Drug Discovery in Low Data Regimes: Leveraging a Computational Pipeline for the Discovery of Novel SARS-CoV-2 Nsp14-MTase Inhibitors (preprint)

The COVID-19 pandemic, caused by the SARS-CoV-2 virus, has led to significant global morbidity and mortality. A crucial viral protein, the non-structural protein 14 (nsp14), catalyzes the methylation of viral RNA and plays a critical role in viral genome replication and transcription. Due to the low mutation rate in the nsp region among various SARS-CoV-2 variants, nsp14 has emerged as a promising therapeutic target. However, discovering potential inhibitors remains a challenge. In this work, we introduce a computational pipeline for the rapid and efficient identification of potential nsp14 inhibitors by leveraging virtual screening and the NCI open compound collection, which contains 250,000 freely available molecules for researchers worldwide. The introduced pipeline provides a cost-effective and efficient approach for early-stage drug discovery by allowing researchers to evaluate promising molecules without incurring synthesis expenses. Our pipeline successfully identified seven promising candidates after experimentally validating only 40 compounds. Notably, we discovered NSC620333, a compound that exhibits a strong binding affinity to nsp14 with a dissociation constant of 427 {+/-} 84 nM. In addition, we gained new insights into the structure and function of this protein through molecular dynamics simulations. We identified new conformational states of the protein and determined that residues Phe367, Tyr368, and Gln354 within the binding pocket serve as stabilizing residues for novel ligand interactions. We also found that metal coordination complexes are crucial for the overall function of the binding pocket. Lastly, we present the solved crystal structure of the nsp14-MTase complexed with SS148, a potent inhibitor of methyltransferase activity at the nanomolar level (IC50 value of 70 {+/-} 6 nM). Our computational pipeline accurately predicted the binding pose of SS148, demonstrating its effectiveness and potential in accelerating drug discovery efforts against SARS-CoV-2 and other emerging viruses.

Structure-based discovery of inhibitors of the SARS-CoV-2 Nsp14 N7-methyltransferase (preprint)

An under-explored target for SARS-CoV-2 is non-structural protein 14 (Nsp14), a crucial enzyme for viral replication that catalyzes the methylation of N7-guanosine of the viral RNA at 5-prime-end; this enables the virus to evade the host immune response by mimicking the eukaryotic post-transcriptional modification mechanism. We sought new inhibitors of the S-adenosyl methionine (SAM)-dependent methyltransferase (MTase) activity of Nsp14 with three large library docking strategies. First, up to 1.1 billion make-on-demand (tangible) lead-like molecules were docked against the enzyme SAM site, seeking reversible inhibitors. On de novo synthesis and testing, three inhibitors emerged with IC50 values ranging from 6 to 43 M, each with novel chemotypes. Structure-guided optimization and in vitro characterization supported their non-covalent mechanism. In a second strategy, docking a library of 16 million tangible fragments revealed nine new inhibitors with IC50 values ranging from 12 to 341 M and ligand efficiencies from 0.29 to 0.42. In a third strategy, a newly created library of 25 million tangible, virtual electrophiles were docked to covalently modify Cys387 in the SAM binding site. Seven inhibitors emerged with IC50 values ranging from 3.2 to 39 M, the most potent being a reversible aldehyde. Initial optimization of a second series yielded a 7 M acrylamide inhibitor. Three inhibitors characteristic of the new series were tested for selectivity against 30 human protein and RNA MTases, with one showing partial selectivity and one showing high selectivity. Overall, 32 inhibitors encompassing eleven chemotypes had IC50 values <50 M and 5 inhibitors in four chemotypes had IC50 values <10 M. These molecules are among the first non-SAM-like inhibitors of Nsp14, providing multiple starting points for optimizing towards antiviral activity.

The Structure-Based Design of SARS-CoV-2 Nsp14 Methyltransferase Ligands Yields Nanomolar Inhibitors (preprint)

COVID-19, caused by the SARS-CoV-2 virus, is responsible for a global pandemic that has paralyzed the normal life in many countries around the globe. Therefore, the preparation of both effective vaccines and potential therapeutics has become a major research priority in the biotechnology sector. Both viral proteins and selected host factors are important targets for the treatment of this disease. Suitable targets for antiviral therapy include i.a. viral methyltransferases, which allow the viral mRNA to be efficiently translated and protect the viral RNA from the innate immune system. In this study, we have focused on the structure-based design of the inhibitors of one of the two SARS-CoV-2 methyltransferases, nsp14. This methyltransferase 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). 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. Our results will serve as an important source of information for the subsequent development of new antivirals to combat COVID-19.

Probing the SAM Binding Site of SARS-CoV-2 nsp14 in vitro Using SAM Competitive Inhibitors Guides Developing Selective bi-substrate Inhibitors (preprint)

The COVID-19 pandemic has clearly brought the healthcare systems world-wide 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. Non-structural 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 (MT) activity, we developed and employed a radiometric MT assay to screen a library of 161 in house synthesized S-adenosylmethionine (SAM) competitive methyltransferase inhibitors and SAM analogs. Among seven identified screening hits, SS148 inhibited nsp14 MT activity with an IC50 value of 70 {+/-} 6 nM and was selective against 20 human protein lysine methyltransferases indicating significant differences in SAM binding sites. Interestingly, DS0464 with IC50 value of 1.1 {+/-} 0.2 M showed a bi-substrate competitive inhibitor mechanism of action. Modeling the binding of this compound to nsp14 suggests that the terminal phenyl group extends into the RNA binding site. DS0464 was also selective against 28 out of 33 RNA, DNA, and protein methyltransferases. The structure-activity relationship provided by these compounds should guide the optimization of selective bi-substrate nsp14 inhibitors and may provide a path towards a novel class of antivirals against COVID-19, and possibly other coronaviruses.

A high-throughput radioactivity-based assay for screening SARS-CoV-2 nsp10-nsp16 complex (preprint)

Frequent outbreaks of novel coronaviruses (CoVs), highlighted by the current SARS-CoV-2 pandemic, necessitate the development of therapeutics that could be easily and effectively administered world-wide. 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, 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 methyltransferase activity of nsp10-nsp16 complex in a 384-well format, and 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 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 towards developing therapeutics for COVID-19 (preprint)

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 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, product of the reaction, 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 nsp10-nsp16 complex for RNA-competitive inhibitors towards developing COVID-19 therapeutics.