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.

Global landscape of the host response to SARS-CoV-2 variants reveals viral evolutionary trajectories (preprint)

A series of SARS-CoV-2 variants of concern (VOCs) have evolved in humans during the COVID-19 pandemic: Alpha, Beta, Gamma, Delta, and Omicron. Here, we used global proteomic and genomic analyses during infection to understand the molecular responses driving VOC evolution. We discovered VOC-specific differences in viral RNA and protein expression levels, including for N, Orf6, and Orf9b, and pinpointed several viral mutations responsible. An analysis of the host response to VOC infection and comprehensive interrogation of altered virus-host protein-protein interactions revealed conserved and divergent regulation of biological pathways. For example, regulation of host translation was highly conserved, consistent with suppression of VOC replication in mice using the translation inhibitor plitidepsin. Conversely, modulation of the host inflammatory response was most divergent, where we found Alpha and Beta, but not Omicron BA.1, antagonized interferon stimulated genes (ISGs), a phenotype that correlated with differing levels of Orf6. Additionally, Delta more strongly upregulated proinflammatory genes compared to other VOCs. Systematic comparison of Omicron subvariants revealed BA.5 to have evolved enhanced ISG and proinflammatory gene suppression that similarly correlated with Orf6 expression, effects not seen in BA.4 due to a mutation that disrupts the Orf6-nuclear pore interaction. Our findings describe how VOCs have evolved to fine-tune viral protein expression and protein-protein interactions to evade both innate and adaptive immune responses, offering a likely explanation for increased transmission in humans.

Bat pluripotent stem cells reveal unique entanglement between host and viruses (preprint)

Bats have evolved features unique amongst mammals, including flight, laryngeal echolocation, and certain species have been shown to have a unique immune response that may enable them to tolerate viruses such as SARS-CoVs, MERS-CoVs, Nipah, and Marburg viruses. Robust cellular models have yet to be developed for bats, hindering our ability to further understand their special biology and handling of viral pathogens. To establish bats as new model study species, we generated induced pluripotent stem cells (iPSCs) from a wild greater horseshoe bat ( Rhinolophus ferrumequinum ) using a modified Yamanaka protocol. Rhinolophids are amongst the longest living bat species and are asymptomatic carriers of coronaviruses, including one of the viruses most closely related to SARS-CoV-2. Bat induced pluripotent stem (BiPS) cells were stable in culture, readily differentiated into all three germ layers, and formed complex embryoid bodies, including organoids. The BiPS cells were found to have a core pluripotency gene expression program similar to that of other species, but it also resembled that of cells attacked by viruses. The BiPS cells produced a rich set of diverse endogenized viral sequences and in particular retroviruses. We further validated our protocol by developing iPS cells from an evolutionary distant bat species Myotis myotis (greater mouse-eared bat) non-lethally sampled in the wild, which exhibited similar attributes to the greater horseshoe bat iPS cells, suggesting that this unique pluripotent state evolved in the ancestral bat lineage. Although previous studies have suggested that bats have developed powerful strategies to tame their inflammatory response, our results argue that they have also evolved mechanisms to accommodate a substantial load of endogenous viral sequences and suggest that the natural history of bats and viruses is more profoundly intertwined than previously thought. Further study of bat iPS cells and their differentiated progeny should advance our understanding of the role bats play as virus hosts, provide a novel method of disease surveillance, and enable the functional studies required to ascertain the molecular basis of bats’ unique traits.

Large library docking for novel SARS-CoV-2 main protease non-covalent and covalent inhibitors (preprint)

Antiviral therapeutics to treat SARS-CoV-2 are much desired for the on-going pandemic. A well-precedented viral enzyme is the main protease (MPro), which is now targeted by an approved drug and by several investigational drugs. With the inevitable liabilities of these new drugs, and facing viral resistance, there remains a call for new chemical scaffolds against MPro. We virtually docked 1.2 billion non-covalent and a new library of 6.5 million electrophilic molecules against the enzyme structure. From these, 29 non-covalent and 11 covalent inhibitors were identified in 37 series, the most potent having an IC 50 of 29 μM and 20 μM, respectively. Several series were optimized, resulting in inhibitors active in the low micromolar range. Subsequent crystallography confirmed the docking predicted binding modes and may template further optimization. Together, these compounds reveal new chemotypes to aid in further discovery of MPro inhibitors for SARS-CoV-2 and other future coronaviruses.

