The 2019 coronavirus disease pandemic (COVID-19), caused by the newly emerged SARS-CoV-2 virus is rapidly transitioning through the dangerous acute phase of its evolution in the United States. The absence of a 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 [1
]. Recent analyses of the structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein revealed the key role of the ACE2
genes in facilitating the high-affinity binding of viral particles and their entry into human cells [3
]. The efficient invasion of host cells by SARS-CoV-2 is further enhanced by the presence of the unexpected FURIN cleavage site, which is cleaved during biosynthesis [3
]. 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 SARS-CoV-2 [3
]. The crystal structure and high-resolution cryo-electron microscopy of the SARS-CoV-2 receptor-binding domain (RBD) in complex with human ACE2 revealed specific structural features of the SARS-CoV-2 RBD that appear to enhance its binding affinity to human ACE2 [4
]. Collectively, these observations firmly established protein products of the human genes ACE2
as the principal mediators of the SARS-CoV-2 invasion into human cells, acting as the high-affinity receptor (ACE2) and invasion-promoting protease (FURIN), respectively.
In this contribution, genomic screens were performed employing the ACE2 and FURIN genes as baits to build genomic-guided human tissues-tailored maps of upstream regulatory elements, their expression and functions. To identify the high-priority list of potential candidate mitigation agents, 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; HIF1a) and activators (HMGA2; INSIG1; RUNX1; HNF4a; JNK1/c-FOS) of 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 the coronavirus pandemic. Collectively, observations reported in this contribution indicate that highly structurally similar quercetin, luteolin, and eriodictyol could serve as scaffolds for the development of efficient inhibitors of SARS-CoV-2 infection. Unexpectedly, present analyses revealed discordant patterns of testosterone versus estradiol impacts on SARS-CoV-2 targets with the former manifesting potential coronavirus infection-promoting activities, which is consistent with the apparently higher male mortality across all age groups during the coronavirus pandemic. Significantly, in agreement with findings reported herein, numerous observational studies suggest that age-associated vitamin D insufficiency and/or deficiency may contribute to the high mortality of older adults and elderly individuals during the COVID-19 pandemic. Consequently, vitamin D supplementation may mitigate the severity of the disease.
The main motivation of this work was to identify human genes implicated in regulatory cross talks affecting expression and functions of the ACE2
genes to build a model of genomic regulatory interactions potentially affecting SARS-CoV-2 infection. A panel of genes acting as activators and/or repressors of the ACE2
expression then could be employed to search for existing drugs and medicinal substances that, based on their mechanisms of action, could be defined as candidate coronavirus infection mitigation agents. After experimental and clinical validation, these existing drugs and/or medicinal agents could be utilized to ameliorate the clinical severity of the pandemic. This knowledge could also be exploited in an ongoing effort to discover novel targeted therapeutics tailored to prevent SARS-CoV-2 infection and block the entry of the virus into human cells. Observations reported in this contribution are in agreement with recent studies describing numerous beneficial clinical effects of the vitamin D supplementations, emphasizing many detrimental effects of the vitamin D insufficiency and deficiency, and underscoring the significant COVID-19 mitigation potential of vitamin D [29
]. Importantly, two recent interventional randomized clinical trials aiming to evaluate effects of vitamin D on the prevention and treatment of COVID-19 were listed on the ClinicalTrials.gov website (https://www.clinicaltrials.gov/ct2/show/NCT04334005
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 study, it has been concluded that any definitive recommendations regarding the potential clinical utility of the herein identified putative coronavirus infection-mitigating agents, namely vitamin D, quercetin, and estradiol, should be made only after preclinical studies and randomized controlled clinical trials have been appropriately designed, carefully executed, and the desired outcomes have been reached.
