Tripartite Combination of Candidate Pandemic Mitigation Agents: Vitamin D, Quercetin, and Estradiol Manifest Properties of Medicinal Agents for Targeted Mitigation of the COVID-19 Pandemic Defined by Genomics-Guided Tracing of SARS-CoV-2 Targets in Human Cells

Abstract

Genes required for SARS-CoV-2 entry into human cells, ACE2 and FURIN, were employed as baits to build genomic-guided molecular maps of upstream regulatory elements, their expression and functions in the human body, and pathophysiologically relevant cell types. 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; RUNX1; HNF4a; JNK1/c-FOS) were then employed to identify existing drugs manifesting in their effects on gene expression signatures of potential coronavirus infection mitigation agents. Using this strategy, vitamin D and quercetin have been identified as putative 2019 coronavirus disease (COVID-19) mitigation agents. Quercetin has been identified as one of top-scoring candidate therapeutics in the supercomputer SUMMIT drug-docking screen and Gene Set Enrichment Analyses (GSEA) of expression profiling experiments (EPEs), indicating that highly structurally similar quercetin, luteolin, and eriodictyol could serve as scaffolds for the development of efficient inhibitors of SARS-CoV-2 infection. In agreement with this notion, quercetin alters the expression of 98 of 332 (30%) of human genes encoding protein targets of SARS-CoV-2, thus potentially interfering with functions of 23 of 27 (85%) of the SARS-CoV-2 viral proteins in human cells. Similarly, Vitamin D may interfere with functions of 19 of 27 (70%) of the SARS-CoV-2 proteins by altering expression of 84 of 332 (25%) of human genes encoding protein targets of SARS-CoV-2. Considering the potential effects of both quercetin and vitamin D, the inference could be made that functions of 25 of 27 (93%) of SARS-CoV-2 proteins in human cells may be altered. GSEA and EPEs identify multiple drugs, smoking, and many disease conditions that appear to act as putative coronavirus infection-promoting agents. Discordant patterns of testosterone versus estradiol impacts on SARS-CoV-2 targets suggest a plausible molecular explanation of the apparently higher male mortality during the coronavirus pandemic. Estradiol, in contrast with testosterone, affects the expression of the majority of human genes (203 of 332; 61%) encoding SARS-CoV-2 targets, thus potentially interfering with functions of 26 of 27 SARS-CoV-2 viral proteins. A hypothetical tripartite combination consisting of quercetin/vitamin D/estradiol may affect expression of 244 of 332 (73%) human genes encoding SARS-CoV-2 targets. Of major concern is the ACE2 and FURIN expression in many human cells and tissues, including immune cells, suggesting that SARS-CoV-2 may infect a broad range of cellular targets in the human body. Infection of immune cells may cause immunosuppression, long-term persistence of the virus, and spread of the virus to secondary targets. Present analyses and numerous observational studies indicate that age-associated vitamin D deficiency may contribute to the high mortality of older adults and the elderly. Immediate availability for targeted experimental and clinical interrogations of potential COVID-19 pandemic mitigation agents, namely vitamin D and quercetin, as well as of the highly selective (Ki, 600 pm) intrinsically specific FURIN inhibitor (a1-antitrypsin Portland (a1-PDX), is considered an encouraging factor. Observations reported in this contribution are intended to facilitate follow-up targeted experimental studies and, if warranted, randomized clinical trials to identify and validate therapeutically viable interventions to combat the COVID-19 pandemic. Specifically, gene expression profiles of vitamin D and quercetin activities and their established safety records as over-the-counter medicinal substances strongly argue that they may represent viable candidates for further considerations of their potential utility as COVID-19 pandemic mitigation agents. In line with the results of present analyses, a randomized interventional clinical trial evaluating effects of estradiol on severity of the coronavirus infection in COVID19+ and presumptive COVID19+ patients and two interventional randomized clinical trials evaluating effects of vitamin D on prevention and treatment of COVID-19 were listed on the ClinicalTrials.gov website.

