A structurally biased combinatorial approach for discovering new anti-picornaviral compounds.

BACKGROUND: Picornaviruses comprise a family of small, non-enveloped RNA viruses. A common feature amongst many picornaviruses is a hydrophobic pocket in the core of VP1, one of the viral capsid proteins. The pocket is normally occupied by a mixture of unidentified, fatty acid-like moieties, which can be competed out by a family of capsid-binding, antiviral compounds. Many members of the Picornaviridae family are pathogenic to both humans and livestock, yet no adequate therapeutics exist despite over a decade's worth of research in the field. To address this challenge, we developed a strategy for rapid identification of capsid-binding anti-picornaviral ligands. The approach we took involved synthesizing structurally biased combinatorial libraries that had been targeted to the VP1 pocket of poliovirus and rhinovirus. The libraries are screened for candidate ligands with a high throughput mass spectrometry assay. RESULTS: Using the mass spectrometry assay, we were able to identify eight compounds from a targeted library of 75 compounds. The antiviral activity of these candidates was assessed by (i) measuring the effect on the kinetics of viral uncoating and (ii) the protective effect of each drug in traditional cell-based assays. All eight of the candidates exhibited antiviral activity, but three of them were particularly effective against poliovirus and rhinovirus. CONCLUSIONS: The results illustrate the utility of combining structure-based design with combinatorial chemistry. The success of our approach suggests that assessment of small, targeted libraries, which query specific chemical properties, may be the best strategy for surveying all of chemical space for ideal anti-picornaviral compounds.

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.

Activity of pleconaril against enteroviruses.

The activity of pleconaril in cell culture against prototypic enterovirus strains and 215 clinical isolates of the most commonly isolated enterovirus serotypes was examined. The latter viruses were isolated by the Centers for Disease Control and Prevention during the 1970s and 1980s from clinically ill subjects. Pleconaril at a concentration of

Attenuated virulence of pleconaril-resistant coxsackievirus B3 variants.

Pleconaril (VP 63843) is a novel orally bioavailable small molecule with broad antipicornavirus (enterovirus and rhinovirus) activity. Ten independently derived pleconaril-resistant variants of coxsackievirus B3 were isolated from cell culture. The molecular basis of drug resistance and the biologic properties of the drug-resistant viruses were investigated. RNA sequence analysis revealed amino acid changes in the drug-binding pocket of the resistant variants. Thermal stability studies showed the drug-resistant viruses to be significantly less stable than wild type virus. When evaluated in a murine model in which wild type virus infection is 100% lethal, the drug-resistant viruses showed attenuated virulence with both reduced mortality and delayed time to death. Virus titers in heart and spleen were dramatically lower in drug-resistant virus-infected mice than in wild type virus-infected animals. The study results indicate that pleconaril-resistant virus variants are attenuated and significantly less virulent than drug-sensitive wild type virus.

Human rhinovirus 3 at 3.0 A resolution.

BACKGROUND: The over 100 serotypes of human rhinoviruses (HRV) are major causative agents of the common cold in humans. These HRVs can be roughly divided into a major and minor group according to their cellular receptors. They can also be divided into two antiviral groups, A and B, based on their sensitivity to different capsid-binding antiviral compounds. The crystal structures of HRV14 and HRV16, major-receptor group rhinoviruses, as well as HRV1A, a minor-receptor group rhinovirus, were determined previously. Sequence comparisons had shown that HRV14 seemed to be an outlier among rhinoviruses. Furthermore, HRV14 was the only virus with no cellular 'pocket factor' in a hydrophobic pocket which is targeted by many capsid-binding antiviral compounds and is thought to regulate viral stability. HRV3, another major-receptor group virus, was chosen for study because it is one of a subset of serotypes that best represents the drug sensitivity of most rhinovirus serotypes. Both HRV3 and HRV14 belong to antiviral group A, while HRV16 and HRV1A belong to antiviral group B. RESULTS: HRV3 was found to be very similar to HRV14 in sequence and structure. Like HRV14, crystallized HRV3 also has no bound pocket factor. The structure of HRV3 complexed with an antiviral compound, WIN56291, was also determined and found to be similar to the same antiviral compound complexed with HRV14. CONCLUSIONS: The amino-acid sequence and structural similarity between HRV3 and HRV14 suggests that rhinoviruses in the same antiviral group have similar amino-acid sequences and structures. The similar amino-acid composition in the pocket region and the viral protein VP1 N termini in all known group B HRV sequences suggests that these viruses may all contain pocket factors and ordered N-terminal amphipathic helices in VP1. Both of these factors contribute to viral stability, which is consistent with the observations that group B rhinoviruses have a higher chance of successful transmission from one host to another and is a possible explanation for the observed higher pathogenicity of these rhinoviruses.