A supercomputer modeling study using the world’s most powerful supercomputer, SUMMIT, identified several candidate small molecule drugs which bind to either the isolated SARS-CoV-2 Viral S-protein at its host receptor region or to the S protein-human ACE2 interface [42
]. Interestingly, in this study, quercetin was identified among the top five scoring ligands for viral S-protein-human ACE2 receptor interface. Thus, quercetin also appears to be a potentially promising therapeutic molecule that may directly interfere with the binding of SARS-CoV-2 to human cells. Previously reported experiments demonstrated that quercetin appears to inhibit SARS-CoV entry into host cells [43
]. Since SARS-CoV-2 utilizes, for the entry into human cells, the same receptor (ACE2) and the accessory protease FURIN as the SARS-CoV coronavirus [3
], these observations suggest that quercetin may, indeed, possess antiviral activity against SARS-CoV-2 as well. Significantly, both quercetin and luteolin have been identified among the top five ligands for the viral S-protein–human ACE2 receptor interface–ligand-binding complex [42
], suggesting that these highly structurally similar compounds (Figure 7
) could serve as efficient inhibitors of SARS-CoV-2 infection. Consistent with this hypothesis, it has been reported that both quercetin and luteolin significantly inhibit the SARS-CoV virus infection [43
It has been observed that administration of testosterone appears to manifest clearly defined patterns of altered gene expression, consistent with testosterone being identified as the potential coronavirus infection-promoting agent, particularly in some cell types that may play a role in the virus entry into the human body and the respiratory system (Supplementary Figure S11
). This is in contrast to estradiol, which seems to manifest complex cell type-specific effects on gene expression consistent with either infection-inhibiting or infection-promoting patterns of gene expression changes. It would be of interest to determine whether these discordant effects may contribute to the apparently higher mortality among men with coronavirus infection. In line with present analyses, a randomized interventional clinical trial entitled “Phase II Clinical Trial of estradiol to Reduce Severity of COVID19 Infection in COVID19+ and Presumptive COVID19+ Patients” has been posted on ClinicalTrials.gov with the start date April 20, 2020 and completion date November 20, 2020 (https://clinicaltrials.gov/ct2/show/NCT04359329
). Interestingly, a hypothetical tripartite combination of pandemic mitigation agents consisting of vitamin D/quercetin/estradiol may affect the expression of 244 out of 332 (73%) human genes encoding SARS-CoV-2 targets (Table 1
and Table 2
). It has been observed that both bipartite (vitamin D and quercetin) and tripartite (vitamin D/quercetin/estradiol) combinations of candidate pandemic mitigation agents manifest statistically more robust effects on expression of SARS-CoV-2 target genes compared to monotherapies (Table 1
and Table 2
). These differences were documented for comparisons of either the numbers of affected human genes encoding SARS-CoV-2 targets (Table 1
) or the numbers of SARS-CoV-2 viral proteins presumed to be functionally blocked by the prospective therapeutics (Table 2
The present analyses highlight the major uncertainty regarding the outcomes of the current pandemic associated with the potential of SARS-CoV-2 in the expansion of the cellular tropism [3
] based on its access to genetically vulnerable host cells due to the nearly ubiquitous expression of the ACE2
genes in the human body. A particular dangerous factor that must be noted in this contribution is the potential ability of SARS-CoV-2 to infect the immune cells, because the infection of immune cells may cause immunosuppression, the long-term persistence of the virus in the body, and the spread of the virus to secondary targets.
It has been reported that both SARS-CoV and SARS-CoV-2 utilize the ACE2 as the entry receptor to infect human cells [3
]. The coronaviruses SARS-CoV and SARS-CoV-2 have comparable binding affinities, achieved by balancing the energetics and dynamics of interactions with the ACE2 receptor [44
]. However, the SARS-CoV-2–ACE2 complex contains a higher number of contacts, a larger interface area, and decreased interface residue fluctuations compared to the SARS-CoV-ACE2 complex [44
], suggesting a markedly distinct and more efficient strategy for SARS-CoV-2 interactions with the human ACE2 receptor. It seems remarkable to attribute this level of adaptation toward the highly efficient human receptor recognition of the natural evolutionary exploration of coronaviruses in non-human hosts.
Taken together with predominantly cell type-specific patterns of expression of genetic repressors and activators of the ACE2
expression, this may complicate the development of universally effective therapeutics. The availability of many genetically relevant transgenic mouse models—in particular, FURIN-null mice—should be regarded as a considerable advantage for the preclinical development of drug candidates tailored to target the coronavirus infection. Specifically, the potential therapeutic utility of the highly selective (Ki, 600 pm) and intrinsically specific FURIN inhibitor (a1-antitrypsin Portland (a1-PDX); [45
]) should be tested in the immediate future.
It has been noted that observations and conclusions derived from the genomic-guided tracing of SARS-CoV-2 targets in human cells appear logistically compatible with relevant experimental and clinical observations. Importantly, observations reported in this contribution seem to provide a mechanistic basis for a better understanding of SARS-CoV-2′s biology and pathophysiology, as well as a diverse spectrum of clinical manifestations of the COVID-19 pandemic, including the potential to inflict the extensive damage to the endothelium, brain, and the cardio-vascular system. Collectively, these findings and conclusions strongly support the feasibility of the proposed bipartite and tripartite combinations of candidate pandemic mitigation agents for the prophylaxis and treatment of COVID-19 (Figure 8
). It is considered a highly encouraging factor that clinical trials have been announced for all three candidate pandemic mitigation agents identified in this study, albeit aiming to evaluate their effectiveness as a monotherapy for the COVID-19 pandemic. Additionally, see the note added in Appendix