Keywords: COVID-19; SARS-CoV-2 coronavirus; drugs and medicinal substance repurposing; eriodictyol; estradiol; genomics; luteolin; mitigation approaches; quercetin; vitamin D.

Figure A1

Similarity of chemical structures of the Quercetin, Taxifolin, and Rhemnetin. Taxifolin and rhemnetin have been identified in the recent a chemical library of over 687 million compounds for binding at the recently solved crystal structure of the main protease of SARS-CoV-2 coronavirus (Fischer et al., 2020. https://doi.org/10.26434/chemrxiv.11923239.v2 ) among the top-scoring candidate inhibitors of the SARS-CoV-2 main protease.

Figure 1

Genomic-guided mapping of regulatory networks affecting expression of human genes encoding protein targets of SARS-CoV-2 enables identification of putative 2019 coronavirus disease (COVID-19) mitigation agents. (A). Flow chart of a decision-making process during the identification of candidate pandemic mitigation agents employing genomic-guided tracing of genetic regulators and biological and chemical effectors of SARS-CoV-2 targets in human cells. (B) and (C). Effects of viral challenges on expression of the ACE2 and FURIN genes. (B). Gene Set Enrichment Analyses (GSEA) of the Virus Perturbations from GEO focused on upregulated genes (Enrichr bioinformatics platform). SARS-CoV p value = 0.0002; q value = 0.072). Star denotes the SARS-CoV record at the 96 hrs. (C). Increased FURIN expression in peripheral blood mononuclear cells (PBMC) of patients with severe acute respiratory syndrome (SARS). p value = 0.002; q value = 0.126.

Figure 1

Genomic-guided mapping of regulatory networks affecting expression of human genes encoding protein targets of SARS-CoV-2 enables identification of putative 2019 coronavirus disease (COVID-19) mitigation agents. (A). Flow chart of a decision-making process during the identification of candidate pandemic mitigation agents employing genomic-guided tracing of genetic regulators and biological and chemical effectors of SARS-CoV-2 targets in human cells. (B) and (C). Effects of viral challenges on expression of the ACE2 and FURIN genes. (B). Gene Set Enrichment Analyses (GSEA) of the Virus Perturbations from GEO focused on upregulated genes (Enrichr bioinformatics platform). SARS-CoV p value = 0.0002; q value = 0.072). Star denotes the SARS-CoV record at the 96 hrs. (C). Increased FURIN expression in peripheral blood mononuclear cells (PBMC) of patients with severe acute respiratory syndrome (SARS). p value = 0.002; q value = 0.126.

Figure 2

Pathways and genes affecting the newly emerged SARS-CoV-2 virus-related host targets. (A). JNK1/c-FOS pathway-associated activation of the ACE2 and FURIN expression may trigger the auto-regulatory negative feed-back loop of the FURIN-mediated repression of the expression of JUN, JUNB, JUND, and c-FOS genes. (B). RUNX1 pathway-associated activation of the ACE2 and FURIN expression may trigger the auto-regulatory negative feed-back loop of the FURIN-mediated repression of the RUNX1 gene expression (C). HNF4a pathway-associated activation of the ACE2 and FURIN expression may trigger the auto-regulatory positive feed-back loop of the FURIN-mediated activation of the HNF4a expression.

Figure 3

Effects of the VDR gene and vitamin D on pathways and genes affecting the newly emerged SARS-CoV-2 virus-related host targets. (A). JNK1/c-FOS pathway-associated activation of the ACE2 and FURIN expression may trigger the auto-regulatory negative feed-back loop of the FURIN-mediated repression of the expression of JUN, JUNB, JUND, and c-FOS genes. (B). HNF4a pathway-associated activation of the ACE2 and FURIN expression may trigger the auto-regulatory positive feed-back loop of the FURIN-mediated activation of the HNF4a expression. Activation of VDR gene would block JNK1/c-FOS- and HNF4a-mediated increased expression of both ACE2 and FURIN genes.

