EDP-938, a novel nucleoprotein inhibitor of respiratory syncytial virus, demonstrates potent antiviral activities in vitro and in a non-human primate model.

EDP-938 is a novel non-fusion replication inhibitor of respiratory syncytial virus (RSV). It is highly active against all RSV-A and B laboratory strains and clinical isolates tested in vitro in various cell lines and assays, with half-maximal effective concentrations (EC50s) of 21, 23 and 64 nM against Long (A), M37 (A) and VR-955 (B) strains, respectively, in the primary human bronchial epithelial cells (HBECs). EDP-938 inhibits RSV at a post-entry replication step of the viral life cycle as confirmed by time-of-addition study, and the activity appears to be mediated by viral nucleoprotein (N). In vitro resistance studies suggest that EDP-938 presents a higher barrier to resistance compared to viral fusion or non-nucleoside L polymerase inhibitors with no cross-resistance observed. Combinations of EDP-938 with other classes of RSV inhibitors lead to synergistic antiviral activity in vitro. Finally, EDP-938 has also been shown to be efficacious in vivo in a non-human primate model of RSV infection.

Preclinical Profile and Clinical Efficacy of a Novel Hepatitis C Virus NS5A Inhibitor, EDP-239.

EDP-239, a novel hepatitis C virus (HCV) inhibitor targeting nonstructural protein 5A (NS5A), has been investigated in vitro and in vivo EDP-239 is a potent, selective inhibitor with potency at picomolar to nanomolar concentrations against HCV genotypes 1 through 6. In the presence of human serum, the potency of EDP-239 was reduced by less than 4-fold. EDP-239 is additive to synergistic with other direct-acting antivirals (DAAs) or host-targeted antivirals (HTAs) in blocking HCV replication and suppresses the selection of resistance in vitro Furthermore, EDP-239 retains potency against known DAA- or HTA-resistant variants, with half-maximal effective concentrations (EC50s) equivalent to those for the wild type. In a phase I, single-ascending-dose, placebo-controlled clinical trial, EDP-239 demonstrated excellent pharmacokinetic properties that supported once daily dosing. A single 100-mg dose of EDP-239 resulted in reductions in HCV genotype 1a viral RNA of >3 log10 IU/ml within the first 48 h after dosing and reductions in genotype 1b viral RNA of >4-log10 IU/ml within 96 h. (This study has been registered at ClinicalTrials.gov under identifier NCT01856426.).

Preclinical and Clinical Resistance Profile of EDP-239, a Novel Hepatitis C Virus NS5A Inhibitor.

EDP-239, a potent and selective hepatitis C virus (HCV) nonstructural protein 5A (NS5A) inhibitor developed for the treatment of HCV infection, has been investigated in vitro and in vivo This study sought to characterize genotypic changes in the HCV NS5A sequence of genotype 1 (GT1) replicons and to compare those changes to GT1 viral RNA mutations isolated from clinical trial patients. Resistance selection experiments in vitro using a subgenomic replicon identified resistance-associated mutations (RAMs) at GT1a NS5A amino acid positions 24, 28, 30, 31, and 93 that confer various degrees of resistance to EDP-239. Key RAMs were similarly identified in GT1b NS5A at amino acid positions 31 and 93. Mutations F36L in GT1a and A92V in GT1b do not confer resistance to EDP-239 individually but were found to enhance the resistance of GT1a K24R and GT1b Y93H. RAMs were identified in GT1 patients at baseline or after dosing with EDP-239 that were similar to those detected in vitro Baseline RAMs identified at NS5A position 93 in GT1, or positions 28 or 30 in GT1a only, correlated with a reduced treatment response. RAMs at additional positions were also detected and may have contributed to reduced EDP-239 efficacy. The most common GT1a and GT1b RAMs found to persist up to weeks 12, 24, or 48 were those at NS5A positions 28, 30, 31, 58 (GT1a only), and 93. Those RAMs persisting at the highest frequencies up to weeks 24 or 48 were L31M and Q30H/R for GT1a and L31M and Y93H for GT1b. (This study has been registered at ClinicalTrials.gov under identifier NCT01856426.).

An extensive network of information flow through the B1b/c intersubunit bridge of the yeast ribosome.

