Toward the identification of viral cap-methyltransferase inhibitors by fluorescence screening assay

Abstract

Two highly pathogenic human coronaviruses associated with severe respiratory syndromes emerged since the beginning of the century. The severe acute respiratory syndrome SARS-coronavirus (CoV) spread first in southern China in 2003 with about 8000 infected cases in few months. Then in 2012, the Middle East respiratory syndrome (MERS-CoV) emerged from the Arabian Peninsula giving a still on-going epidemic associated to a high fatality rate. CoVs are thus considered a major health threat. This is especially true as no vaccine nor specific therapeutic are available against either SARS- or MERS-CoV. Therefore, new drugs need to be identified in order to develop antiviral treatments limiting CoV replication. In this study, we focus on the nsp14 protein, which plays a key role in virus replication as it methylates the RNA cap structure at the N7 position of the guanine. We developed a high-throughput N7-MTase assay based on Homogenous Time Resolved Fluorescence (HTRF) and screened chemical libraries (2000 compounds) on the SARS-CoV nsp14. 20 compounds inhibiting the SARS-CoV nsp14 were further evaluated by IC determination and their specificity was assessed toward flavivirus- and human cap N7-MTases. Our results reveal three classes of compounds: 1) molecules inhibiting several MTases as well as the dengue virus polymerase activity unspecifically, 2) pan MTases inhibitors targeting both viral and cellular MTases, and 3) inhibitors targeting one viral MTase more specifically showing however activity against the human cap N7-MTase. These compounds provide a first basis towards the development of more specific inhibitors of viral methyltransferases.

Direct High‐Throughput Screening Assay for mRNA Cap Guanine‐N7 Methyltransferase Activity

“…Quercetin and myricetin have already been found to inhibit activity of SARS-nsp14 guanine-N7 methyltransferase, and human RNMT-RAM enzymes. [35] An interesting finding was that reactive blue 2a lso potently inhibited N7-MTases. Based on the comparison of our hits with structurally similarl ibrary compounds with lower or no activity,w ec onclude that the presence of sulfonic groups tends to increase the inhibitory potential.…”

“…[34] At ime-resolved fluorescence method for studies on SARS-nsp14 enzymeh as also been developed. [35] All of these methods provided insightsi nto N7-MTase activity and inhibition. However,t ot he besto fo ur knowledge, there is no direct method allowing N7-MTase activity to be monitored in real-time by using smallm olecular probes.…”

RNA Cap Methyltransferase Activity Assay

“…We note that an alternative, nonradioactive assay has been reported for the analysis of cap methyltransferase reactions (Peyrane et al , 2007), but this method requires HPLC instrumentation that may not be available to all labs and may differ from the one presently reported in terms of sensitivity and sample compatibility. Additionally, a fluorescent assay for measuring cap methyltransferase activity was recently described (Aouadi et al , 2017), but this assay indirectly measures activity by monitoring the accumulation of S-adenosylhomocysteine (SAH) and may be incompatible with biological samples containing SAH. We hope that the level of detail in the protocol presented here enables future investigators to easily repeat and build upon our work.…”

An updated roadmap for MERS-CoV research and product development: focus on diagnostics

“…Effective MERS therapeutics are still in the early stages of research and evaluation. Several broad-spectrum antiviral agents including nitazoxanide,56 viral methyltransferase inhibition57 and nucleotide prodrugs58 have shown in vitro activity against MERS-CoV. Early results for novel MERS-specific therapeutics that inhibit viral replication or have specific neutralising activity are promising 47 59 60…”