Predicting Broad-Spectrum Antiviral Drugs against RNA Viruses Using Transcriptional Responses to Exogenous RNA

All RNA viruses deliver their genomes into target host cells through processes distinct from normal trafficking of cellular RNA transcripts. The delivery of viral RNA into most cells hence triggers innate antiviral defenses that recognize viral RNA as foreign. In turn, viruses have evolved mechanisms to subvert these defenses, allowing them to thrive in target cells. Therefore, drugs activating defense to exogenous RNA could serve as broad-spectrum antiviral drugs. Here we show that transcriptional signatures associated with cellular responses to the delivery of a non-viral exogenous RNA sequence into human cells predict small molecules with broad-spectrum antiviral activity. In particular, transcriptional responses to the delivery of Cas9 mRNA into human hematopoietic stem and progenitor cells (HSPCs) highly matches those triggered by small molecules with broad-spectrum antiviral activity such as emetine, homoharringtonine, pyrvinium pamoate and anisomycin, indicating that these drugs are potentially active against other RNA viruses. Furthermore, these drugs have been approved for other indications and could thereby be repurposed to novel viruses. We propose that the antiviral activity of these drugs to SARS-CoV-2 should therefore be determined as they have been shown as active against other coronaviruses including SARS-CoV-1 and MERS-CoV. Indeed, two of these drugs- emetine and homoharringtonine- were independently shown to inhibit SARS-CoV-2 as this article was in preparation. These drugs could also be explored as potential adjuvants to COVID-19 vaccines in development due to their potential effect on the innate antiviral defenses that could bolster adaptive immunity when delivered alongside vaccine antigens.


Introduction
Invasion by viruses presents one of the biggest challenges faced by both prokaryotic and eukaryotic cells. As a consequence, a wide range of mechanisms to counter viral infections have evolved across all life forms. In higher vertebrates including mammals, both innate and acquired immunity to viruses are employed as a defense against viral infections. While acquired immunity is driven by highly specialized cell types, for example B and T-cells, innate immunity against viral infections is possessed by nearly all cell types in mammals. Unlike acquired immunity which involves antigen-specific receptors generated through gene rearrangements, innate defense against a broad range of viruses relies on germline encoded pattern recognition receptors (PRRs). Antiviral defenses by PRRs recognize specific pathogen associated molecular patterns (PAMPs) that are crucial for viral replication and transmission, for example viral nucleic acid (1), and hence difficult for the target virus to change. However, many viruses circumvent this challenge by targeting PRPs. Thus, activating the intrinsic antiviral defenses in individual cells could provide a means for developing broad-spectrum antiviral drugs.
Here, we hypothesized that the delivery of an exogenous RNA sequence into human cells elicits antiviral defense characterized by a specific transcriptional response, and that small molecules that enhance this response have potential for broad-spectrum antiviral activity. In contrast to viruses which subvert innate cellular defenses to foreign RNA, nonviral exogenous RNA should evoke antiviral responses but fail to subvert these defenses.
Thus, studying cellular responses to non-viral exogenous RNA could provide an unbiased view of exogenous RNA defenses. Using the Connectivity Map database of thousands of genetic and chemical perturbations of human cell lines (2), we show that several small molecules with broad-spectrum antiviral activity elicit transcriptional responses that are highly similar to those of human cells perturbed by the delivery of an exogenous, synthetic RNA sequence.

