The HCV IRES RNA is an attractive drug target due to its central role in regulating viral protein synthesis and pan-genotypic sequence conservation. In this study, we screened a small molecule library for specific inhibitors of HCV IRES function. The results of this work highlight both successes in high-throughput assay development and challenges inherent in identifying compounds that recognize RNA targets.
Previous work established that direct IRES binding by an antisense oligonucleotide can block IRES-mediated protein synthesis.14
Although this nucleic acid based candidate (ISIS 14803) reached Phase I clinical trials, limited on-target activity and aminotransferase flares led to a halt of clinical development.15
Additionally, there have been other attempts to identify IRES-specific inhibitors by high-throughput screens with smaller libraries. Fragment-based screening using mass spectrometry has identified small molecule binders to domain II of the IRES which are inhibitors of an HCV replicon assay.25
A dual luciferase-based HTS assay in Krebs extracts with a bicistronic reporter did not reveal any IRES-specific compounds.26
We sought to identify new chemical scaffolds that directly target IRES function and could be further optimized to yield potent oral antiviral agents.
A screening assay was developed using RRL supplemented with additional salts, which improved the fidelity of both cap-dependent and HCV IRES-mediated translation. These results are analogous to what has previously been seen with the encephalomyocarditis virus (EMCV) IRES (although the EMCV IRES requires a different set of translation initiation factors from the HCV IRES).27
The use of two monocistronic reporters (one IRES-driven and one 5′ cap-driven) enabled each reporter’s signal to be maximized independently, while avoiding competition between the two reporter templates, which cannot be achieved using a single bicistronic mRNA. We noted that in general, many more compounds in the secondary screen inhibited the RN enzyme than the FF enzyme, and no compounds inhibited both. This result suggests that the primary screen was effective at excluding general luciferase inhibitors, such as competitive inhibitors of the ATP substrate or protein aggregants. However, ~3% of the primary hits appeared to be general translation inhibitors (in the upper left-hand quadrant of ) which would have ideally been eliminated in the primary screen.
Due to issues with luciferase interference, an [35S]methionine-incorporation validation assay was essential to verify the inhibition activity of the fifty-nine compounds which appeared to be selective IRES inhibitors in the secondary screen with either the firefly or Renilla reporter. Two of these compounds were found to be general translation inhibitors and, to the best of our knowledge, represent a new chemical scaffold of general translation inhibitors. The compound GS-036984 was selected from the primary screen as a potent hit, consistent with it inhibiting both 5′ cap- and IRES-dependent translation. The selectivity indices determined in the secondary screen for 5′ cap/IRES were barely above the cut-off of 3.0 (3.9 with the Renilla reporter and 3.3 with the firefly reporter). While this low selectivity suggested a potential preference for inhibiting the IRES, the direct visualization of translation products without interference of luciferase reporters clearly showed that this compound inhibits both 5′ cap-dependent and IRES-dependent translation ().
In the [35
S]methionine-incorporation validation assay, 57 out of 59 of the compounds identified in the secondary luciferase screen showed no inhibition against either IRES- or 5′ cap-dependent translation. This observation suggests that these compounds are likely to be direct inhibitors of Renilla
luciferase enzyme. Several scaffolds emerged from the compounds showing selective Renilla
luciferase inhibition, such as para-amino-sulfonamides, sulfanyltriazoles, and dihydropyrrolones. The ten most potent Renilla-specific inhibitors identified in the screen are shown in Supplementary Table 2
. These Renilla
luciferase inhibitors may not have been eliminated in the secondary screen due to differences in the assay conditions, such as the temperature of incubation and the higher final luciferase protein levels in the enzymatic interference assay due to the lack of DMSO during translation. A subset of compounds that showed no inhibition at the HTS conditions for the enzymatic interference assay (incubation at room temperature for 30 minutes) demonstrated enhanced inhibitory activity against the luciferase enzymes when the incubation conditions were changed to more closely match the primary assay (30°C for 90 minutes). Additionally, when the interference assay was performed using recombinant Renilla
luciferase enzyme rather than in vitro
translated enzyme, an even larger subset of compounds (~80% of the 54 hits) showed inhibition when incubated at 30°C for 90 minutes.
The relative abundance of luciferase inhibitors identified in this study reflects the fact that it is much simpler chemically for a compound to block an enzyme active site than to bind to a macromolecular surface and inhibit a conformational change or an intermolecular interaction (note that a large fraction (~40%) of current drug targets are enzymes and many other drug targets also naturally bind to small molecules).28
Our secondary counter screens eliminated most, but not all, of the facile and prevalent direct inhibitors of Renilla
luciferase. The high level of Renilla
luciferase inhibitors selected in this screen suggests there is an inherent problem in using an enzymatic reporter assay as a means to screen for compounds that interact with a particularly challenging target, such as a structured RNA, as the reporter enzyme is itself a classical target of small molecules.
In order to expand our ability to devise new and diverse drugs, the scope of small molecule activities must be expanded to non-traditional targets, such as influencing macromolecular interactions and conformations, and efforts are underway to expand the diversity of small molecules in screening libraries.29
Based on our experience with enzyme inhibition masking the desired RNA-based inhibition, assays for new functions would do well to avoid traditional small molecule targets as read-outs for activity. Fluorescence, as opposed to luminescence, may be an alternative approach to couple translation activity to a spectroscopic signal. For example, members of the green fluorescence protein family do not have a defined active site for binding small molecules and may therefore be less subject to direct reporter inhibition. A dual reporter screen could be conducted with a pair of red and green fluorescent proteins, though spectroscopic interference from heterocyclic compounds in the library would need to be considered.30
Additionally, in an effort to reduce enzyme interference, the active site of firefly luciferase has been redesigned and evolved to discourage small molecules binding to the luciferin and ATP pockets. This new enzyme, ‘Ultra-Glo’, shows a marked loss of inhibition from several scaffolds that inhibited the original firefly luciferase.31
Other strategies for identifying RNA-binding small molecules as inhibitors that do not require enzymatic read-outs, such as fragment-based or in silico
screening, could also be considered as alterative approaches.25,32,33
A FRET-based screen for small molecule binders to RNA has previously been used to find compounds that bind to and stabilize the HCV IRES IIId loop,34
but whether these compounds are effective inhibitors of HCV IRES translation has yet to be determined. As a complement to such small molecule binding screens, it is certainly attractive to envision direct functional screens to look for inhibitors of IRES-mediated translation. However, the present study shows that such assays are not as straightforward as they may first appear.
In this study we validated the use of rabbit reticulocyte lysate with increased salt concentrations as a system to study the effects of mutations and inhibitors of the HCV IRES. The current screen has provided insights into how deleterious direct enzyme inhibition can be to an otherwise robust high-throughput assay for a difficult target, such as the HCV IRES. Additionally, a new scaffold has been identified of a general eukaryotic translation inhibitor, the mechanism of which will be interesting to investigate in the future.