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Genomics-guided molecular maps of coronavirus targets in human cells: a path toward the repurposing of existing drugs to mitigate the pandemic

Human genes required for SARS-CoV-2 entry into human cells, ACE2 and FURIN, were employed as baits to build genomics-guided maps of up-stream regulatory elements, their expression and functions in human body, including pathophysiologically-relevant cell types. Genes acting as repressors and activators of the ACE2 and FURIN genes were identified based on the analyses of gene silencing and overexpression experiments as well as relevant transgenic mouse models. Panels of repressors (VDR; GATA5; SFTPC; HIF1a) and activators (HMGA2; INSIG1) were then employed to identify existing drugs that could be repurposed to mitigate the coronavirus infection. Present analyses identify Vitamin D and Quercetin as promising pandemic mitigation agents. Gene expression profiles of Vitamin D and Quercetin activities and their established safety records as over-the-counter medicinal substances suggest that they may represent viable candidates for further assessment and considerations of their potential as coronavirus pandemic mitigation agents. Notably, gene set enrichment analyses and expression profiling experiments identify multiple drugs, most notably testosterone, dexamethasone, and doxorubicin, smoking, and many disease conditions that appear to act as putative coronavirus infection-promoting agents. Discordant patterns of Testosterone versus Estradiol impacts on SCARS-CoV-2 targets suggest a plausible molecular explanation of the apparently higher male mortality during coronavirus pandemic.


Introduction
Coronavirus pandemic 2020 caused by the newly emerged SARS-CoV-2 virus is rapidly entering the most dangerous acute phase of its evolution in the United States. Absence of the vaccine and lack of efficient targeted therapeutic approaches emphasizes the urgent need for identification of candidate pandemic mitigation agents among existing drugs and medicinal substances.
SARS-CoV-2 virus was discovered in December 2019 and shortly thereafter it was isolated and sequenced (Zhou et al., 2020;Zhu et al., 2020). Recent analyses of the structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein revealed the key role of the ACE2 and FURIN genes in facilitating the high-affinity binding of viral particles and their entry into human cells (Walls et al., 2020). The efficient invasion of host cells by the SARS-CoV-2 is further enhanced by the presence of the unexpected furin cleavage site, which is cleaved during the biosynthesis (Walls et al., 2020). This novel feature distinguishes the previously known SARS-CoV and the newly emerged SARS-CoV-2 viruses and possibly contributes to the expansion of the cellular tropism of the SARS-CoV-2 (Walls et al., 2020). Collectively, these observations identified protein products of the human genes ACE2 and FURIN as the highaffinity receptor (ACE2) and invasion-promoting protease (FURIN) acting as the principal mediators of the SARS-CoV-2 invasion into human cells.
In this contribution, genomic screens were performed employing the ACE2 and FURIN genes as baits to build genomics-guided human tissues-tailored maps of up-stream regulatory elements, their expression and functions. To identify the high-priority list of potential candidate mitigation agents, the validation analyses were performed using gene silencing and overexpression experiments as well as relevant transgenic mouse models with the emphasis on pathophysiologically-relevant cell types. Panels of repressors (VDR; GATA5; SFTPC) and activators (HMGA2; INSIG1) of the ACE2 and FURIN expression were identified and then employed to identify existing drugs and medicinal substances that could be repurposed to ameliorate the outcomes of the coronavirus infection. Two of the most promising candidate mitigation agents, namely Vitamin D and Quercetin, manifest gene expression-altering activities and have established safety records as over-the-counter medicinal substances that seem sufficient for further assessment and considerations of their potential utility for amelioration of the clinical course of coronavirus pandemic. Unexpectedly, present analyses revealed discordant patterns of Testosterone versus Estradiol impacts on SCARS-CoV-2 targets with the former manifesting the potential coronavirus infection-promoting activities, which is consistent with the apparently higher male mortality across all age groups during the coronavirus pandemic.

FURIN genes
One of the goals of this work was to identify human genes implicated in regulatory cross-talks affecting expression and functions of the ACE2 and FURIN genes to build a model of genomic regulatory interactions potentially affecting the SCARS-CoV-2 coronavirus infection. To this end, GSEA were carried out using the ACE2 and FURIN genes as baits applied to a broad spectrum of genomic databases reflecting the current state of knowledge regarding the structural, functional, regulatory, and pathophysiological features that could be statistically linked to these genes. Expression profiling experiments and GSEA revealed ubiquitous patterns of both ACE2 and FURIN genes across human tissues (Supplemental Figure S1 Figure S8). These findings were corroborated by observations that HIF1a overexpression in human embryonic kidney cells significantly inhibits the ACE2 expression (Supplemental Figure S8). Notably, Vitamin D significantly increases expression of the HIF1a gene in human bronchial smooth muscle cells (Supplemental Figure S8), suggesting that VDR and HIF1A genes may cooperate as repressors of the ACE2 expression.

GSEA identify Estradiol and Quercetin as putative candidate coronavirus infection mitigation agents.
GSEA of the Drug Perturbations from GEO database focused on down-regulated genes identified Estradiol and Quercetin among the top significantly enriched records (Supplemental Figure S9). Estradiol appears to affect both FURIN and ACE2 expression, while Quercetin seems to target the ACE2 expression. Consistently, GSEA of the Ligand Perturbations from GEO focused on down-regulated genes identified five of Estradiol administration records (50%) among top ten significantly enriched ligand perturbations records (Supplemental Figure S9).  Figure   S9). However, Quercetin administration appears to increase c-Fos expression in cultured rat cardiomyocytes (Supplementary Figure S9).

