227G and indicate the importance of understanding ligand-enzyme interactions at the molecular level for the rational design of new and more potent leads.

Pharmacophore modeling, resistant mutant isolation, docking, and MM-PBSA analysis: Combined experimental/computer-assisted approaches to identify new inhibitors of the bovine viral diarrhea virus (BVDV).

Starting from a series of our new 2-phenylbenzimidazole derivatives, shown to be selectively and potently active against the bovine viral diarrhea virus (BVDV), we developed a hierarchical combined experimental/molecular modeling strategy to explore the drug leads for the BVDV RNA-dependent RNA-polymerase. Accordingly, a successful 3D pharmacophore model was developed, characterized by distinct chemical features that may be responsible for the activity of the inhibitors. BVDV mutants resistant to lead compounds in our series were then isolated, and the mutant residues on the viral molecular target, the RNA-dependent RNA-polymerase, were identified. Docking procedures upon pharmacophoric constraints and mutational data were carried out, and the binding affinity of all active compounds for the RdRp were estimated. Given the excellent agreement between in silico and in vitro data, this procedure is currently being employed in the design a new series of more selective and potent BVDV inhibitors.

Synergistic experimental/computational studies on arylazoenamine derivatives that target the bovine viral diarrhea virus RNA-dependent RNA polymerase.

Starting from a series of arylazoenamine derivatives, shown to be selectively and potently active against the bovine viral diarrhea virus (BVDV), we developed a hierarchical combined experimental/molecular modeling strategy to explore the drug leads for the BVDV RNA-dependent RNA polymerase. Accordingly, BVDV mutants resistant to lead compounds in our series were isolated, and the mutant residues on the viral molecular target, the RNA-dependent RNA polymerase, were identified. Docking procedures upon previously identified pharmacophoric constraints and actual mutational data were carried out, and the binding affinity of all active compounds for the RdRp was estimated. Given the excellent agreement between in silico and in vitro data, this procedure is currently being employed in the design a new series of more selective and potent BVDV inhibitors.

Conformational Characterization of Linker Revealed the Mechanism of Cavity Formation by 227G in BVDV RDRP.

RNA-dependent RNA polymerase (RdRp) is a relevant antiviral drug target. We investigated a potent benzimidazole inhibitor (227G; IC50 = 0.002 µM) against bovine viral diarrhea virus (BVDV) RdRp; however, its inhibition action was completely impaired in the presence of a resistant mutation, I261M. The binding of 227G in mutant RdRp affected the binding site loop conformations (especially Linker) that increased the volume of the binding site. It was also observed that the innate Linker's flexibility was retained, which was otherwise completely frozen in the wild-type complex. The functional role of Linker was hypothesized that it is a multidocking site for RNA template, inhibitors, and the other proteins involved in replication complex formation. The binding phenomenon requires significant molecular flexibility and the large-amplitude conformational dynamics of Linker, which is currently unknown. We observed a bidirectional "hinge"-like motion of Linker from crystal position, indicating its pronounced flexible behavior. This study underscores the importance of Linker's flexibility in the functionality of BVDV RdRp and proposes the template entrance site for selective anti-BVDV drug discovery.

The structure of bovine viral diarrhea virus RNA-dependent RNA polymerase and its amino-terminal domain.

Viral RNA-dependent RNA polymerases (RdRp) differ from DNA-dependent RNA polymerases, DNA-dependent DNA polymerases, and reverse transcriptases in that RdRps contain "fingertips" consisting of several polypeptide strands in the fingers domain interacting with the thumb domain. The crystal structure of bovine viral diarrhea virus (BVDV) RdRp containing an Asn438 duplication shows that the "N-terminal domain," which occurs only in pestiviruses such as BVDV, interacts with the polymerase component of the same polypeptide chain. This contrasts with the domain swapping observed in the previously determined structure of the BVDV NADL strain RdRp. By comparison with the NADL structure and through the use of biochemical data, it is possible that the N-terminal domain, in conjunction with the fingertips, is required to bind and assist the translocation of the RNA template. The partial disorder of the loop containing the additional Asn438 residue may explain the low replication rate of the recombinant compared with the wild-type virus.