Structural studies on human rhinovirus 14 drug-resistant compensation mutants.

Structures have been determined of three human rhinovirus 14 (HRV14) compensation mutants that have resistance to the antiviral capsid binding compounds WIN 52035 and WIN 52084. In addition, the structure of HRV14 is reported, with a site-directed mutation at residue 1219 in VP1. A spontaneous mutation occurs at the same site in one of the compensation mutants. Some of the mutations are on the viral surface in the canyon and some lie within the hydrophobic binding pocket in VP1 below the ICAM footprint. Those mutant virus strains with mutations on the surface bind better to cells than does wild-type virus. The antiviral compounds bind to the mutant viruses in a manner similar to their binding to wild-type virus. The receptor and WIN compound binding sites overlap, causing competition between receptor attachment and antiviral compound binding. The compensation mutants probably function by shifting the equilibrium in favor of receptor binding. The mutations in the canyon increase the affinity of the virus for the receptor, while the mutations in the pocket probably decrease the affinity of the WIN compounds for the virus by reducing favorable hydrophobic contacts and constricting the pore through which the antiviral compounds are thought to enter the pocket. This is in contrast to the resistant exclusion mutants that block compounds from binding by increasing the bulk of residues within the hydrophobic pocket in VP1.

Structures of four methyltetrazole-containing antiviral compounds in human rhinovirus serotype 14.

Four novel antiviral WIN compounds, that contain a methyl tetrazole ring as well as isoxazole, pyridazine or acetylfuran rings, have had their structures determined in human rhinovirus serotype 14 at 2.9 A resolution. These compounds bind in the VP1 hydrophobic pocket, but are shifted significantly towards the pocket pore when compared to previously examined WIN compounds. A putative water network at the pocket pore is positioned to hydrogen bond with these four WIN compounds, and this network can account for potency differences seen in structurally similar WIN compounds.

[(Biaryloxy)alkyl]isoxazoles: picornavirus inhibitors.

A series of biphenyl analogs, 6, of 5-[5-(2,6-dichloro-5-oxazolylphenoxy)pentyl]-3-methylisoxazole (2) have been synthesized and tested in vitro against 10 human rhinovirus serotypes in a TCID50 assay. The most potent compound in the series 6s, 3-[3-[2,6-dimethyl-4-(4-fluorophenyl)-phenoxy]propyl]-3-methylisoxazole , was screened against an additional 84 serotypes. It was found to be active against 64 of the serotypes, while 87 serotypes were sensitive to 2 at < 3 micrograms/mL. On comparison of the active serotypes, 6s exhibited greater potency versus 2. Analogs 6a-c,s were examined for in vitro metabolic stability by monkey liver microsomal assay. These analogs exhibited a greater than 7-fold improvement (t1/2 > 200 min) in metabolic stability compared with 2 (t1/2 > 27 min).

The structure of coxsackievirus B3 at 3.5 A resolution.

BACKGROUND: Group B coxsackieviruses (CVBs) are etiologic agents of a number of human diseases that range in severity from asymptomatic to lethal infections. They are small, single-stranded RNA icosahedral viruses that belong to the enterovirus genus of the picornavirus family. Structural studies were initiated in light of the information available on the cellular receptors for this virus and to assist in the design of antiviral capsid-binding compounds for the CVBs. RESULTS: The structure of coxsackievirus B3 (CVB3) has been solved to a resolution of 3.5 A. The beta-sandwich structure of the viral capsid proteins VP1, VP2 and VP3 is conserved between CVB3 and other picornaviruses. Structural differences between CVB3 and other enteroviruses and rhinoviruses are located primarily on the viral surface. The hydrophobic pocket of the VP1 beta-sandwich is occupied by a pocket factor, modeled as a C16 fatty acid. An additional study has shown that the pocket factor can be displaced by an antiviral compound. Myristate was observed covalently linked to the N terminus of VP4. Density consistent with the presence of ions was observed on the icosahedral threefold and fivefold axes. CONCLUSIONS: The canyon and twofold depression, major surface depressions, are predicted to be the primary and secondary receptor-binding sites on CVB3, respectively. Neutralizing immunogenic sites are predicted to lie on the extreme surfaces of the capsid at sites that lack amino acid sequence conservation among the CVBs. The ions located on the icosahedral threefold and fivefold axes together with the pocket factor may contribute to the pH stability of the coxsackieviruses.

Localization of human rhinovirus replication in the upper respiratory tract by in situ hybridization.