Confirmation of the Estradiol and Quercetin activities as potential candidate coronavirus infection mitigation agents.
Results of GSEA suggest that both Estradiol and Quercetin appear to exhibit biological activities consistent with the activity of medicinal compounds expected to mitigate the coronavirus infection. Next, manual curation of the GEO data sets has been carried out to identify further experimental evidence supporting this hypothesis. Administration of Estradiol appears to inhibit ACE2 and/or FURIN expression in rat, mouse, and human cells (Supplemental Figure S10) and the effects of Estradiol seem to be mediated by the estrogen receptor beta. In agreement with the hypothesis on potential therapeutic utility of the Quercetin, administration of Quercetin has resulted in significantly decreased expression of the ACE2 gene during differentiation of human intestinal cells (Supplemental Figure S10).
However, Estradiol administration appears to manifest the cell type-specific effects on c-FOS expression (Supplemental Figure S10)  Figure S12). For example, the HIF1a expression is significantly increased in murine alveolar type I cells deficient in sterol-response element-binding proteins inhibitor Insig1 (Supplemental Figure S6). These data indicate that the INSIG1 gene product, which appears to function as activator of the ACE2 expression, may function as the inhibitor of the HIF1a expression, thus interfering with the HIF1a-mediated ACE2 repression in specific cell types. Additional examples of the potential positive and negative effects on gene expression inferred from transgenic mouse models are reported in the Supplemental Figure S13.

Conclusion
The main motivation of this work was to identify human genes implicated in regulatory cross-  This knowledge could also be exploited in an ongoing effort to discover novel targeted therapeutics tailored to block the SCARS-CoV-2 infection.
One of the important findings documented herein is that identified medicinal compounds with potential coronavirus infection-mitigating effects also appear to induce cell type-specific patterns of gene expression alterations. Therefore, based on all observations reported in this contribution, it has been concluded that any definitive recommendations regarding the potential clinical utility of identified herein potential coronavirus infection mitigating agents, namely Vitamin D and Quercetin, should be made only after appropriately designed and carefully executed preclinical studies and randomized clinical trials.
Present analyses highlighted the major uncertainty regarding the outcomes of the current pandemic associated with the potential of the SCARS-CoV-2 virus for the expansion of the cellular tropism (Walls et al., 2020) based on access to genetically vulnerable host cells due to nearly ubiquitous expression of the ACE2 and FURIN genes in the human body. Particularly dangerous seems the potential ability of the the SCARS-CoV-2 virus to infect the immune cells.
Taken together with predominantly cell type-specific patterns of expression of genetic repressors and activators of the ACE2 and FURIN expression it may complicate the development of universally effective therapeutics. The availability of many genetically-relevant transgenic mouse models, in particular, the Furin null mice, should be regarded as a considerable advantage for preclinical development of drug candidates tailored to target the coronavirus infection. Specifically, the potential therapeutic utility of the highly selective (Ki, 600 pm) intrinsically-specific FURIN inhibitor (a1-antitrypsin Portland (a1-PDX); Jean et al., 1998) should be tested in the immediate future.

Data source and analytical protocols
All data analyzed in this study were obtained from the publicly available sources. Gene set enrichment analyses (GSEA) were carried-out using the Enrichr bioinformatics platform, which enables the interrogation of nearly 200,000 gene sets from more than 100 gene set libraries.
The Enrichr API (January 2020 through March 2020 releases) (Chen et al., 2013;Kuleshov et al., 2016) was used to test genes linked to the ACE2 and FURIN genes (or other genes of interest) for significant enrichment in numerous functional categories. In all tables and plots (unless stated otherwise), in addition to the nominal p values and adjusted p values, the "combined score" calculated by Enrichr is reported, which is a product of the significance estimate and the magnitude of enrichment (combined score c = log(p) * z, where p is the Fisher's exact test p-value and z is the z-score deviation from the expected rank). Validation of the GSEA findings were carried-out employing the computational retrievals and manual curations of the gene expression profiles of the Gene Expression Omnibus (GEO) database.

Statistical Analyses of the Publicly Available Datasets
All statistical analyses of the publicly available genomic datasets, including error rate estimates, background and technical noise measurements and filtering, feature peak calling, feature selection, assignments of genomic coordinates to the corresponding builds of the reference human genome, and data visualization, were performed exactly as reported in the original publications (Glinsky, 2015(Glinsky, -2020Glinsky and Barakat, 2019;Glinsky et al., 2019;Guffanti et al., 2018) and associated references linked to the corresponding data visualization tracks (http://genome.ucsc.edu/). Any modifications or new elements of statistical analyses are described in the corresponding sections of the Results. Statistical significance of the Pearson correlation coefficients was determined using GraphPad Prism version 6.00 software. Both nominal and Bonferroni adjusted p values were estimated. The statistical significance between the mean values was estimated using the Student T-test. The significance of the differences in the numbers of events between the groups was calculated using two-sided Fisher's exact and Chi-square test, and the significance of the overlap between the events was determined using the hypergeometric distribution test (Tavazoie et al., 1999).

Supplemental Information
Supplemental information includes Supplemental Figures S1-S13. Supplemental information is available upon request.

Author Contributions
This is a single author contribution. All elements of this work, including the conception of ideas, formulation, and development of concepts, execution of experiments, analysis of data, and writing of the paper, were performed by the author.