Homodimerisation-independent cleavage of dsRNA by a pestiviral nicking endoribonuclease.

The glycoprotein Erns plays a central role in the biology of the pestivirus bovine viral diarrhea virus (BVDV). This soluble endonuclease mediates the escape from an interferon (IFN) response in the infected fetus, thereby permitting the establishment of persistent infection. Viral single-stranded (ss) and double-stranded (ds) RNA act as potent IFN inducing signals and we previously showed that Erns efficiently cleaves these substrates, thereby inhibiting an IFN response that is crucial for successful fetal infection. Considering that a large variety of RNases and DNases require dimerisation to cleave double-stranded substrates, the activity of Erns against dsRNA was postulated to depend on homodimer formation mediated by disulfide bonds involving residue Cys171. Here, we show that monomeric Erns is equally able to cleave dsRNA and to inhibit dsRNA-induced IFN synthesis as the wild-type form. Furthermore, both forms were able to degrade RNA within a DNA/RNA- as well as within a methylated RNA/RNA-hybrid, with the DNA and the methylated RNA strand being resistant to degradation. These results support our model that Erns acts as 'nicking endoribonuclease' degrading ssRNA within double-stranded substrates. This efficiently prevents the activation of IFN and helps to maintain a state of innate immunotolerance in persistently infected animals.

SAR and mode of action of novel non-nucleoside inhibitors of hepatitis C NS5b RNA polymerase.

Novel non-nucleoside inhibitors of the HCV RNA polymerase (NS5b) with sub-micromolar biochemical potency have been identified which are selective for the inhibition of HCV NS5b over other polymerases. The structures of the complexes formed between several of these inhibitors and HCV NS5b were determined by X-ray crystallography, and the inhibitors were found to bind in an allosteric binding site separate from the active site. Structure-activity relationships and structural studies have identified the mechanism of action for compounds in this series, several of which possess drug-like properties, as unique, reversible, covalent inhibitors of HCV NS5b.

A combined in silico/in vitro approach unveils common molecular requirements for efficient BVDV RdRp binding of linear aromatic N-polycyclic systems.

In this work, we present and discuss a comprehensive set of both newly and previously synthesized compounds belonging to 5 distinct molecular classes of linear aromatic N-polycyclic systems that efficiently inhibits bovine viral diarrhea virus (BVDV) infection. A coupled in silico/in vitro investigation was employed to formulate a molecular rationale explaining the notable affinity of all molecules to BVDV RNA dependent RNA polymerase (RdRp) NS5B. We initially developed a three-dimensional common-feature pharmacophore model according to which two hydrogen bond acceptors and one hydrophobic aromatic feature are shared by all molecular series in binding the viral polymerase. The pharmacophoric information was used to retrieve a putative binding site on the surface of the BVDV RdRp and to guide compound docking within the protein binding site. The affinity of all compounds towards the enzyme was scored via molecular dynamics-based simulations, showing high correlation with in vitro EC50 data. The determination of the interaction spectra of the protein residues involved in inhibitor binding highlighted amino acids R295 and Y674 as the two fundamental H-bond donors, while two hydrophobic cavities HC1 (residues A221, I261, I287, and Y289) and HC2 (residues V216, Y303, V306, K307, P408, and A412) fulfill the third pharmacophoric requirement. Three RdRp (K263, R295 and Y674) residues critical for drug binding were selected and mutagenized, both in silico and in vitro, into alanine, and the affinity of a set of selected compounds towards the mutant RdRp isoforms was determined accordingly. The agreement between predicted and experimental data confirmed the proposed common molecular rationale shared by molecules characterized by different chemical scaffolds in binding to the BVDV RdRp, ultimately yielding compound 6b (EC50 = 0.3 µM; IC50 = 0.48 µM) as a new, potent inhibitor of this Pestivirus.

3-(imidazo[1,2-a:5,4-b']dipyridin-2-yl)aniline inhibits pestivirus replication by targeting a hot spot drug binding pocket in the RNA-dependent RNA polymerase.

