Broad-Spectrum Small-Molecule Inhibitors Targeting the SAM-Binding Site of Flavivirus NS5 Methyltransferase

Abstract

Flavivirus infections, such as those caused by dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV), and Zika virus (ZIKV), pose a rising threat to global health. There are no FDA-approved drugs for flaviviruses, although a small number of flaviviruses have vaccines. For flaviviruses or unknown viruses that may appear in the future, it is particularly desirable to identify broad-spectrum inhibitors. The NS5 protein is regarded as one of the most promising flavivirus drug targets because it is conserved across flaviviruses. In this study, we used FL-NAH, a fluorescent analog of the methyl donor S-adenosyl methionine (SAM), to develop a fluorescence polarization (FP)-based high throughput screening (HTS) assay to specifically target methyltransferase (MTase), a vital enzyme for flaviviruses that methylates the N7 and 2'-O positions of the viral 5'-RNA cap. Pilot screening identified two candidate MTase inhibitors, NSC 111552 and 288387. The two compounds inhibited the FL-NAH binding to the DENV3 MTase with low micromolar IC₅₀. Functional assays verified the inhibitory potency of these molecules for the flavivirus MTase activity. Binding studies indicated that these molecules are bound directly to the DENV3 MTase with similar low micromolar affinity. Furthermore, we showed that these compounds greatly reduced ZIKV replication in cell-based experiments at dosages that did not cause cytotoxicity. Finally, docking studies revealed that these molecules bind to the SAM-binding region on the DENV3 MTase, and further mutagenesis studies verified residues important for the binding of these compounds. Overall, these compounds are innovative and attractive candidates for the development of broad-spectrum inhibitors for the treatment of flavivirus infections.

Keywords: NS5; broad spectrum; flaviviruses; high throughput screening; methyltransferase.

Figure 1.

Binding of FL-NAH to flavivirus MTases. (A) Chemical structure of FL-NAH. (B) Dose-dependent FL-NAH FP assay. FL-NAH (50 nM) was applied to 2-fold diluted concentration series of DENV3, WNV, YFV and ZIKV MTases. FP was calculated by measuring the parallel and perpendicular fluorescence with excitation and emission wavelengths of 485 nm and 528 nm, respectively. N = 3. (C) Dose-dependent inhibition of FL-NAH binding to the DENV3 NS5 MTase by SAM, SAH, and NSC12155. The DENV3 MTase was incubated with concentration series of compounds for 30 min. FL-NAH (50 nM) was added and further incubated for 30 min before fluorescence measurement. N = 3. (D) SAH 25 μM inhibited FL-NAH (50 nM) binding to the DENV3 NS5 MTase (0.5 μM) MTases in 96-well plate. ****, p < 0.0001.

Figure 2.

HTS of the NCI Diversity Set VI. (A) FL-NAH FP HTS assay statistics against the NCI Diversity Set VI compound library. Left axis, Z’-factor and CV; right axis, S/B. (B) Screening flowchat. (C) Structures of NSC 111552 and 288387. (D) Dose-dependent inhibition of FL-NAH binding to the DENV3 NS5 MTase by NSC 111552 and 288387 in the presence and absence of DTT (1 mM). N = 3. Assay was performed similarly as described in Figure 1C. FP values in the presence of compounds were normalized to that of the DMSO control (100%). (E) Dose-dependent inhibition of FL-NAH binding to hRNMT by NSC 111552 and 288387. N = 3.

Figure 3.

Functional DENV3 NS5 MTase activity assays. (A) HTRF analysis of inhibition of the N7 MTase activity of the DENV3 MTase by SAH at 25 μM. ****, p<0.0001. (B) HTRF analyses of dose-dependent inhibition of the N7 MTase activity of viral MTases of DENV3 ZIKV, WNV, and YFV by compounds NSC 111552 and 288387. N = 3. HTRF values in the presence of compounds were reverse normalized to that of the (−) MTase control (0%) and that of the DMSO control (100%). (C) TLC analyses of dose−response inhibition of the N7 methylation activity of the WNV NS5 MTase by compound NSC 111552 and 288387 (left panels). The migration positions of the G*pppA and m⁷G*pppA molecules were labeled on the side of the TLC images. Right panels, curve fitting to determine the ${\text{IC}}_{50}$ values for each compound on the N7 activities of the WNV NS5 MTase. The percentage of activity was determined after quantification of G*pppA and m7G*pppA. The ${\text{IC}}_{50}$ value was determined by fitting of the dose−response curve.

Figure 4.

Analysis of antiviral activity and cytotoxicity of compounds NSC 111552 and 288387. (A) Cytotoxicity of NSC 111552 and 288387 were evaluated in A549 cells. (B) Dose-dependent inhibition of DENV2 replication using a DENV2 replicon cell line by NSC 111552 and 288387. N = 3. (C) IFA analysis of inhibition of viral replication by NSC 111552 and 288387 against DENV2 and ZIKV. N = 3. (D) Dose-dependent inhibition of Zika-Venus by NSC 111552 and 288387. N = 3.

Figure 5.

Binding analysis of NSC 111552 and 288387 to the DENV3 NS5 MTase protein using MST. Dose-response curve was generated by fitting experimental data by titrating NSC 111552 and 288387 from 0.2 mM to 6.1 nM against the DENV3 NS5 MTase (10 nM). N = 3.

Figure 6.

Binding modes of NSC 111552 (A) and 288387 (B) to the DENV3 MTase in the SAM-binding site (A,B). 2D-Interaction maps are shown in right panels where $\mathrm{\pi }$-cation interactions are indicated in red, $\mathrm{\pi }-\mathrm{\pi }$ interactions are indicated in green and hydrogen bonding is indicated in purple with arrow(s) indicating role as donor or acceptor 3D diagram shows dashed lines indicating interaction type by color where hydrogen bonding is yellow, $\mathrm{\pi }$-cation interactions are green, $\mathrm{\pi }-\mathrm{\pi }$ and aromatic hydrogen bonding interactions are shown in blue.

Figure 7.

Functional assays of the DENV3 MTase mutants. (A) SDS-PAGE analysis of the purified WT and mutant DENV3 MTases. (B) The HTRF analysis of the N7 MTase activity of the DENV3 MTase Mutants. All proteins were at 1 μM. The MTase activity of WT was set to 100%. (C) Analysis of binding of FL-NAH to WT and mutant DENV3 MTases. The FP value for the WT MTase in binding FL-NAH was set to 100%. (D) The HTRF analysis of inhibition of the N7 MTase activity of the DENV3 MTase mutants by DMSO, NSC111552 at 10 μM, or NSC288387 at 15 μM. All proteins were at 1 μM. (E) $I{C}_{\mathit{50}\mathit{-disp}}$ fold change of inhibition of FL-NAH binding to the mutant DENV3 MTases relative to those of WT by NSC 111552 and 288387.