Figure 3

Effects of the VDR gene and vitamin D on pathways and genes affecting the newly emerged SARS-CoV-2 virus-related host targets. (A). JNK1/c-FOS pathway-associated activation of the ACE2 and FURIN expression may trigger the auto-regulatory negative feed-back loop of the FURIN-mediated repression of the expression of JUN, JUNB, JUND, and c-FOS genes. (B). HNF4a pathway-associated activation of the ACE2 and FURIN expression may trigger the auto-regulatory positive feed-back loop of the FURIN-mediated activation of the HNF4a expression. Activation of VDR gene would block JNK1/c-FOS- and HNF4a-mediated increased expression of both ACE2 and FURIN genes.

Figure 4

Effects of quercetin and vitamin D on expression of genes encoding human prey proteins of SARS-CoV-2. (A) Quercetin alters the expression of 98 of 332 (30%) of genes encoding human prey proteins for 23 of 27 (85%) of SARS-CoV-2 proteins. Percent values refer to the fractions of targets affected by quercetin based on gene expression profiling experiments. Both downregulated and upregulated genes were scored assuming that expression changes would alter the stoichiometry of viral protein/human prey protein interactions. Numerical values next to each viral protein names indicate the number of affected target protein-encoding genes. (B) Vitamin D alters the expression of 84 of 332 (25%) of genes encoding human prey proteins for 19 of 27 (70%) of SARS-CoV-2 proteins. Percent values refer to the fractions of targets affected by vitamin D based on gene expression profiling experiments. Both downregulated and upregulated genes were scored assuming that expression changes would alter the stoichiometry of viral protein/human prey protein interactions. Numerical values next to each viral protein names indicate the number of affected target protein-encoding genes. Panels C and D report the results of GSEA of the 332 genes in the Virus Perturbations from GEO database for upregulated (C) and downregulated (D) genes. See text and Supplementary Figure S14 for details.

Figure 4

Effects of quercetin and vitamin D on expression of genes encoding human prey proteins of SARS-CoV-2. (A) Quercetin alters the expression of 98 of 332 (30%) of genes encoding human prey proteins for 23 of 27 (85%) of SARS-CoV-2 proteins. Percent values refer to the fractions of targets affected by quercetin based on gene expression profiling experiments. Both downregulated and upregulated genes were scored assuming that expression changes would alter the stoichiometry of viral protein/human prey protein interactions. Numerical values next to each viral protein names indicate the number of affected target protein-encoding genes. (B) Vitamin D alters the expression of 84 of 332 (25%) of genes encoding human prey proteins for 19 of 27 (70%) of SARS-CoV-2 proteins. Percent values refer to the fractions of targets affected by vitamin D based on gene expression profiling experiments. Both downregulated and upregulated genes were scored assuming that expression changes would alter the stoichiometry of viral protein/human prey protein interactions. Numerical values next to each viral protein names indicate the number of affected target protein-encoding genes. Panels C and D report the results of GSEA of the 332 genes in the Virus Perturbations from GEO database for upregulated (C) and downregulated (D) genes. See text and Supplementary Figure S14 for details.

Figure 5

Effects of estradiol on expression of genes encoding human prey proteins of SARS-CoV-2. (A) Estradiol alters the expression of 203 of 332 (61%) of genes encoding human prey proteins for 26 of 27 (96%) of SARS-CoV-2 proteins. Percent values refer to the fractions of targets affected by the estradiol based on gene expression profiling experiments. Both downregulated and upregulated genes were scored assuming that expression changes would alter the stoichiometry of viral protein/human prey protein interactions. Numerical values next to each viral protein names indicate the number of affected target protein-encoding genes. Panels (B) and (C) reports the results of GSEA of the 332 genes in the Ligand Perturbations from GEO of downregulated (B) and upregulated (C) genes. Note marked representations of the estradiol records among top 10 significantly enriched entries of endogenous ligands.