The compound 3-(imidazo[1,2-a:5,4-b']dipyridin-2-yl)aniline (CF02334) was identified as a selective inhibitor of the cytopathic effect (CPE) caused by bovine viral diarrhea virus (BVDV) in a virus-cell-based assay. The EC50-values for inhibition of CPE, viral RNA synthesis and the production of infectious virus progeny were 13.0 ± 0.6 µM, 2.6 ± 0.9 µM and 17.8 ± 0.6 µM, respectively. CF02334 was found to be inactive in the hepatitis C subgenomic replicon system. CF02334-resistant BVDV was obtained and was found to carry the N264D mutation in the viral RNA-dependent RNA polymerase (RdRp). Molecular modeling revealed that N264D is located in a small cavity near the fingertip domain of the pestivirus polymerase. CF02334-resistant BVDV was proven to be cross-resistant to BPIP, AG110 and LZ37, inhibitors that have previously been described to target the same region of the BVDV RdRp. CF02334 did not inhibit the in vitro activity of recombinant BVDV RdRp, but did inhibit the activity of BVDV replication complexes. Taken together, these observations indicate that CF02334 likely interacts with the fingertip of the pestivirus RdRp at the same position as BPIP, AG110 and LZ37, which marks this region of the viral polymerase as a "hot spot" for inhibition of pestivirus replication.

Design, expression, and purification of a Flaviviridae polymerase using a high-throughput approach to facilitate crystal structure determination.

Bovine viral diarrhea virus (BVDV) nonstructural protein 5B is an RNA-dependent RNA polymerase, essential for viral replication. Initial attempts to crystallize a soluble form of the 695-residue BVDV polymerase did not produce any crystals. Limited proteolysis, homology modeling, and mutagenesis data were used to aid the design of polymerase constructs that might crystallize more readily. Limited proteolysis of the polymerase with trypsin identified a domain boundary within the protein. Homology modeling of the polymerase, based on the structure of hepatitis C virus polymerase, indicated that the two polymerases share a 23% identical "core," although overall sequence identity is low. Eighty-four expression clones of the BVDV polymerase were designed by fine-sampling of chain termini at the boundaries of domain and of active truncated forms of the polymerase. The resulting constructs were expressed in Escherichia coli and purified using high-throughput methods. Soluble truncated proteins were subjected to crystallization trials in a 96-well format, and two of these proteins were successfully crystallized.

Selection of a thiazole urea-resistant variant of bovine viral diarrhoea virus that maps to the RNA-dependent RNA polymerase.

By passing wild type bovine viral diarrhoea virus (BVDV) in increasing concentrations of DPC-A69280-29, a thiazole urea class compound that inhibits BVDV replication, we were able to select several variants of BVDV that exhibited decreased susceptibility to this compound. When the non-structural genes of these variants were sequenced and compared with wild type, only one change was common to all the variants that also exhibited resistance to DPC-A69280-29 (>10-fold increase in IC50). This change was a T-to-A transversion at position 11198 of the BVDV genome, which would cause a predicted substitution of isoleucine for phenylalanine at amino acid 78 of the RNA-dependent RNA polymerase (RdRp). This substitution would occur in a region of the BVDV RdRp which has been proposed to be important for the formation of the RdRp homodimer that is essential for the activity of the enzyme. However, since DPC-69280-29 inhibits BVDV replication by interfering with the initiation of viral RNA synthesis, we discuss the possibility that this region of the BVDV RdRp also may play a role in the initiation process. Furthermore, since this region is located fairly close to the template RNA, we also propose that the role it plays may involve either template selection, stabilization or processivity.

Inhibition of the bovine viral diarrhoea virus NS3 serine protease by a boron-modified peptidyl mimetic of its natural substrate.

