A High-Throughput RNA Displacement Assay for Screening SARS-CoV-2 nsp10-nsp16 Complex toward Developing Therapeutics for COVID-19

Abstract

SARS-CoV-2, the coronavirus that causes COVID-19, evades the human immune system by capping its RNA. This process protects the viral RNA and is essential for its replication. Multiple viral proteins are involved in this RNA capping process, including the nonstructural protein 16 (nsp16), which is an S-adenosyl-l-methionine (SAM)-dependent 2'-O-methyltransferase. Nsp16 is significantly active when in complex with another nonstructural protein, nsp10, which plays a key role in its stability and activity. Here we report the development of a fluorescence polarization (FP)-based RNA displacement assay for nsp10-nsp16 complex in a 384-well format with a Z' factor of 0.6, suitable for high-throughput screening. In this process, we purified the nsp10-nsp16 complex to higher than 95% purity and confirmed its binding to the methyl donor SAM, the product of the reaction, S-adenosyl-l-homocysteine (SAH), and a common methyltransferase inhibitor, sinefungin, using isothermal titration calorimetry (ITC). The assay was further validated by screening a library of 1124 drug-like compounds. This assay provides a cost-effective high-throughput method for screening the nsp10-nsp16 complex for RNA competitive inhibitors toward developing COVID-19 therapeutics.

Keywords: COVID-19; SARS-CoV-2; coronavirus; nsp10; nsp16.

Figure 1

Binding assay. Binding of the nsp10-nsp16 complex to (A) SAM, (B) SAH, and (C) sinefungin was confirmed by ITC with K_d values of 3.4 ± 1.5 µM, 5.7 ± 1.9 µM, and 6.8 ± 2.0 µM, respectively. The K_d values are the mean ± standard deviation from three independent experiments.

Figure 2

FP assay optimization for nsp10-nsp16 interaction with N7-meGpppACCCCC-FAM. The FP signal from the interaction of 0.5 µM nsp10-nsp16 complex with 30 nM N7-meGpppACCCCC-FAM was evaluated at (A) a pH range of 7.0 to 9.0 in 10 mM Tris buffer. The effect of additives such as (B) DTT, (C) NaCl, (D) EDTA, (E) Triton X-100, and (F) DMSO was evaluated in 10 mM Tris, pH 7.5. Plotted values are the mean ± standard deviation of three independent experiments. The FP values are blank subtracted and presented as a percentage of the control. In A, pH 7.5 was considered as 100% signal. Data were analyzed using GraphPad Prism software 7.0.4.

Figure 3

FP saturation binding and competitive displacement curves for the nsp10-nsp16 complex. (A) The nsp10-nsp16 complex binds to 5′ N7-meGpppACCCCC-FAM 3′ with a K_d of 0.13 ± 0.002 µM and Bmax of 270 ± 5 mP. Unlabeled methylated cap RNAs, (B) N7-meGpppG and (C) N7-meGpppA, compete with 5′ N7-meGpppACCCCC-FAM 3′ for binding to the nsp10-nsp16 complex. Unlabeled unmethylated cap RNAs, (D) GpppG and (E) GpppA, did not disrupt the interaction between the nsp10-nsp16 complex and RNA-FAM. All values are the mean ± standard deviation of three independent experiments. (F) The Z′ factor (0.6) was determined for evaluation of the assay for high-throughput screening. FAM-labeled RNA (5′ N7-meGpppACCCCC-FAM 3′) at 30 nM was used to generate the FP signal, while 50 µM unlabeled RNA (N7-meGpppA) was used as a positive control. Data were analyzed using GraphPad Prism software 7.0.4.

Figure 4

Screening Prestwick Compound Library. The distribution of the readout for 1124 compounds screened by the RNA displacement assay is shown. Compounds that significantly reduced the polarization signal (>50%) compared with the control were selected as screening hits (highlighted in red). Three compounds (highlighted in green) showed concentration-dependent inhibition. However, none of these compounds showed any binding when tested by ITC (data not shown).