Transcriptomics-based drug repositioning pipeline identifies therapeutic candidates for COVID-19 (preprint)

The novel SARS-CoV-2 virus emerged in December 2019 and has few effective treatments. We applied a computational drug repositioning pipeline to SARS-CoV-2 differential gene expression signatures derived from publicly available data. We utilized three independent published studies to acquire or generate lists of differentially expressed genes between control and SARS-CoV-2-infected samples. Using a rank-based pattern matching strategy based on the Kolmogorov-Smirnov Statistic, the signatures were queried against drug profiles from Connectivity Map (CMap). We validated sixteen of our top predicted hits in live SARS-CoV-2 antiviral assays in either Calu-3 or 293T-ACE2 cells. Validation experiments in human cell lines showed that 11 of the 16 compounds tested to date (including clofazimine, haloperidol and others) had measurable antiviral activity against SARS-CoV-2. These initial results are encouraging as we continue to work towards a further analysis of these predicted drugs as potential therapeutics for the treatment of COVID-19.

Hepatitis C Virus Drugs Simeprevir and Grazoprevir Synergize with Remdesivir to Suppress SARS-CoV-2 Replication in Cell Culture (preprint)

Effective control of COVID-19 requires antivirals directed against SARS-CoV-2 virus. Here we assess ten available HCV protease inhibitor drugs as potential SARS-CoV-2 antivirals. There is a striking structural similarity of the substrate binding clefts of SARS- CoV-2 Mpro and HCV NS3/4A proteases, and virtual docking experiments show that all ten HCV drugs can potentially bind into the Mpro binding cleft. Seven of these HCV drugs inhibit SARS-CoV-2 Mpro protease activity, while four dock well into the PLpro substrate binding cleft and inhibit PLpro protease activity. These same seven HCV drugs inhibit SARS-CoV-2 virus replication in Vero and/or human cells, demonstrating that HCV drugs that inhibit Mpro, or both Mpro and PLpro, suppress virus replication. Two HCV drugs, simeprevir and grazoprevir synergize with the viral polymerase inhibitor remdesivir to inhibit virus replication, thereby increasing remdesivir inhibitory activity as much as 10-fold. HighlightsO_LISeveral HCV protease inhibitors are predicted to inhibit SARS-CoV-2 Mpro and PLpro. C_LIO_LISeven HCV drugs inhibit Mpro enzyme activity, four HCV drugs inhibit PLpro. C_LIO_LISeven HCV drugs inhibit SARS-CoV-2 replication in Vero and/or human cells. C_LIO_LIHCV drugs simeprevir and grazoprevir synergize with remdesivir to inhibit SARS- CoV-2. C_LI eTOC blurbBafna, White and colleagues report that several available hepatitis C virus drugs inhibit the SARS-CoV-2 Mpro and/or PLpro proteases and SARS-CoV-2 replication in cell culture. Two drugs, simeprevir and grazoprevir, synergize with the viral polymerase inhibitor remdesivir to inhibit virus replication, increasing remdesivir antiviral activity as much as 10-fold. O_FIG O_LINKSMALLFIG WIDTH=185 HEIGHT=200 SRC="FIGDIR/small/422511v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1c12181org.highwire.dtl.DTLVardef@7ed993org.highwire.dtl.DTLVardef@1fe56aaorg.highwire.dtl.DTLVardef@ebc34e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Hepatitis C Virus Drugs Simeprevir And Grazoprevir Synergize with Remdesivir to Suppress SARS-CoV2 Replication (preprint)