Figure 5

Effects of estradiol on expression of genes encoding human prey proteins of SARS-CoV-2. (A) Estradiol alters the expression of 203 of 332 (61%) of genes encoding human prey proteins for 26 of 27 (96%) of SARS-CoV-2 proteins. Percent values refer to the fractions of targets affected by the estradiol based on gene expression profiling experiments. Both downregulated and upregulated genes were scored assuming that expression changes would alter the stoichiometry of viral protein/human prey protein interactions. Numerical values next to each viral protein names indicate the number of affected target protein-encoding genes. Panels (B) and (C) reports the results of GSEA of the 332 genes in the Ligand Perturbations from GEO of downregulated (B) and upregulated (C) genes. Note marked representations of the estradiol records among top 10 significantly enriched entries of endogenous ligands.

Figure 6

Effects of the VDR gene, vitamin D, and quercetin on pathways and genes affecting the newly emerged SARS-CoV-2 virus-related host targets. (A) Effects of the VDR gene, vitamin D, and quercetin on repressors of the ACE expression. (B). Effects of the VDR gene, vitamin D, and quercetin on activators of the ACE expression. (C). Effects of the VDR gene, vitamin D, and quercetin on repressors of the ACE expression reflecting GATA5 inhibitory effects on SFTPC expression in the mouse lungs. (D). Effects of the VDR gene, vitamin D, and quercetin on activators of the ACE expression reflecting the cell type-specific effects of vitamin D and quercetin: vitamin D-induced activation of the INSIG1 expression in human bronchial smooth muscle cells and quercetin-induced activation of the INSIG1 expression in human intestinal cells.

Figure 6

Effects of the VDR gene, vitamin D, and quercetin on pathways and genes affecting the newly emerged SARS-CoV-2 virus-related host targets. (A) Effects of the VDR gene, vitamin D, and quercetin on repressors of the ACE expression. (B). Effects of the VDR gene, vitamin D, and quercetin on activators of the ACE expression. (C). Effects of the VDR gene, vitamin D, and quercetin on repressors of the ACE expression reflecting GATA5 inhibitory effects on SFTPC expression in the mouse lungs. (D). Effects of the VDR gene, vitamin D, and quercetin on activators of the ACE expression reflecting the cell type-specific effects of vitamin D and quercetin: vitamin D-induced activation of the INSIG1 expression in human bronchial smooth muscle cells and quercetin-induced activation of the INSIG1 expression in human intestinal cells.

Figure 6

Effects of the VDR gene, vitamin D, and quercetin on pathways and genes affecting the newly emerged SARS-CoV-2 virus-related host targets. (A) Effects of the VDR gene, vitamin D, and quercetin on repressors of the ACE expression. (B). Effects of the VDR gene, vitamin D, and quercetin on activators of the ACE expression. (C). Effects of the VDR gene, vitamin D, and quercetin on repressors of the ACE expression reflecting GATA5 inhibitory effects on SFTPC expression in the mouse lungs. (D). Effects of the VDR gene, vitamin D, and quercetin on activators of the ACE expression reflecting the cell type-specific effects of vitamin D and quercetin: vitamin D-induced activation of the INSIG1 expression in human bronchial smooth muscle cells and quercetin-induced activation of the INSIG1 expression in human intestinal cells.

Figure 7

Similarity between chemical structures of the luteolin, quercetin, and eriodictyol. Luteolin, quercetin, and eriodictyol have been identified in the recent supercomputer SUMMIT drug-docking screen as top candidate inhibitors of the SARS-CoV-2 spike-protein–human ACE2 receptor interface–ligand binding complex [42], while Luteolin and quercetin have been identified as potent inhibitors of the SARS-CoV infection [43].

Figure 8

Proposed mitigation approaches for prophylaxis and treatment of the COVID-19 pandemic based on prescriptions of bipartite (vitamin D and quercetin) and tripartite (vitamin D/quercetin/estradiol) combinations of candidate therapeutics.