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

Abstract

In eukaryotes, mature mRNA is formed through modifications of precursor mRNA, one of which is 5’ cap biosynthesis, involving RNA cap guanine‐N7 methyltransferase (N7‐MTase). N7‐MTases are also encoded by some eukaryotic viruses and facilitate their replication. N7‐MTase inhibitors have therapeutic potential, but their discovery is difficult because long RNA substrates are usually required for activity. Herein, we report a universal N7‐MTase activity assay based on small‐molecule fluorescent probes. We synthesized 12 fluorescent substrate analogues (GpppA and GpppG derivatives) varying in the dye type, dye attachment site, and linker length. GpppA labeled with pyrene at the 3’‐O position of adenosine acted as an artificial substrate with the properties of a turn‐off probe for all three tested N7‐MTases (human, parasite, and viral). Using this compound, a N7‐MTase inhibitor assay adaptable to high‐throughput screening was developed and used to screen synthetic substrate analogues and a commercial library. Several inhibitors with nanomolar activities were identified.

Identification and Evaluation of Potential SARS-CoV-2 Antiviral Agents Targeting mRNA Cap Guanine N7-Methyltransferase

“…Based on the determined KM value (3.5 ± 1.1 μM), the optimal probe concentration was set at 1 μM (approximate to KM). The catalytic efficiency (kcat/KM) 42 of nsp14-catalyzed N7-methylation was 0.025 s -1 μM -1 , which was almost two-fold higher than previously calculated for the RNMT-RAM enzyme (0.014 s -1 μM -1 ), 40 indicating that the Py-FLINT probe is a slightly better substrate for SARS-CoV-2 than human N7-MTase.…”

“…Herein, we sought to apply our previously developed N7-MTase Py-FLINT assay 40 for the identification of inhibitors against the nsp14 N7-methyltansferase, which is involved in mRNA capping of SARS-CoV-2. We first confirmed that our assay could be used for monitoring nsp14 activity in real time.…”

“…We confirmed specific N7-methylation by nsp14 and observed that p-benzoquinone (IC50 1.93 ± 0.42 μM) was an even more potent inhibitor than sinefungin . 40 For all compounds, the IC50 values were calculated and compared to the values determined for the viral nsp14 enzyme (Table 1; Figure S11). The correlation between determined IC50 values is illustrated in Figure 5.…”

“…To target the nucleotide-binding site, we used the previously synthesized Gp3A labeled with ( 5)-carboxyfluorescein via the 3'-Oposition of adenosine (Gp3A-3'-O-( 5)FAM). 40 The second probe, designed for the SAM-binding site, was synthesized in four steps, starting from 6-chloropurine riboside (Figure S12).…”

“…We have applied the assay in studies on model N7-MTases, including Ecm1 from E. cuniculi, human RNMT-RAM, and Vaccinia capping enzyme (VCE) from Vaccinia virus. 40 In the current work, we adapted the assay to explore the N7-MTase activity of nsp14 from SARS-CoV-2 in order to identify potent inhibitors of viral mRNA N7-methylation with antiviral drug potential. The method allows for the direct monitoring of nsp14 activity in real time and is suitable for the exploration of inhibitors of both the nucleotideand SAM-binding sites of N7-MTase.…”