To localize the sites and determine the extent of human rhinovirus (HRV) replication in the upper respiratory tract, biopsies of nasal and nasopharyngeal epithelia were collected from 26 HRV- or 7 sham-inoculated volunteers on days 1, 3, and 5 and on days 12, 20, or 33 after inoculation and analyzed by in situ hybridization. HRV-infected cells were detected on at least 1 day in 22 of the 23 HRV-infected subjects and in 1 of the 7 sham-inoculated subjects who developed a cold and had nasal secretions positive for a picornavirus by polymerase chain reaction. Low numbers of in situ hybridization-positive ciliated cells were present in nasal biopsies. In the nasopharynx, most HRV-infected cells were ciliated, but infected nonciliated epithelial cells were also detected. Our results indicate that HRV replicates in a very small proportion of cells in the nasal epithelium and in both ciliated and nonciliated cells in the nasopharynx of experimentally infected humans.

Picornavirus inhibitors: trifluoromethyl substitution provides a global protective effect against hepatic metabolism.

Several modifications of the oxazoline ring of WIN 54954, a broad spectrum antipicornavirus compound, have been prepared in order to address the acid lability and metabolic instability of this compound. We have previously shown that the oxadiazole analogue 3 displayed comparable activity against a variety of rhinoviruses and appeared to be stable to acid. A monkey liver microsomal assay was developed to examine the metabolic stability in vitro of both compounds, and it was determined that WIN 54954 displayed 18 metabolic products while 3 was converted to 8 products. Two major products of 3 were determined by LC-MS/MS to be monohydroxylated at each of the terminal methyl groups. Replacement of the methyl on the isoxazole ring with a trifluoromethyl group, while preventing hydroxylation at this position, did not reduce the sensitivity of the molecule to microsomal metabolism at other sites. However, the (trifluoromethyl)oxadiazole 9 not only prevented hydroxylation at this position but also provided protection at the isoxazole end of the molecule, resulting in only two minor products to the extent of 4%. The major product was identified as the monohydroxylated compound 23. The global metabolic protective effect of trifluoromethyl group on the oxadiazole ring was further demonstrated by examining a variety of analogues including heterocyclic replacements of the isoxazole ring. In each case, the trifluoromethyl analogue displayed a protective effect when compared to the corresponding methyl analogue.

An evaluation of the antirhinoviral activity of acetylfuran replacements for 3-methylisoxazoles. Are 2-acetylfurans bioisosteres for 3-methylisoxazoles?

As a probe of the 3-methylisoxazole portion of our broad-spectrum antipicornaviral series, a panel of 2-acetylfuran analogues was prepared as replacements for the 3-methylisoxazole ring. Comparison of the two series showed remarkable similarity in potency, spectrum of activity, logP, and electrostatic parameters. X-ray studies of 21b bound to human rhinovirus-14 showed that the 2-acetyl group adopted a syn conformation and the carbonyl oxygen acts as a hydrogen bond acceptor with ASN219 in much the same way as the nitrogen of the isoxazole. The importance of the syn conformation and the hydrogen-bonding capability was confirmed by the reduced antiviral activity of the 2-methylfuran and 2-formylfuran analogues. From the results of this study, it is apparent that the syn-2-acetylfuran ring is acting as a bioisostere for the 3-methylisoxazole.

Oxadiazoles as ester bioisosteric replacements in compounds related to disoxaril. Antirhinovirus activity.

A series of 1,2,4-oxadiazoles has been prepared as ester bioisosteres and tested against 15 human rhinovirus serotypes, and the MIC80, the concentration which inhibits 80% or 12 of the serotypes tested, was determined. Homologation of the alkyl group attached to the oxadiazole ring resulted in a reduction in activity with increased chain length. Introduction of hydrophilic groups in this position rendered the compounds inactive. Increasing the length of the side chain attached to the isoxazole ring resulted in an increase in activity. Replacement of the methyl with alkoxyalkyl substituents retained activity; however, introduction of a hydroxyl group on to the side chain reduced activity. Compound 8a, where both the isoxazole and oxadiazole rings were substituted with methyl groups, was one of the most active compounds in the series. A comparison was made between 8a and the two isomeric oxadiazoles 41 and 46, and an attempt was made to explain the difference in activity by examining electrostatic potential maps and by an energy profiling study. No conclusive results were obtained from these studies.

Human rhinovirus 14 complexed with fragments of active antiviral compounds.

Crystallographic studies of human rhinovirus 14 (HRV14) crystals soaked with fragments of antiviral WIN compounds, at high concentrations (82-200 micrograms/ml), show the compounds bind into the hydrophobic beta-barrel (WIN pocket) of VP1. Two of these short compounds (5-[3,5-dimethyl-4-hydroxyphenyl]-2-methyltetrazole and phenol oxazoline) cause conformational changes in the virus similar to the active, longer WIN compounds. In addition, thermostabilization studies suggest these short WIN compounds provide some stability to the HRV14 capsid. We conclude that the short compounds appear to mimic the cellular cofactors observed in the hydrophobic pocket of VP1 for some picornaviruses. Both cofactors and short WIN compounds bind into the pocket, cause conformational changes in VP1, and provide a small degree of virion stabilization but are unlikely to inhibit attachment. Three specific binding sites for dimethyl sulfoxide (DMSO), used as solvent, were also identified. One of the DMSO molecules binds into the drug binding pocket near the pocket opening, while the other two bind in the canyon near the VP1 protomer-protomer interface.

