The RNA template channel of the RNA-dependent RNA polymerase as a target for development of antiviral therapy of multiple genera within a virus family.

The genus Enterovirus of the family Picornaviridae contains many important human pathogens (e.g., poliovirus, coxsackievirus, rhinovirus, and enterovirus 71) for which no antiviral drugs are available. The viral RNA-dependent RNA polymerase is an attractive target for antiviral therapy. Nucleoside-based inhibitors have broad-spectrum activity but often exhibit off-target effects. Most non-nucleoside inhibitors (NNIs) target surface cavities, which are structurally more flexible than the nucleotide-binding pocket, and hence have a more narrow spectrum of activity and are more prone to resistance development. Here, we report a novel NNI, GPC-N114 (2,2'-[(4-chloro-1,2-phenylene)bis(oxy)]bis(5-nitro-benzonitrile)) with broad-spectrum activity against enteroviruses and cardioviruses (another genus in the picornavirus family). Surprisingly, coxsackievirus B3 (CVB3) and poliovirus displayed a high genetic barrier to resistance against GPC-N114. By contrast, EMCV, a cardiovirus, rapidly acquired resistance due to mutations in 3Dpol. In vitro polymerase activity assays showed that GPC-N114 i) inhibited the elongation activity of recombinant CVB3 and EMCV 3Dpol, (ii) had reduced activity against EMCV 3Dpol with the resistance mutations, and (iii) was most efficient in inhibiting 3Dpol when added before the RNA template-primer duplex. Elucidation of a crystal structure of the inhibitor bound to CVB3 3Dpol confirmed the RNA-binding channel as the target for GPC-N114. Docking studies of the compound into the crystal structures of the compound-resistant EMCV 3Dpol mutants suggested that the resistant phenotype is due to subtle changes that interfere with the binding of GPC-N114 but not of the RNA template-primer. In conclusion, this study presents the first NNI that targets the RNA template channel of the picornavirus polymerase and identifies a new pocket that can be used for the design of broad-spectrum inhibitors. Moreover, this study provides important new insight into the plasticity of picornavirus polymerases at the template binding site.

Toward antiviral therapy/prophylaxis for rhinovirus-induced exacerbations of chronic obstructive pulmonary disease: challenges, opportunities, and strategies.

Chronic obstructive pulmonary disease (COPD) is a life-threatening lung illness characterized by persistent and progressive airflow limitation. Exacerbations of COPD contribute to the severity of this pathology and accelerate disease progression. To date, pharmacological treatment of both stable COPD patients and patients experiencing exacerbations is mainly symptomatic with bronchodilators and steroids as the mainstay of therapy. Bacteria trigger such exacerbations in a number of cases; hence, antibiotics might be included in the treatment as well. Several respiratory viruses are frequently detected in sputum from patients during COPD exacerbations. These include influenza viruses, respiratory syncytial virus, and, most often, rhinoviruses. In this review, we discuss the potential use of an anti-rhinovirus drug for the treatment and prophylaxis of rhinovirus-induced COPD exacerbations and the path forward toward the development and use of such a drug. Copyright © 2015 John Wiley & Sons, Ltd.

Selective serotonin reuptake inhibitor fluoxetine inhibits replication of human enteroviruses B and D by targeting viral protein 2C.

Although the genus Enterovirus contains many important human pathogens, there is no licensed drug for either the treatment or the prophylaxis of enterovirus infections. We report that fluoxetine (Prozac)--a selective serotonin reuptake inhibitor--inhibits the replication of human enterovirus B (HEV-B) and HEV-D but does not affect the replication of HEV-A and HEV-C or human rhinovirus A or B. We show that fluoxetine interferes with viral RNA replication, and we identified viral protein 2C as the target of this compound.

Novel substituted 9-norbornylpurines and their activities against RNA viruses.