Relationship between transcriptional responses to exogenous RNA and viral infection
As a proxy to cellular response to the delivery of exogenous RNA, we considered recently published transcriptional responses of a human cell type to delivery of non-viral RNA sequences encoding cas9 protein and CRISPR gRNA to hemoglobin B locus (HBB), 4 hereafter referred to as 'cas9 mRNA' (3). Specifically, the delivery of cas9 mRNA into hematopoietic stem and progenitor cells (HSPCs) was found to induce a strong antiviral transcriptional response (3). Several genes involved in innate antiviral response such as interferon stimulatory genes (ISGs), interferon regulatory factors (IRF1, IRF7, IRF9) and the cytosolic foreign RNA sensor RIG-1 were upregulated by cas9 mRNA delivery (3).
The activation of innate antiviral defenses upon delivery of exogenous RNA into cells has also been reported for in vitro transcribed CRISPR guide RNAs (4). As little as 1nm of gRNA triggered a 30 to 50 fold increase in interferon-beta1 (IFNB1) in HEK293T cells and 50nm gRNA led to a 1000-fold induction of IFNB1 (4), an effect that is equal to that induced by RNA from Sendai virus or hepatitis C virus PAMP (5,6).
To explore the potential regulatory mechanisms underlying transcriptional response to cas9 mRNA delivery and their association with key antiviral response regulators, we Interestingly, we found that the overexpression of IFNB1 across several cell lines in the CMap database has a transcriptional signature that is the most similar to the delivery of cas9 mRNA (Fig. 1), consistent with previous reports that CRISPR gRNAs induce IFNB1 (4,7). In addition, several of the top ranked signatures are of genes involved in innate immunity to viruses such as B-cell lymphoma-2-like protein (BCL2L2), TNFRFS1A, KLF6 and TIRAP. For example, B-cell lymphoma-2-like protein (BCL2L2) inhibition by the 5 anticancer compound ABT-263 accelerates apoptosis of influenza A virus (IAV) infected cells and lowers survival of infected mice (8); Kruppel-like factor 6 (KLF6) is a transcription factor that induces the production of the antiviral compound nitric oxide during infection by negative-sense, single stranded RNA viruses such as IAV (9) or respiratory syncytial virus (RSV) (10); BCL10 is required for antiviral response mediated by the retinoic acid inducible gene 1 (RIG-1) which senses foreign RNA in the cytosol (11,12), while the tollinterleukin-1 domain adaptor receptor containing protein (TIRAP) is required for innate immune signaling to ligands such as viral nucleic acids in multiple subcellular compartments (13).

Similarity between transcriptional signatures to exogenous RNA and small molecules
Next, we investigated the similarity between transcriptional response to cas9 mRNA delivery and small molecules in the CMap database. We have previously shown that the transcriptional signatures of several small molecules in various cell lines are highly similar to those associated with the delivery of CRISPR/Cas9 components, including the delivery of cas9 mRNA sequence (14). Given that transcriptional response to cas9 mRNA is associated with antiviral transcriptional signatures, we reasoned that some of the small molecules with transcriptional signatures similar to that of cas9 mRNA could bear broadspectrum antiviral activity. Since the delivery of cas9 mRNA may also lead to transcriptional responses unrelated to foreign RNA sensing, we filtered out small molecules whose transcriptional signatures are highly similar to that of the delivery of cas9 protein pre-complexed with gRNA to form a ribonucleoprotein, RNP (Fig. 2). This activation of interferon regulatory factor 3 (IRF3), a transcription factor that regulates multiple IFN-inducing pathways triggered by DNA and RNA viruses (21). Thapsigargin has antiviral activity at low concentrations without cellular toxicity to several viruses including Newcastle disease virus, peste des petits ruminant virus, murine norovirus and flaviviruses (22)(23)(24). Narciclasine, another top small molecule (Fig. 2) has been shown to be active against the RNA flaviviruses (Japanese encephalitis, yellow fever and dengue viruses) as well as to the bunyaviruses Punta Toro and Rift Valley Fever virus (25).

