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 IC₅₀ 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.

Keywords: Antiviral; Coronavirus; Flavivirus; HTRF; Inhibitor; Methyltransferase.

Fig. 1

Characterization of the recombinant SARS-CoV nsp14. A) 2.5 μg of SARS-CoV nsp14 on a 10% SDS-PAGE gel stained by Coomassie blue. MW, molecular size markers. B) Bar graph representing the N7-MTase activity of SARS-nsp14 determined by monitoring the transfer of tritiated methyl from [³H] SAM onto GpppA and ^7mGpppA. The tritiated RNA was quantified by DEAE filter binding assay (FBA).

Fig. 2

HTRF assay validation using SARS-nsp14. A) Time course analysis of SARS-CoV nsp14 MTase activity by HTRF assay: 0 or 5 nM of nsp14 (empty or full triangles, respectively) are incubated with 8 μM GpppA and 2 μM SAM at 30 °C for increasing time periods. The reaction by-product (SAH) was quantified by measuring the ratio of emission and excitation fluorescence at 665 and 620 nm, on a PolarStar reader as described in materials and methods. B) Sinefungin IC_{50 (HTRF)} determination. Increasing concentrations of sinefungin (0–0.5 μM) were incubated with 5 nM nsp14, 2 μM SAM, 8 μM GpppA at 30 °C during 20 min and the SAH by-product was detected as described in panel A. The IC_{50 (HTRF)} was then calculated using graphPad Prism equation (n = 3; Mean value ± SD).

Fig. 3

Distribution of inhibition percentage values obtained by HTRF screening on Prestwick Chemical libraries. The 2000 compounds were screened at 50 μM, 5% DMSO on SARS CoV nsp14 MTase activity using HTRF assay (5 nM nsp14, 8 μM GpppA, 2 μM SAM, SAH-d2 (1/16) and anti-SAH (1/150)). The grey line corresponds to an increase of 20% of the HTRF signal that was used as a threshold for the selection of the primary hits.