We report on the synthesis and the study of the structure-activity relationship of novel 9-norbornyl-6-chloropurine derivatives, which exert selective antiviral activity on the replication of Coxsackievirus B3. In particular, the synthetic approaches towards norbornyl derivatives bearing diverse side chains were studied. The main goal of the study was to determine the influence of the norbornane moiety substitution at positions 5' and 6' on selective antiviral activity with special regard to the liphophilicity profile of the substituent.

SAR studies of 9-norbornylpurines as Coxsackievirus B3 inhibitors.

Coxsackievirus and related enteroviruses are important human pathogens that cause various diseases with clinical manifestations ranging from trivial flu-like syndromes to dangerous or even fatal diseases such as myocarditis, meningitis and encephalitis. Here, we report on our continuous SAR study focused on 9-(bicyclo[2.2.1]hept-2-yl)-9H-purines as anti-enteroviral inhibitors. The purine moiety was modified at positions 2, 6 and 8. Several analogues inhibited Coxsackievirus B3 as well as other enteroviruses at low-micromolar concentrations. The 6-chloropurine derivative was confirmed as the most active compound in this series.

Inflammatory rather than infectious insults play a role in exocrine tissue damage in a mouse model for coxsackievirus B4-induced pancreatitis.

Infection with coxsackievirus B4 (CVB4) may result in an acute severe necrotizing pancreatitis that mostly remains restricted to the acini of the exocrine parenchyma. The mechanisms responsible for this tissue damage, however, remain poorly understood. We here report that COAM, a polyanionic carboxylic acid, provides marked protection against CVB4-induced pancreatitis in a mouse model. Despite the fact that COAM largely reduced disease severity, as detected by serum amylase and lipase levels as well as histologically, titres of replicating CVB4 in the pancreas were virtually unaffected. COAM treatment diminished the infection-associated MMP-9 levels and also resulted in a decreased influx of neutrophils into the infected pancreas. Moreover, we demonstrate that titres of replicating virus in the pancreas did not directly correlate with the severity of disease. In conclusion, our data suggest that immunopathological effects, rather than direct virus-induced destruction, are responsible for the damage to acinar tissue in CVB4-induced pancreatitis.

Design, synthesis, and biological evaluation of novel coxsackievirus B3 inhibitors.

The synthesis and SAR study of a novel class of coxsackievirus B3 (CVB3) inhibitors are reported. These compounds could be considered as the 6-chloropurines substituted at position 9 with variously substituted bicyclic scaffolds (bicyclo[2.2.1]heptane/ene-norbornane or norbornene). The synthesis and biological evaluation of 31 target compounds are described. Several of the analogues inhibited CVB3 in the low micromolar range (0.66-2muM). Minimal or no cytotoxicity was observed.

Mutations in the nonstructural protein 3A confer resistance to the novel enterovirus replication inhibitor TTP-8307.

A novel compound, TTP-8307, was identified as a potent inhibitor of the replication of several rhino- and enteroviruses. TTP-8307 inhibits viral RNA synthesis in a dose-dependent manner, without affecting polyprotein synthesis and/or processing. Drug-resistant variants of coxsackievirus B3 were all shown to carry at least one amino acid mutation in the nonstructural protein 3A. In particular, three mutations located in a nonstructured region preceding the hydrophobic domain (V45A, I54F, and H57Y) appeared to contribute to the drug-resistant phenotype. This region has previously been identified as a hot sport for mutations that resulted in resistance to enviroxime, the sole 3A-targeting enterovirus inhibitor reported thus far. This was corroborated by the fact that TTP-8307 and enviroxime proved cross-resistant. It is hypothesized that TTP-8307 and enviroxime disrupt proper interactions of 3A(B) with other viral or cellular proteins that are required for efficient replication.

The crystal structure of coxsackievirus B3 RNA-dependent RNA polymerase in complex with its protein primer VPg confirms the existence of a second VPg binding site on Picornaviridae polymerases.