Effective control of COVID-19 requires antivirals directed against SARS-CoV-2 virus. Here we assess ten available HCV protease inhibitor drugs as potential SARS-CoV-2 antivirals. There is a striking structural similarity of the substrate binding clefts of SARS-CoV-2 Mpro and HCV NS3/4A proteases, and virtual docking experiments show that all ten HCV drugs can potentially bind into the Mpro binding cleft. Seven of these HCV drugs inhibit SARS-CoV-2 Mpro protease activity, while four dock well into the PLpro substrate binding cleft and inhibit PLpro protease activity. These same seven HCV drugs inhibit SARS-CoV-2 virus replication in Vero and/or human cells, demonstrating that HCV drugs that inhibit Mpro, or both Mpro and PLpro, suppress virus replication. Two HCV drugs, simeprevir and grazoprevir synergize with the viral polymerase inhibitor remdesivir to inhibit virus replication, thereby increasing remdesivir inhibitory activity as much as 10-fold.Funding: This research was supported by grants from the National Institutes of Health (R01-GM120574 to GTM) and RPI Center for Computational Innovations (to KB and GTM). This research was also partly funded by CRIP (Center for Research for Influenza Pathogenesis), a NIAID supported Center of Excellence for Influenza Research and Surveillance (CEIRS, contract #,HHSN272201400008C), by DARPA grant HR0011-19-2-0020, by supplements to NIAID grant U19AI142733 U19AI135972 and DoD grant W81XWH-20-1-0270, and by the generous support of the JPB Foundation, the Open Philanthropy Project (research grant 2020-215611 (5384)), and anonymous donors to AG-S.Conflict of Interest: A provisional patent application related to these, studies has been filed. GTM is a founder of Nexomics Biosciences, Inc. This, relationship has no conflict of interest with respect to this study. GTM and RMK are inventors in patents owned jointly by Rutgers University and the University of Texas at Austin concerning the use of specific compounds as antivirals against influenza virus. These patents have no conflict of interest for this study. AG-S is inventor in patents and patent application owned by the Icahn School of Medicine concerning the use of specific antiviral compounds. This inventorship has no conflict of interest with respect to this study.

Topoisomerase 1 inhibition therapy protects against SARS-CoV-2-induced inflammation and death in animal models. (preprint)

The ongoing pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is currently affecting millions of lives worldwide. Large retrospective studies indicate that an elevated level of inflammatory cytokines and pro-inflammatory factors are associated with both increased disease severity and mortality. Here, using multidimensional epigenetic, transcriptional, in vitro and in vivo analyses, we report that Topoisomerase 1 (Top1) inhibition suppresses lethal inflammation induced by SARS-CoV-2. Therapeutic treatment with two doses of Topotecan (TPT), a FDA-approved Top1 inhibitor, suppresses infection-induced inflammation in hamsters. TPT treatment as late as four days post-infection reduces morbidity and rescues mortality in a transgenic mouse model. These results support the potential of Top1 inhibition as an effective host-directed therapy against severe SARS-CoV-2 infection. TPT and its derivatives are inexpensive clinical-grade inhibitors available in most countries. Clinical trials are needed to evaluate the efficacy of repurposing Top1 inhibitors for COVID-19 in humans.

A Large-scale Drug Repositioning Survey for SARS-CoV-2 Antivirals (preprint)

The emergence of novel SARS coronavirus 2 (SARS-CoV-2) in 2019 has triggered an ongoing global pandemic of severe pneumonia-like disease designated as coronavirus disease 2019 (COVID-19). To date, more than 2.1 million confirmed cases and 139,500 deaths have been reported worldwide, and there are currently no medical countermeasures available to prevent or treat the disease. As the development of a vaccine could require at least 12-18 months, and the typical timeline from hit finding to drug registration of an antiviral is >10 years, repositioning of known drugs can significantly accelerate the development and deployment of therapies for COVID-19. To identify therapeutics that can be repurposed as SARS-CoV-2 antivirals, we profiled a library of known drugs encompassing approximately 12,000 clinical-stage or FDA-approved small molecules. Here, we report the identification of 30 known drugs that inhibit viral replication. Of these, six were characterized for cellular dose-activity relationships, and showed effective concentrations likely to be commensurate with therapeutic doses in patients. These include the PIKfyve kinase inhibitor Apilimod, cysteine protease inhibitors MDL-28170, Z LVG CHN2, VBY-825, and ONO 5334, and the CCR1 antagonist MLN-3897. Since many of these molecules have advanced into the clinic, the known pharmacological and human safety profiles of these compounds will accelerate their preclinical and clinical evaluation for COVID-19 treatment.