To confirm the observed inhibition of some adenine dinucleosides against SARS-CoV nsp14 N7-MTase, we further tested 10 compounds in a dose-response assay (Table 2 , Supporting Information Fig. S2). The selection of these compounds was based on their percentage inhibition higher than 50% against SARS-CoV nsp14 (Table 1). After pre-incubation with increasing concentrations of dinucleosides, the MTase activity was measured by FBA. The IC50 of compounds, deduced from Hill slope equation (Y = 100/(1+((X/IC50)^Hillslope) curve-fitting, ranged from 0.6 μM to 70.4 μM on SARS-CoV nsp14 and 10.9 μM to > 500 μM on hRNMT activity. Among all the potential inhibitors, three compounds 2, 6 and 11 showed the lowest inhibitory activity on SARS-CoV nsp14 with IC50 > 40 μM, the others have IC50 values in the micromolar range except 13 that showed an IC50 value in the submicromolar range. The potency of the derivative 13 to inhibit SARS-CoV nsp14 was comparable to the broad spectrum inhibitor sinefungin. However, it is noteworthy that compound 13 specifically inhibits the N7-MTase of SARS-CoV with a high 413-fold of specificity in dose-response testing whereas sinefungin is also active against hRNMT [11]. Remarkably 13 bearing a chlorine atom in “para” position and a nitro group in “meta” position displayed the best inhibition which was 6.5–9.5 fold more potent than the derivatives 10 and 12, respectively that contain the nitro in “ortho” position and a lipophilic group CF3 or MeO in “para” position. The comparison of IC50 values of 14 and 13 indicates that the addition of a nitro group in the phenyl ring increased the inhibitory activity by 2.5 fold in 13. These results confirm that the chlorine atom in “para” position and the presence of a nitro group seem crucial for submicromolar SARS-CoV nsp14 inhibitory activity.
Table 2 Comparison of IC50 values of 10 most active compounds on SARS-CoV nsp14 and human RNMT activities.
Compounds SARS-CoV nsp14 IC50a (μM) hRNMTIC50 (μM)
Sinefunginb 0.36 <0.05
2 40.6 ± 3.2 10.9 ± 1.0
6 55.5 ± 5.1 >500
9 2.6 ± 0.2 >500
10 3.9 ± 0.4 >500
11 70.4 ± 4.9 169.3 ± 30.4
12 5.7 ± 0.5 275.9 ± 28.7
13 0.6 ± 0.1 247.5 ± 14.9
14 1.5 ± 0.1 n.d
15 2.4 ± 0.2 n.i
16 9.9 ± 0.9 n.d
a Concentration inhibiting MTase activity by 50%; mean value from three independent experiments.
b values from the literature [11]. The MTase activity for IC50 determinations was measured using a filter binding assay as described above. n.i: no inhibition detected at 50 μM; n.d: not determined due to inhibition lower than 50% at 50 μM.