The RNA-dependent RNA polymerase (RdRp) is a central piece in the replication machinery of RNA viruses. In picornaviruses this essential RdRp activity also uridylates the VPg peptide, which then serves as a primer for RNA synthesis. Previous genetic, binding, and biochemical data have identified a VPg binding site on poliovirus RdRp and have shown that is was implicated in VPg uridylation. More recent structural studies have identified a topologically distinct site on the closely related foot-and-mouth disease virus RdRp supposed to be the actual VPg-primer-binding site. Here, we report the crystal structure at 2.5-A resolution of active coxsackievirus B3 RdRp (also named 3D(pol)) in a complex with VPg and a pyrophosphate. The pyrophosphate is situated in the active-site cavity, occupying a putative binding site either for the coproduct of the reaction or an incoming NTP. VPg is bound at the base of the thumb subdomain, providing first structural evidence for the VPg binding site previously identified by genetic and biochemical methods. The binding mode of VPg to CVB3 3D(pol) at this site excludes its uridylation by the carrier 3D(pol). We suggest that VPg at this position is either uridylated by another 3D(pol) molecule or that it plays a stabilizing role within the uridylation complex. The CVB3 3D(pol)/VPg complex structure is expected to contribute to the understanding of the multicomponent VPg-uridylation complex essential for the initiation of genome replication of picornaviruses.

The thiazolobenzimidazole TBZE-029 inhibits enterovirus replication by targeting a short region immediately downstream from motif C in the nonstructural protein 2C.

TBZE-029 {1-(2,6-difluorophenyl)-6-trifluoromethyl-1H,3H-thiazolo[3,4-a]benzimidazole} is a novel selective inhibitor of the replication of several enteroviruses. We show that TBZE-029 exerts its antiviral activity through inhibition of viral RNA replication, without affecting polyprotein processing. To identify the viral target of TBZE-029, drug-resistant coxsackievirus B3 (CVB3) was selected. Genotyping of resistant clones led to the identification of three amino acid mutations in nonstructural protein 2C, clustered at amino acid positions 224, 227, and 229, immediately downstream of NTPase/helicase motif C. The mutations were reintroduced, either alone or combined, into an infectious full-length CVB3 clone. In particular the mutations at positions 227 and 229 proved essential for the altered sensitivity of CVB3 to TBZE-029. Resistant virus exhibited cross-resistance to the earlier-reported antienterovirus agents targeting 2C, namely, guanidine hydrochloride, HBB [2-(alpha-hydroxybenzyl)-benzimidazole], and MRL-1237 {1-(4-fluorophenyl)-2-[(4-imino-1,4-dihydropyridin-1-yl)methyl]benzimidazole hydrochloride}. The ATPase activity of 2C, however, remained unaltered in the presence of TBZE-029.

Selective inhibitors of picornavirus replication.

Picornaviruses cover a large family of pathogens that have a major impact on human but also on veterinary health. Although most infections in man subside mildly or asymptomatically, picornaviruses can also be responsible for severe, potentially life-threatening disease. To date, no therapy has been approved for the treatment of picornavirus infections. However, efforts to develop an antiviral that is effective in treating picornavirus-associated diseases are ongoing. In 2007, Schering-Plough, under license of ViroPharma, completed a phase II clinical trial with Pleconaril, a drug that was originally rejected by the FDA after a New Drug Application in 2001. Rupintrivir, a rhinovirus protease inhibitor developed at Pfizer, reached clinical trials but was recently halted from further development. Finally, Biota's HRV drug BTA-798 is scheduled for phase II trials in 2008. Several key steps in the picornaviral replication cycle, involving structural as well as non-structural proteins, have been identified as valuable targets for inhibition. The current review aims to highlight the most important developments during the past decades in the search for antivirals against picornaviruses.

Potential use of antiviral agents in polio eradication.

In 1988, the World Health Assembly launched the Global Polio Eradication Initiative, which aimed to use large-scale vaccination with the oral vaccine to eradicate polio worldwide by the year 2000. Although important progress has been made, polio remains endemic in several countries. Also, the current control measures will likely be inadequate to deal with problems that may arise in the postpolio era. A panel convoked by the National Research Council concluded that the use of antiviral drugs may be essential in the polio eradication strategy. We here report on a comparative study of the antipoliovirus activity of a selection of molecules that have previously been reported to be inhibitors of picornavirus replication and discuss their potential use, alone or in combination, for the treatment or prophylaxis of poliovirus infection.

