Compounds 1–16 were tested for their ability to inhibit the methylation of the RNA cap structure. The inhibition induced by each compound (50 μM) was determined by a radioactive MTase assay (filter binding assay) which consists in measuring the [3H] radiolabeled methyl transferred from the methyl donor SAM onto RNA substrate (GpppAC4) synthetized by using T7 primase [33]. Compounds 1–16 designed as mimics of the transition state of RNA 2′-O-methylation were screened against several viral RNA 2′-O-MTases from SARS-CoV (nsp10/nsp16 complex), Zika, West-Nile, Dengue, Vaccinia (VP39) viruses. At the same time, the compounds were tested against human RNA N7-MTase (hRNMT) and selected viral N7-MTases such as SARS-CoV nsp14 and Vaccinia D1-D12 complex to evaluate their specificity (Table 1 ).
Table 1 Screening for inhibitory activity of sinefungin and compounds 1–16 at 50 μM on N7-MTases.
Compounds Percentage of inhibition at 50 μM (%)a
SARS-CoV nsp14 Vaccinia virusD1-D12 hRNMT
Sinefungin 98.3 ± 0.2 99.8 ± 0.1 99.8 ± 0.2
1 31.0 ± 6.8 20.3 ± 0.8 35.2 ± 4.9
2 72.0 ± 1.2 85.8 ± 2.5 77.4 ± 1.2
3 30.6 ± 9.3 32.1 ± 2.4 33.2 ± 4.3
4 13.1 ± 13.3 53.2 ± 2.6 12.2 ± 2.1
5 n.i n.i 27.5 ± 6.6
6 38.4 ± 11.7 11.6 ± 7.1 23.1 ± 9.7
7 n.i 69.2 ± 1.9 32.8 ± 16.1
8 43.0 ± 4.0 n.i n.i
9 88.6 ± 1.3 49.8 ± 3.2 66.0 ± 6.1
10 96.6 ± 0.9 4.6 ± 0.3 31.8 ± 3.3
11 47.6 ± 2.8 5.3 ± 4.3 44.2 ± 8.5
12 94.6 ± 1.1 10.1 ± 5.5 23.3 ± 3.6
13 97.2 ± 2.7 2.8 ± 0.8 33.9 ± 3.3
14 96.2 ± 1.5 19.7 ± 3.8 20.2 ± 9.4
15 94.0 ± 1.1 4.3 ± 3.9 n.i
16 75.9 ± 2.5 4.5 ± 15.1 14.7 ± 1.3
aValues are the mean of three independent experiments. The MTase activity was measured using a filter binding assay. Assays were carried out in reaction mixture [40 mM Tris-HCl (pH 8.0), 1 mM DTT, 1 mM MgCl2, 2 μM SAM and 0.1 μM 3H-SAM] in the presence of 0.7 μM GpppAC4 synthetic RNA and incubated at 30 °C. SARS-CoV nsp14 (50 nM), vaccinia virus capping enzyme (D1-D12) (41 U), human RNA N7 MTase (hRNMT) (50 nM). Compounds were previously dissolved in 100% DMSO. n.i: no inhibition detected at 50 μM.