Probing the SAM Binding Site of SARS-CoV-2 Nsp14 In Vitro Using SAM Competitive Inhibitors Guides Developing Selective Bisubstrate Inhibitors

Abstract

The COVID-19 pandemic has clearly brought the healthcare systems worldwide to a breaking point, along with devastating socioeconomic consequences. The SARS-CoV-2 virus, which causes the disease, uses RNA capping to evade the human immune system. Nonstructural protein (nsp) 14 is one of the 16 nsps in SARS-CoV-2 and catalyzes the methylation of the viral RNA at N7-guanosine in the cap formation process. To discover small-molecule inhibitors of nsp14 methyltransferase (MTase) activity, we developed and employed a radiometric MTase assay to screen a library of 161 in-house synthesized S-adenosylmethionine (SAM) competitive MTase inhibitors and SAM analogs. Among six identified screening hits, SS148 inhibited nsp14 MTase activity with an IC₅₀ value of 70 ± 6 nM and was selective against 20 human protein lysine MTases, indicating significant differences in SAM binding sites. Interestingly, DS0464 with an IC₅₀ value of 1.1 ± 0.2 µM showed a bisubstrate competitive inhibitor mechanism of action. DS0464 was also selective against 28 out of 33 RNA, DNA, and protein MTases. The structure-activity relationship provided by these compounds should guide the optimization of selective bisubstrate nsp14 inhibitors and may provide a path toward a novel class of antivirals against COVID-19, and possibly other coronaviruses.

Keywords: COVID-19; SARS-CoV-2; coronavirus; nsp14.

Figure 1

Kinetic characterization and screening of nsp14 MTase activity. The optimized radiometric MTase assay was used to determine the K_m^app and k_cat^app values for (a) RNA and (b) SAM at saturating concentrations of SAM and RNA, respectively. (c) The linearity of the initial velocity of the nsp14 MTase reaction was tested at K_m for both substrates. (d) The Z′ factor was determined at the same conditions as in c. Nsp14 activity was assessed in the absence (100% activity) () and presence () of 1 µM sinefungin. (e) Nsp14 was screened against a library of SAM analogs. A collection of 161 SAM competitive inhibitors and analogs was tested at 50 µM for inhibition of nsp14 activity under conditions described for Z′-factor determination. Compounds with higher than 75% inhibition () were selected for follow-up experiments. Values in a, b, and c are presented as the mean ± standard deviation of three independent experiments (n = 3). Values in d and e are from single screening.

Figure 2

Structures of nsp14 screening hits.

Figure 3

Dose–response curves of nsp14 screening hits. The IC₅₀ values were determined for (a) SS148 (0.07 ± 0.006 µM), (b) WZ16 (0.19 ± 0.04 µM), (c) DS0464 (1.1 ± 0.2 µM), (d) DS0466 (3.4 ± 1.4 µM), (e) JL27-56A1 (0.26 ± 0.13 µM), (f) compound 8 (95 ± 6 µM), (g) SAH (0.13 ± 0.03 µM), and (h) MTTR025495 (17 ± 2 µM) by testing their inhibitory effects ranging from 3 nM to 200 µM final concentration. IC₅₀ values are also presented in Table 1. All values are presented as the mean ± standard deviation of three independent experiments (n = 3).

Figure 4

Docking model of DS0464. (a) A large and enclosed binding pocket (yellow) in the MTase domain of nsp14 accommodates both the methyl-donating cofactor (gray; SAH is shown instead) and the methyl-accepting RNA cap GpppA (purple) (PDB code 5C8S). Black arrow, site of methyl transfer. (b) The docked inhibitor DS0464 occupies the cofactor site and extends into the substrate binding site.

Figure 5

MOA of DS0464. IC₅₀ values were determined for DS0464 at (a) varying concentrations of RNA at 1.25 µM SAM and (b) varying concentrations of SAM at 250 nM RNA. DS0464 competes against both RNA and SAM. All values are presented as the mean ± standard deviation of three independent experiments (n = 3).

Figure 6

Structural determinant for SS148 selectivity. A nitrile group (stick) at position 7 of the cofactor adenine ring is compatible with the crystal structure of SAH (wire) bound to (a) nsp14 and (b) BCDIN3D but not (c) G9a. The Van der Waals surface of the cofactor binding pocket is shown as gray mesh. Dose–response curves for SS148 against these proteins with their IC₅₀ values are also shown.

Figure 7

Selectivity of SS148 and DS0464. Both SS148 and DS0464 were screened for selectivity against 33 human RNA, DNA, and protein MTases at 50 µM. All experiments were performed in duplicate. When significant inhibition was observed, IC₅₀ values were determined and are presented in Table 2.