R75761, a lead compound for the development of antiviral drugs in late stage poliomyelitis eradication strategies and beyond.

In this study the antiviral activity of a panel of 18 out of 240 pyridazinamine analogues was evaluated against the Sabin strains of the three poliovirus types. We found one compound, R75761 which had a comparable 50% effective concentration (EC50) value against all three poliovirus Sabin strains. Virus multiplication was reduced by 10(4.0)-fold, 10(6.2)-fold and 10(6.6)-fold for poliovirus type 1, type 2 and type 3, respectively. R75761 could be considered as a lead compound for development of anti-poliovirus drugs to be used during the late stage of poliovirus eradication and beyond.

Synthesis and anti-CVB 3 evaluation of substituted 5-nitro-2-phenoxybenzonitriles.

The synthesis and SAR of a series of 60 substituted 2-phenoxy-5-nitrobenzonitriles (analogues of MDL-860) as inhibitors of enterovirus replication (in particular of coxsackievirus B3 (CVB 3)) are reported. Several of the analogues inhibited CVB 3 and other enteroviruses at low-micromolar concentrations.

Improved crystallization of the coxsackievirus B3 RNA-dependent RNA polymerase.

The Picornaviridae virus family contains a large number of human pathogens such as poliovirus, hepatitis A virus and rhinoviruses. Amongst the viruses belonging to the genus Enterovirus, several serotypes of coxsackievirus coexist for which neither vaccine nor therapy is available. Coxsackievirus B3 is involved in the development of acute myocarditis and dilated cardiomyopathy and is thought to be an important cause of sudden death in young adults. Here, the first crystal of a coxsackievirus RNA-dependent RNA polymerase is reported. Standard crystallization methods yielded crystals that were poorly suited to X-ray diffraction studies, with one axis being completely disordered. Crystallization was improved by testing crystallization solutions from commercial screens as additives. This approach yielded crystals that diffracted to 2.1 A resolution and that were suitable for structure determination.

Combating enterovirus replication: state-of-the-art on antiviral research.

Enteroviruses form an important genus within the large family of Picornaviridae. They are small, non-enveloped (+)RNA viruses, many of which are important pathogens in human and veterinary science. Despite their huge medical and socio-economical impact, there is still no approved antiviral therapy at hand for the treatment of these infections. Three capsid-targeting molecules (pleconaril, BTA-798 and V-073) are in clinical development. Pleconaril and BTA-798 are in phase II clinical trials for the treatment of enterovirus-induced sepsis syndrome and rhinovirus-induced aggravation of pre-existing asthma or COPD respectively. V-073 is in preclinical development for the treatment of poliovirus infections in the context of the worldwide polio eradication program. The capsid binding molecules have shown good in vitro potency against a number of enterovirus species, but lack activity against others. Another potential drawback of capsid inhibitors in the clinical setting could be the rapid emergence of drug resistance. It will therefore be important to develop inhibitors that affect other stages in the viral replication cycle. Several viral proteins, such as the viral 3C protease, the putative 2C helicase and the 3D RNA-dependent RNA polymerase may be/are excellent targets for inhibition of viral replication. Also host cell factors that are crucial in viral replication may be considered as potential targets for an antiviral approach. Unraveling these complex virus-host interactions will also provide better insights into the replication of enteroviruses. This review aims to summarize and discuss known inhibitors and potential viral and cellular targets for antiviral therapy against enteroviruses.

Picornavirus non-structural proteins as targets for new anti-virals with broad activity.