Yeast ribosomal proteins L11 and S18 form a dynamic intersubunit interaction called the B1b/c bridge. Recent high resolution images of the ribosome have enabled targeting of specific residues in this bridge to address how distantly separated regions within the large and small subunits of the ribosome communicate with each other. Mutations were generated in the L11 side of the B1b/c bridge with a particular focus on disrupting the opposing charge motifs that have previously been proposed to be involved in subunit ratcheting. Mutants had wide-ranging effects on cellular viability and translational fidelity, with the most pronounced phenotypes corresponding to amino acid changes resulting in alterations of local charge properties. Chemical protection studies of selected mutants revealed rRNA structural changes in both the large and small subunits. In the large subunit rRNA, structural changes mapped to Helices 39, 80, 82, 83, 84, and the peptidyltransferase center. In the small subunit rRNA, structural changes were identified in helices 30 and 42, located between S18 and the decoding center. The rRNA structural changes correlated with charge-specific alterations to the L11 side of the B1b/c bridge. These analyses underscore the importance of the opposing charge mechanism in mediating B1b/c bridge interactions and suggest an extensive network of information exchange between distinct regions of the large and small subunits.

A rapid, inexpensive yeast-based dual-fluorescence assay of programmed--1 ribosomal frameshifting for high-throughput screening.

Programmed -1 ribosomal frameshifting (-1 PRF) is a mechanism that directs elongating ribosomes to shift-reading frame by 1 base in the 5' direction that is utilized by many RNA viruses. Importantly, rates of -1 PRF are fine-tuned by viruses, including Retroviruses, Coronaviruses, Flavivriuses and in two endogenous viruses of the yeast Saccharomyces cerevisiae, to deliver the correct ratios of different viral proteins for efficient replication. Thus, -1 PRF presents a novel target for antiviral therapeutics. The underlying molecular mechanism of -1 PRF is conserved from yeast to mammals, enabling yeast to be used as a logical platform for high-throughput screens. Our understanding of the strengths and pitfalls of assays to monitor -1 PRF have evolved since the initial discovery of -1 PRF. These include controlling for the effects of drugs on protein expression and mRNA stability, as well as minimizing costs and the requirement for multiple processing steps. Here we describe the development of an automated yeast-based dual fluorescence assay of -1 PRF that provides a rapid, inexpensive automated pipeline to screen for compounds that alter rates of -1 PRF which will help to pave the way toward the discovery and development of novel antiviral therapeutics.

The central core region of yeast ribosomal protein L11 is important for subunit joining and translational fidelity.

Yeast ribosomal protein L11 is positioned at the intersubunit cleft of the large subunit central protuberance, forming an intersubunit bridge with the small subunit protein S18. Mutants were engineered in the central core region of L11 which interacts with Helix 84 of the 25S rRNA. Numerous mutants in this region conferred 60S subunit biogenesis defects. Specifically, many mutations of F96 and the A66D mutant promoted formation of halfmers as assayed by sucrose density ultracentrifugation. Halfmer formation was not due to deficiency in 60S subunit production, suggesting that the mutants affected subunit-joining. Chemical modification analyses indicated that the A66D mutant, but not the F96 mutants, promoted changes in 25S rRNA structure, suggesting at least two modalities for subunit joining defects. 25S rRNA structural changes were located both adjacent to A66D (in H84), and more distant (in H96-7). While none of the mutants significantly affected ribosome/tRNA binding constants, they did have strong effects on cellular growth at both high and low temperatures, in the presence of translational inhibitors, and promoted changes in translational fidelity. Two distinct mechanisms are proposed by which L11 mutants may affect subunit joining, and identification of the amino acids associated with each of these processes are presented. These findings may have implications for our understanding of multifaceted diseases such as Diamond--Blackfan anemia which have been linked in part with mutations in L11.

A flexible loop in yeast ribosomal protein L11 coordinates P-site tRNA binding.

High-resolution structures reveal that yeast ribosomal protein L11 and its bacterial/archael homologs called L5 contain a highly conserved, basically charged internal loop that interacts with the peptidyl-transfer RNA (tRNA) T-loop. We call this the L11 'P-site loop'. Chemical protection of wild-type ribosome shows that that the P-site loop is inherently flexible, i.e. it is extended into the ribosomal P-site when this is unoccupied by tRNA, while it is retracted into the terminal loop of 25S rRNA Helix 84 when the P-site is occupied. To further analyze the function of this structure, a series of mutants within the P-site loop were created and analyzed. A mutant that favors interaction of the P-site loop with the terminal loop of Helix 84 promoted increased affinity for peptidyl-tRNA, while another that favors its extension into the ribosomal P-site had the opposite effect. The two mutants also had opposing effects on binding of aa-tRNA to the ribosomal A-site, and downstream functional effects were observed on translational fidelity, drug resistance/hypersensitivity, virus maintenance and overall cell growth. These analyses suggest that the L11 P-site loop normally helps to optimize ribosome function by monitoring the occupancy status of the ribosomal P-site.