Bovine viral diarrhoea virus (BVDV) is closely related to hepatitis C virus (HCV), and has been used as a surrogate virus in drug development for HCV infection. Similar to HCV, BVDV-encoded NS3 serine proteinase is responsible for multiple cleavages in the viral polyprotein, generating mature NS4A, NS4B, NS5A and NS5B proteins. NS3-dependent cleavage sites of BVDV contain a strictly conserved leucine at P1, and either serine or alanine at P1'. The full length BVDV NS3/4A serine protease has been cloned and expressed in bacterial cells. The enzyme has been purified from the soluble portion of Escherichia coli via a two-step purification procedure employing chromatography on heparin resin and gel filtration. The protease activity was characterized using in vitro translated BVDV NS4A/B and NS5A/B polyprotein substrates. A boronic acid analogue of the BVDV NS4A/NS4B cleavage site was synthesized and shown to be an efficient inhibitor of the NS3 serine protease in vitro. The compound, designated DPC-AB9144-00, inhibited approximately 75% of the NS3/4 activity at 10 microM with the NS4A/B substrate. However, no antiviral activity was detected with DPC-AB9144-00 in BVDV-infected Madin-Darby bovine kidney cells at concentrations as great as 90 pM, suggesting permeability or that other cellular-derived limitations were present.

Action of celgosivir (6 O-butanoyl castanospermine) against the pestivirus BVDV: implications for the treatment of hepatitis C.

Alpha-glucosidase I inhibitors have been shown to inhibit the replication of a broad range of enveloped viruses by preventing the correct folding of their envelope glycoproteins. This study assesses the potential of 6 O-butanoyl castanospermine (celgosivir) as a treatment for hepatitis C virus (HCV). In the absence of an adequate culture system for HCV, the closely related virus, bovine viral diarrhoea virus (BVDV), was used as a surrogate model. Using both a plaque assay and a cytopathic effect assay, celgosivir (IC50 16 and 47 microM respectively) was shown to be more potent than N-nonyl DNJ (105 and 74 microM), castanospermine (110 and 367 microM) and N-butyl DNJ (> 250 and 550 microM). Of the alpha-glucosidase inhibitors tested, only N-nonyl DNJ showed evidence of toxicity (CC50 > or = 120 microM). Two-way combinations of interferon-alpha, ribavirin and either celgosivir or castanospermine demonstrated that each could enhance the antiviral efficacy of the others, either additively or synergistically. The observation that the number of viral genomes released from BVDV-infected cells was inhibited by either castanospermine or celgosivir in parallel with the number of infectious units was taken as confirmation that these alpha-glucosidase I inhibitors block the production or release of flavivirus particles.

Pestiviral E(rns) blocks TLR-3-dependent IFN synthesis by LL37 complexed RNA.

The ribonuclease activity of the soluble glycoprotein E(rns) of pestiviruses represents a unique mechanism to circumvent the host's innate immune system by blocking interferon type-I synthesis in response to extracellularly added single- (ss) and double-stranded (ds) RNA. However, the reason why pestiviruses encode a ribonuclease in addition to the abundant serum RNases remained elusive. Here, we show that the 5' UTR and NS5B regions of various strains of the RNA genome of the pestivirus bovine viral diarrhea virus (BVDV) are resistant to serum RNases and are potent TLR-3 agonists. Inhibitory activity of E(rns) was restricted to cleavable RNA products, and did not extend to the synthetic TLR-7/8 agonist R-848. RNA complexed with the antimicrobial peptide LL37 was protected from degradation by E(rns)in vitro but was fully inhibited by E(rns) in its ability to induce IFN in cell cultures, suggesting that the viral protein is mainly active in cleaving RNA in an intracellular compartment. We propose that secreted E(rns) represents a potent IFN antagonist, which degrades viral RNA that is resistant to the ubiquitous host RNases in the extracellular space. Thus, the viral RNase prevents its own pathogen-associated molecular pattern (PAMP) to inadvertently activate the IFN response that might break innate immunotolerance required for persistent pestivirus infections.

Stability of the resistance to the thiosemicarbazone derived from 5,6-dimethoxy-1-indanone, a non-nucleoside polymerase inhibitor of bovine viral diarrhea virus.

