Structure-Based Discovery of Inhibitors of the SARS-CoV-2 Nsp14 N7-Methyltransferase.

An under-explored target for SARS-CoV-2 is the S-adenosyl methionine (SAM)-dependent methyltransferase Nsp14, which methylates the N7-guanosine of viral RNA at the 5'-end, allowing the virus to evade host immune response. We sought new Nsp14 inhibitors with three large library docking strategies. First, up to 1.1 billion lead-like molecules were docked against the enzyme's SAM site, leading to three inhibitors with IC50 values from 6 to 50 µM. Second, docking a library of 16 million fragments revealed 9 new inhibitors with IC50 values from 12 to 341 µM. Third, docking a library of 25 million electrophiles to covalently modify Cys387 revealed 7 inhibitors with IC50 values from 3.5 to 39 µM. Overall, 32 inhibitors encompassing 11 chemotypes had IC50 values < 50 µM and 5 inhibitors in 4 chemotypes had IC50 values < 10 µM. These molecules are among the first non-SAM-like inhibitors of Nsp14, providing starting points for future optimization.

SS148 and WZ16 inhibit the activities of nsp10-nsp16 complexes from all seven human pathogenic coronaviruses.

Seven coronaviruses have infected humans (HCoVs) to-date. SARS-CoV-2 caused the current COVID-19 pandemic with the well-known high mortality and severe socioeconomic consequences. MERS-CoV and SARS-CoV caused epidemic of MERS and SARS, respectively, with severe respiratory symptoms and significant fatality. However, HCoV-229E, HCoV-NL63, HCoV-HKU1, and HCoV-OC43 cause respiratory illnesses with less severe symptoms in most cases. All coronaviruses use RNA capping to evade the immune systems of humans. Two viral methyltransferases, nsp14 and nsp16, play key roles in RNA capping and are considered valuable targets for development of anti-coronavirus therapeutics. But little is known about the kinetics of nsp10-nsp16 methyltransferase activities of most HCoVs, and reliable assays for screening are not available. Here, we report the expression, purification, and kinetic characterization of nsp10-nsp16 complexes from six HCoVs in parallel with previously characterized SARS-CoV-2. Probing the active sites of all seven by SS148 and WZ16, the two recently reported dual nsp14 / nsp10-nsp16 inhibitors, revealed pan-inhibition. Overall, our study show feasibility of developing broad-spectrum dual nsp14 / nsp10-nsp16-inhibitor therapeutics.

Tetrahedral DNA framework assisted rotational paper-based analytical device for differential detection of SARS-CoV-2 and influenza A H1N1 virus.

Coronavirus disease 2019 (COVID-19) and influenza A are two respiratory infectious diseases with similar clinical manifestations. Because of the complex global epidemic situation of COVID-19, the distinction and diagnosis of COVID-19 and influenza A infected persons is crucial for epidemic prevention and control. In this study, tetrahedral DNA framework (TDF) was combined with a rotational paper-based analytical device, and the color change generated by the reaction between horseradish peroxidase (HRP) and 3,3'5,5'-tetramethylbenzidine (TMB)-H2O2 was used for grayscale signal analysis by ImageJ software. The quantitative detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A H1N1 virus were realized simultaneously. Under the optimal conditions, the paper-based analytical device showed a good linear relationship between the two viruses in the range of 10-14-10-8g/mL, and the two viruses were not affected by cross reaction. This sensor provides a convenient and reliable method for clinical rapid differentiation and diagnosis of COVID-19 and influenza A.

Crystal structure of SARS-CoV-2 nsp10-nsp16 in complex with small molecule inhibitors, SS148 and WZ16.