Picornaviridae is one of the largest viral families and is composed of 14 genera, six of which include human pathogens. The best known picornaviruses are enteroviruses (including polio, PV, and rhinoviruses), foot-and-mouth disease virus (FMDV), and hepatitis A virus (HAV). Although infections often are mild, certain strains may cause pandemic outbreaks accompanied with meningitis and/or paralysis. Vaccines are available for PV, HAV and FMDV. When the oral vaccines are given to immunocompromised individuals, they may be chronically infected, and remain secretors of vaccine-derived variants of virus for years. There is no effective prophylaxis available for these or other picornaviruses. So far, only the 3C protease from viruses in three genera has been fully characterized as an anti-viral target, whereas the mode of action of compounds targeting other non-structural proteins have remained largely unaddressed. Within the EU-supported FP6 project-VIZIER (Comparative Structural Genomics of Viral Enzymes Involved in Replication), the non-structural proteins were studied to identify conserved binding sites for broadly reactive anti-virals. The putative 2C helicase from echovirus-30 was shown to form ring-shaped hexamers typical for DNA-encoded SF3 helicases, and to possess ATPase activity. Hexamer formation of 2C from enterovirus 76 was in vitro shown to be dependent on the 44 N-terminal residues. Crystal structures of three enterovirus 3C proteases were solved and shown to be similar to those of other picornaviruses. A new binding site of VPg to the bottom of the thumb domain of CV-B3 3D polymerase was identified as a potential target. Broad anti-enterovirus compounds against 2C and 3A proteins were also identified, including thiazolobenzimidazoles (active against 2C) and TTP-8307 (targeting 3A). There is a need for more potent inhibitors against PV and other picornaviruses, which are potential silent reservoirs for re-emerging PV-like disease.

Increased gelatinase B/matrix metalloproteinase 9 (MMP-9) activity in a murine model of acute coxsackievirus B4-induced pancreatitis.

Infection of mice with coxsackievirus B4 results within days in a severe acute necrotizing pancreatitis, which resolves completely within weeks. Gelatinase B or matrix metalloproteinase 9 (MMP-9) has previously been shown to be involved in several models of pancreatitis, but its role in virus-induced pancreatitis has never been investigated. We here report that MMP-9 levels are markedly increased in the pancreas of mice that developed acute pancreatitis following infection with coxsackievirus B4. Moreover, using in situ zymography, we demonstrated that MMP-9 is active in vivo. Double immunohistochemical analysis revealed that macrophages and neutrophils were the cellular source of MMP-9. Extensive tissue rearrangements involving collagen turnover were observed, and these were associated with extensive pathology and resolution of the disease. In summary, this report demonstrates that acute coxsackievirus B4-induced pancreatitis involves the action of MMP-9, which is mainly originating from macrophages and neutrophils.

Anti-enterovirus activity and structure-activity relationship of a series of 2,6-dihalophenyl-substituted 1H,3H-thiazolo[3,4-a]benzimidazoles.

Despite the fact that enteroviruses are implicated in a variety of human diseases, there is no approved therapy for the treatment of enteroviral infections. Here, a series of 2,6-dihalophenyl-substituted 1H,3H-thiazolo[3,4-a]benzimidazoles with anti-enterovirus activity is reported. The compounds elicit potent activity against coxsackievirus A9, echovirus 9 and 11 and all six strains of coxsackievirus B. A structure-activity relationship analysis revealed that the presence of substituents at position 6 of the tricyclic system positively influences the antiviral activity, whereas substitutions at position 7 are less favorable. In particular a 6-trifluoromethyl substitution leads to a substantial improvement of the antiviral activity as compared to the unsubstituted structure. Furthermore, an additional introduction of a 2-Cl, 6-F substitution on the phenyl at C-1 results in a further increase of the antiviral activity. Hence, 1-(2-chloro-6-fluorophenyl)-6-trifluoromethyl-1H,3H-thiazolo[3,4-a]benzimidazole results in a dose-dependent inhibition of viral replication with a 50% effective concentration (EC50) of 0.41 microg/ml without any detectable cytotoxicity at the highest concentration (100 microg/ml) tested.