Armando M. De Palma

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, Biotas 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.

Inhibition of Urokinase-Type Plasminogen Activator or Matrix Metalloproteinases Prevents Cardiac Injury and Dysfunction During Viral Myocarditis

Background— Acute viral myocarditis is an important cause of cardiac failure in young adults for which there is no effective treatment apart from general heart failure therapy. The present study tested the hypothesis that increased expression of the proteinases urokinase-type plasminogen activator (uPA) and matrix metalloproteinases (MMPs) is implicated in cardiac inflammation, injury, and subsequent failure during Coxsackievirus-B3 (CVB3)–induced myocarditis. Methods and Results— First, we showed increased expression and activity of uPA and MMP-9 in wild-type mice at 7 days of CVB3-induced myocarditis. Targeted deletion of uPA, which resulted in reduced MMP activity and cytokine expression or inhibition of MMPs by adenoviral gene overexpression of tissue inhibitor of metalloproteinases-1, decreased cardiac inflammation and reduced myocardial necrosis at 7 days and decreased cardiac fibrosis at 35 days after CVB3 infection. Importantly, loss of uPA or MMP activity prevented CVB3-induced cardiac dilatation and dysfunction, as determined by serial echocardiography. Conclusions— Loss of uPA or MMP activity reduces the cardiac inflammatory response after CVB3 infection, thereby protecting against cardiac injury, dilatation, and failure during CVB3-induced myocarditis.

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.

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

ABSTRACT 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-Å resolution of active coxsackievirus B3 RdRp (also named 3Dpol) 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 3Dpol at this site excludes its uridylation by the carrier 3Dpol. We suggest that VPg at this position is either uridylated by another 3Dpol molecule or that it plays a stabilizing role within the uridylation complex. The CVB3 3Dpol/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.

A Novel, Broad-Spectrum Inhibitor of Enterovirus Replication That Targets Host Cell Factor Phosphatidylinositol 4-Kinase IIIβ

ABSTRACT Despite their high clinical and socioeconomic impacts, there is currently no approved antiviral therapy for the prophylaxis or treatment of enterovirus infections. Here we report on a novel inhibitor of enterovirus replication, compound 1, 2-fluoro-4-(2-methyl-8-(3-(methylsulfonyl)benzylamino)imidazo[1,2-a]pyrazin-3-yl)phenol. This compound exhibited a broad spectrum of antiviral activity, as it inhibited all tested species of enteroviruses and rhinoviruses, with 50% effective concentrations ranging between 4 and 71 nM. After a lengthy resistance selection process, coxsackievirus mutants resistant to compound 1 were isolated that carried substitutions in their 3A protein. Remarkably, the same substitutions were recently shown to provide resistance to inhibitors of phosphatidylinositol 4-kinase IIIβ (PI4KIIIβ), a lipid kinase that is essential for enterovirus replication, suggesting that compound 1 may also target this host factor. Accordingly, compound 1 directly inhibited PI4KIIIβ in an in vitro kinase activity assay. Furthermore, the compound strongly reduced the PI 4-phosphate levels of the Golgi complex in cells. Rescue of coxsackievirus replication in the presence of compound 1 by a mutant PI4KIIIβ carrying a substitution in its ATP-binding pocket revealed that the compound directly binds the kinase at this site. Finally, we determined that an analogue of compound 1, 3-(3-fluoro-4-methoxyphenyl)-2-methyl-N-(pyridin-4-ylmethyl)imidazo[1,2-a]pyrazin-8-amine, is well tolerated in mice and has a dose-dependent protective activity in a coxsackievirus serotype B4-induced pancreatitis model.

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.

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.

The Interferon Inducer Ampligen [Poly(I)-Poly(C12U)] Markedly Protects Mice against Coxsackie B3 Virus-Induced Myocarditis

ABSTRACT Viral replication, as well as an immunopathological component, is assumed to be involved in coxsackie B virus-induced myocarditis. We evaluated the efficacy of the interferon inducer Ampligen on coxsackie B3 virus-induced myocarditis in C3H/HeNHsd mice. The efficacy of Ampligen was compared with that of the interferon inducer poly(inosinic acid)-poly(cytidylic acid) [poly(IC)], alpha interferon 2b (INTRON A), and pegylated alpha interferon 2b (PEG-INTRON-α-2b). Ampligen at 20 mg/kg of body weight/day was able to reduce the severity of virus-induced myocarditis, as assessed by morphometric analysis, by 98% (P = 3.0 × 10−8). When poly(IC) was administered at 15 mg/kg/day, it reduced the severity of virus-induced myocarditis by 93% (P = 5.6 × 10−5). Alpha interferon 2b (1 × 105 U/day) and pegylated alpha interferon 2b (5 × 105 U/day) were less effective and reduced the severity of virus-induced myocarditis by 66% (P = 0.0009) and 78% (P = 0.0002), respectively. The observed efficacies of Ampligen and poly(IC) were corroborated by the observation that the drugs also markedly reduced the virus titers in the heart, as detected by (i) quantitative real-time reverse transcription-PCR and (ii) titration for infectious virus content. Whereas the electrocardiograms for untreated mice with myocarditis were severely disturbed, the electrocardiographic parameters were normalized in Ampligen- and poly(IC)-treated mice. Even when start of treatment with Ampligen was delayed until day 2 postinfection, a time at which lesions had already appeared in untreated control animals, a marked protective effect on the development of viral myocarditis (as assessed at day 6 postinfection) was still noted.

The Thiazolobenzimidazole TBZE-029 Inhibits Enterovirus Replication by Targeting a Short Region Immediately Downstream from Motif C in the Nonstructural Protein 2C

ABSTRACT 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.

Mutations in the Nonstructural Protein 3A Confer Resistance to the Novel Enterovirus Replication Inhibitor TTP-8307

ABSTRACT 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.