The structure of human rhinovirus 16.

BACKGROUND: Rhinoviruses and the homologous polioviruses have hydrophobic pockets below their receptor-binding sites, which often contain unidentified electron density ('pocket factors'). Certain antiviral compounds also bind in the pocket, displacing the pocket factor and inhibiting uncoating. However, human rhinovirus (HRV)14, which belongs to the major group of rhinoviruses that use intercellular adhesion molecule-1 (ICAM-1) as a receptor, has an empty pocket. When antiviral compounds bind into the empty pocket of HRV14, the roof of the pocket, which is also the floor of the receptor binding site (the canyon), is deformed, preventing receptor attachment. The role of the pocket in viral infectivity is not known. RESULTS: We have determined the structure of HRV16, another major receptor group rhinovirus serotype, to atomic resolution. Unlike HRV14, the pockets contain electron density resembling a fatty acid, eight or more carbon atoms long. Binding of the antiviral compound WIN 56291 does not cause deformation of the pocket, although it does prevent receptor attachment. CONCLUSIONS: We conjecture that the binding of the receptor to HRV16 can occur only when the pocket is temporarily empty, when it is possible for the canyon floor to be deformed downwards into the pocket. We further propose that the role of the pocket factor is to stabilize virus in transit from one host cell to the next, and that binding of ICAM-1 traps the pocket in the empty state, destabilizing the virus as required for uncoating.

Conformationally restricted analogues of disoxaril: a comparison of the activity against human rhinovirus types 14 and 1A.

A series of conformationally restricted analogs of disoxaril has been synthesized and evaluated against human rhinovirus types (HRV) 14 and 1A. The sensitivity of these serotypes to this series varied and was dependent upon the length of the molecule as well as upon the flexibility of the aliphatic chain. Minimum energy conformations of these compounds were overlaid with the X-ray structure of a closely related analog 9 bound to the capsid protein of both HRV-14 and -1A and then modeled in the compound-binding site of both serotypes. A comparative sweep volume of these compounds about the isoxazole ring revealed an inaccessible region of space for the cis-olefin 8b, which is not the case for either the trans-olefin 8a or the acetylene 5. This region may be important to the binding of the compounds to the HRV-14 site particularly during entry into the pocket.

Antirhinoviral activity of heterocyclic analogs of Win 54954.

A variety of heterocyclic analogs of Win 54954 have been synthesized and tested in vitro against human rhinovirus type 14 (HRV-14) in a plaque reduction assay. The more active compounds were tested against 14 additional serotypes, and the concentration which inhibited 80% of the serotypes tested (MIC80) was measured. One compound, 37, exhibited activity comparable to Win 59454. Physicochemical as well as electrostatic parameters were calculated and the results subjected to a QSAR analysis in an effort to explain differences in activity observed between these compounds; however, no meaningful correlation with biological activity was found with any of these parameters.

CoMFA analysis of the interactions of antipicornavirus compounds in the binding pocket of human rhinovirus-14.

A CoMFA analysis of eight compounds related to disoxaril whose X-ray structures bound to HRV-14 had been determined resulted in a strong positive correlation of activity with steric effects of the compounds, particularly toward the pore end of the compound binding site, and no correlation with electrostatic effects. These results confirm what had been previously found, that the activity of these compounds was highly dependent upon their hydrophobic nature as expressed by log p. The CoMFA study also confirmed the results from the comparison of a series of active and inactive compounds using volume maps which showed that bulk at the pore end of the molecule was conducive to high levels of antiviral activity while excessive bulk around the ring led to poor activity.

Treatment of the picornavirus common cold by inhibitors of viral uncoating and attachment.

The human rhinoviruses are the leading cause of the ubiquitous, mild, and self-limiting infections generally referred to as the common cold. Considerable research effort has been expended in the search for well tolerated antiviral agents capable of preventing and treating the common cold. Although no antirhinovirus drug is yet commercially available, considerable progress has been made in the discovery and development of novel, viral specific inhibitors of rhinovirus replication. This report reviews the history and current status of the research that has focused on inhibitors of the early steps in the virus life cycle: attachment to the cellular receptor and uncoating of the viral RNA. Molecules directed at these targets currently possess the greatest potential for generating a safe and efficacious treatment for the rhinovirus common cold.