Bovine viral diarrhea virus (BVDV) is the prototype Pestivirus. BVDV infection is distributed worldwide and causes serious problems for the livestock industry. The thiosemicarbazone of 5,6-dimethoxy-1-indanone (TSC) is a non-nucleoside polymerase inhibitor (NNI) of BVDV. All TSC-resistant BVDV variants (BVDV-TSCr T1-5) present an N264D mutation in the NS5B gene (RdRp) whereas the variant BVDV-TSCr T1 also presents an NS5B A392E mutation. In the present study, we carried out twenty passages of BVDV-TSCr T1-5 in MDBK cells in the absence of TSC to evaluate the stability of the resistance. The viral populations obtained (BVDV R1-5) remained resistant to the antiviral compound and conserved the mutations in NS5B associated with this phenotype. Along the passages, BVDV R2, R3 and R5 presented a delay in the production of cytopathic effect that correlated with a decrease in cell apoptosis and intracellular accumulation of viral RNA. The complete genome sequences that encode for NS2 to NS5B, Npro and Erns were analyzed. Additional mutations were detected in the NS5B of BVDV R1, R3 and R4. In both BVDV R2 and R3, most of the mutations found were localized in NS5A, whereas in BVDV R5, the only mutation fixed was NS5A V177A. These results suggest that mutations in NS5A could alter BVDV cytopathogenicity. In conclusion, the stability of the resistance to TSC may be due to the fixation of different compensatory mutations in each BVDV-TSCr. During their replication in a TSC-free medium, some virus populations presented a kind of interaction with the host cell that resembled a persistent infection: decreased cytopathogenicity and viral genome synthesis. This is the first report on the stability of antiviral resistance and on the evolution of NNI-resistant BVDV variants. The results obtained for BVDV-TSCr could also be applied for other NNIs.

Space constrained homology modelling: the paradigm of the RNA-dependent RNA polymerase of dengue (type II) virus.

Protein structure is more conserved than sequence in nature. In this direction we developed a novel methodology that significantly improves conventional homology modelling when sequence identity is low, by taking into consideration 3D structural features of the template, such as size and shape. Herein, our new homology modelling approach was applied to the homology modelling of the RNA-dependent RNA polymerase (RdRp) of dengue (type II) virus. The RdRp of dengue was chosen due to the low sequence similarity shared between the dengue virus polymerase and the available templates, while purposely avoiding to use the actual X-ray structure that is available for the dengue RdRp. The novel approach takes advantage of 3D space corresponding to protein shape and size by creating a 3D scaffold of the template structure. The dengue polymerase model built by the novel approach exhibited all features of RNA-dependent RNA polymerases and was almost identical to the X-ray structure of the dengue RdRp, as opposed to the model built by conventional homology modelling. Therefore, we propose that the space-aided homology modelling approach can be of a more general use to homology modelling of enzymes sharing low sequence similarity with the template structures.

Evidence of a humoral immune response against the prokaryotic expressed N-terminal autoprotease (N(pro)) protein of bovine viral diarrhoea virus.

Bovine viral diarrhoea virus (BVDV) is an economically important pathogen of cattle and sheep belonging to the genus Pestivirus of the family Flaviviridae. Although the BVDV non-structural N-terminal protease (N(pro)) acts as an interferon antagonist and subverts the host innate immunity, little is known about its immunogenicity. Hence, we expressed a recombinant BVDV N(pro)-His fusion protein (28 kDa) in E. coli and determined the humoral immune response generated by it in rabbits. The antigenicity of the N(pro) protein was confirmed by western blot using anti-BVDV hyperimmune cattle, sheep and goat serum, and anti-N(pro) rabbit serum. When rabbits were immunized with the N(pro) protein, a humoral immune response was evident by 4 weeks and persisted till 10 weeks post immunization as detected by ELISA and western blot. Despite N(pro)-specific antibodies remaining undetectable in 80 serum samples from BVDV-infected sheep and goats, BVDV hyperimmune sera along with some of the field cattle, sheep and goat sera with high BVDV neutralizing antibody titres were found positive for N(pro) antibodies. Our results provide evidence that despite the low immunogenicity of the BVDV N(pro) protein, a humoral immune response is induced in cattle, sheep and goats only with repeated BVDV exposure.