Implications for broad-spectrum antivirals to emerging viruses, SARS-CoV-2
There is an urgent need to identify broad-spectrum antiviral drugs to emerging viral  (30). Thus, the predicted molecules appear to have high activity to these RNA viruses (Fig.2). Chloroquine, a onetime successful anti-malarial drug, has also shown activity against SARS-CoV-2 and other viruses (32,34). We compared the broad-spectrum antiviral range of these drugs to those of the small molecules identified in this study (Fig. 3). Unlike remdesivir which is undergoing a clinical trial for SARS-CoV-2, the drugs we identified in this study have a number of advantages: i) they target host antiviral defenses instead of viral proteins, and may therefore avoid evolution of drug resistance which is rampant in RNA viruses, ii) they are likely to have activity across viruses that have little genetic similarity and infect different cell types, and iii) compared to other drugs that are only active in cells they diffuse in, small molecules that activate antiviral defenses could result into secretion of antiviral effector proteins, cytokines and chemokines that diffuse to other cells and attract/ activate specialized immune cells. cancer cell lines exposed to small molecules in the CMap database, it is evident some of the genes involved in innate antiviral defenses are highly coregulated across cell types (Fig. 2). Secondly, CD34+ HSPCs have a strong restriction to lentiviruses such as HIV-1 (36,37), making it possible that the transcriptional responses to exogenous RNA in these cells could be stronger than that in other cells. While this may be the case, there appears to be a considerable extent of co-regulation of the genes involved across cell types.
Finally, whether any of the identified drugs can be repurposed to novel viruses like SARS-CoV-2 requires a deeper understanding of the drug bioavailability in relevant tissues, the relative tolerability given the disease severity, the cost and scalability in manufacturing which are not addressed in this study.
At least two mRNA-based vaccines are under development for COVID-19 by the companies Moderna Therapeutics and CureVac. These vaccines aim to elicit adaptive immunity which is commonly viewed as independent from the innate antiviral defenses considered in this study. Yet, it is becoming increasingly clear that long-lasting adaptive humoral and cellular immunity involves an interplay with innate immunity (38)(39)(40). While innate antiviral responses to foreign RNA can negatively impact expression of the encoded antigens in mRNA vaccines, some of the small molecules identified in this study may act as adjuvants to these vaccines. Since PRRs including those that detect foreign RNA are targets of many vaccine adjuvants (38,39), these small molecules may increase the quantity and quality of immune responses to mRNA vaccines when co-delivered.
Hence, we propose that the small molecules identified in this study should be explored both for inhibitory activity against SARS-CoV-2/ COVID-19 as well as for potential enhancement of immunological responses to mRNA vaccines including for COVID-19.

GHS is supported by the Center for Research Computing and the Eck Institute for Global
Health at the University of Notre Dame.

Author contributions
GHS conceived and designed the study. Both GHS and AVN performed analysis. GHS wrote the manuscript. Both authors reviewed the manuscript.

Conflict of interest:
The authors declare no competing financial interests in the work presented.

Matching cas9 mRNA transcriptional responses to small molecules
We obtained differentially expressed genes in CD34+ hematopoietic stem cells (HSPCs) exposed to cas9 mRNA and sgRNA targeting the hemoglobin B locus (HBB) from Cromer et al (3). Briefly, the study by Cromer at al delivered cas9 mRNA containing 5methylcytidine and pseudouridine modifications which limit innate immune responses whereas the sgRNA had 2'-o-methyl-3'-phosphothioate modification to enhance tolerance and activity. The RNAs were delivered into cells using electroporation.
Differential gene expression analysis was performed by computing log2 fold changes between cas9 mRNA treated samples and those exposed to mock electroporation. We We performed two independent queries on CMap: i) the first query was to assess the similarity between the cas9 mRNA signature and overexpression of different genes in human cells lines, ii) the second query was to assess the similarity between the cas9 mRNA signature and several small molecules. We performed queries using the Clue.io API data version 1.1.1.2 and software version 1.1.1.43.

Filtering of predicted small molecules for downstream mRNA delivery effects
To filter out small molecules whose transcriptional signatures may have matched that of cas9 mRNA delivery due to downstream effects (e.g. translation of the mRNA into cas9 protein that mediates double strand breaks), we also queried CMap using transcriptional signatures constructed from genes differentially expressed after delivery of cas9 protein combined with sgRNA targeting HBB (ribonucleoprotein, RNP). The RNP signatures were constructed in a similar way as described above for cas9 mRNA and used to query CMap for small molecules with matching signatures. We then excluded small molecules whose signatures matched both cas9 mRNA and RNP signatures.

Broad-spectrum antiviral analysis
To determine broad-antiviral potential of the predicted small molecules on coronaviruses, we used public data obtained from high-throughput screens of 2000 compounds (29) and a smaller library of 290 approved drugs (30). We also obtained broad-spectrum activity data to other viruses from a recent study (28) and its associated database (drugvirus.info).