SARS-CoV-2 nsp10-nsp16 complex is a 2'-O-methyltransferase (MTase) involved in viral RNA capping, enabling the virus to evade the immune system in humans. It has been considered a valuable target in the discovery of antiviral therapeutics, as the RNA cap formation is crucial for viral propagation. Through cross-screening of the inhibitors that we previously reported for SARS-CoV-2 nsp14 MTase activity against nsp10-nsp16 complex, we identified two compounds (SS148 and WZ16) that also inhibited nsp16 MTase activity. To further enable the chemical optimization of these two compounds towards more potent and selective dual nsp14/nsp16 MTase inhibitors, we determined the crystal structure of nsp10-nsp16 in complex with each of SS148 and WZ16. As expected, the structures revealed the binding of both compounds to S-adenosyl-L-methionine (SAM) binding pocket of nsp16. However, our structural data along with the biochemical mechanism of action determination revealed an RNA-dependent SAM-competitive pattern of inhibition for WZ16, clearly suggesting that binding of the RNA first may help the binding of some SAM competitive inhibitors. Both compounds also showed some degree of selectivity against human protein MTases, an indication of great potential for chemical optimization towards more potent and selective inhibitors of coronavirus MTases.

Kinetic Characterization of SARS-CoV-2 nsp13 ATPase Activity and Discovery of Small-Molecule Inhibitors.

SARS-CoV-2 non-structural protein 13 (nsp13) is a highly conserved helicase and RNA 5'-triphosphatase. It uses the energy derived from the hydrolysis of nucleoside triphosphates for directional movement along the nucleic acids and promotes the unwinding of double-stranded nucleic acids. Nsp13 is essential for replication and propagation of all human and non-human coronaviruses. Combined with its defined nucleotide binding site and druggability, nsp13 is one of the most promising candidates for the development of pan-coronavirus therapeutics. Here, we report the development and optimization of bioluminescence assays for kinetic characterization of nsp13 ATPase activity in the presence and absence of single-stranded DNA. Screening of a library of 5000 small molecules in the presence of single-stranded DNA resulted in the discovery of six nsp13 small-molecule inhibitors with IC50 values ranging from 6 ± 0.5 to 50 ± 6 µM. In addition to providing validated methods for high-throughput screening of nsp13 in drug discovery campaigns, the reproducible screening hits we present here could potentially be chemistry starting points toward the development of more potent and selective nsp13 inhibitors, enabling the discovery of antiviral therapeutics.

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

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.

The Structure-Based Design of SARS-CoV-2 nsp14 Methyltransferase Ligands Yields Nanomolar Inhibitors.

In this study, we have focused on the structure-based design of the inhibitors of one of the two SARS-CoV-2 methyltransferases (MTases), nsp14. This MTase catalyzes the transfer of the methyl group from S-adenosyl-l-methionine (SAM) to cap the guanosine triphosphate moiety of the newly synthesized viral RNA, yielding the methylated capped RNA and S-adenosyl-l-homocysteine (SAH). As the crystal structure of SARS-CoV-2 nsp14 is unknown, we have taken advantage of its high homology to SARS-CoV nsp14 and prepared its homology model, which has allowed us to identify novel SAH derivatives modified at the adenine nucleobase as inhibitors of this important viral target. We have synthesized and tested the designed compounds in vitro and shown that these derivatives exert unprecedented inhibitory activity against this crucial enzyme. The docking studies nicely explain the contribution of an aromatic part attached by a linker to the position 7 of the 7-deaza analogues of SAH.

A High-Throughput Radioactivity-Based Assay for Screening SARS-CoV-2 nsp10-nsp16 Complex.

Frequent outbreaks of novel coronaviruses (CoVs), highlighted by the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, necessitate the development of therapeutics that could be easily and effectively administered worldwide. The conserved mRNA-capping process enables CoVs to evade their host immune system and is a target for antiviral development. Nonstructural protein (nsp) 16 in complex with nsp10 catalyzes the final step of coronaviral mRNA capping through its 2'-O-methylation activity. Like other methyltransferases, the SARS-CoV-2 nsp10-nsp16 complex is druggable. However, the availability of an optimized assay for high-throughput screening (HTS) is an unmet need. Here, we report the development of a radioactivity-based assay for the methyltransferase activity of the nsp10-nsp16 complex in a 384-well format, kinetic characterization, and optimization of the assay for HTS (Z' factor = 0.83). Considering the high conservation of nsp16 across known CoV species, the potential inhibitors targeting the SARS-CoV-2 nsp10-nsp16 complex may also be effective against other emerging pathogenic CoVs.

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

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.