COVID-19 is caused by SARS-CoV-2 infection and characterized by diverse clinical symptoms. Type I interferon (IFN-I) production is impaired and severe cases lead to ARDS and widespread coagulopathy. We propose that COVID-19 pathophysiology is initiated by SARS-CoV-2 gene products, the NSP1 and ORF6 proteins, leading to a catastrophic cascade of failures. These viral components induce signal transducer and activator of transcription 1 (STAT1) dysfunction and compensatory hyperactivation of STAT3. In SARS-CoV-2-infected cells, a positive feedback loop established between STAT3 and plasminogen activator inhibitor-1 (PAI-1) may lead to an escalating cycle of activation in common with the interdependent signaling networks affected in COVID-19. Specifically, PAI-1 upregulation leads to coagulopathy characterized by intravascular thrombi. Overproduced PAI-1 binds to TLR4 on macrophages, inducing the secretion of proinflammatory cytokines and chemokines. The recruitment and subsequent activation of innate immune cells within an infected lung drives the destruction of lung architecture, which leads to the infection of regional endothelial cells and produces a hypoxic environment that further stimulates PAI-1 production. Acute lung injury also activates EGFR and leads to the phosphorylation of STAT3. COVID-19 patients’ autopsies frequently exhibit diffuse alveolar damage (DAD) and increased hyaluronan (HA) production which also leads to higher levels of PAI-1. COVID-19 risk factors are consistent with this scenario, as PAI-1 levels are increased in hypertension, obesity, diabetes, cardiovascular diseases, and old age. We discuss the possibility of using various approved drugs, or drugs currently in clinical development, to treat COVID-19. This perspective suggests to enhance STAT1 activity and/or inhibit STAT3 functions for COVID-19 treatment. This might derail the escalating STAT3/PAI-1 cycle central to COVID-19.
We hypothesize that COVID-19 disease is due in large part to the actions of the SARS-CoV-2 NSP1 and ORF6 proteins, which cripple STAT1 function and predominantly promote STAT3 activation. STAT3 in turn upregulates PAI-1, and together these molecules serve as a central hub of reactions that perpetuate a catastrophic cascade. Our understanding of immune responses, coupled with lessons from SARS-CoV-1 and recent research on SARS-CoV-2, point to STAT1 and STAT3 as enticing drug targets because they function upstream of the cytokine storm and thrombosis. Developing vaccines will take some time, and attacking the downstream cytokine storm is difficult due to its many targets. Hence, in the short term, the manipulation of STAT1 and/or STAT3 may be the most practical strategy for treating COVID-19. Fortunately, many therapeutic regulators of these STATs have been, or are being, developed.
The first consideration in treating SARS-CoV-2 infection should be to preserve STAT1 function in its earliest stages. However, patients may be asymptomatic or presymptomatic for days while the virus replicates, spreads, and evades the IFN response. If the virus has already compromised STAT1 when symptoms arise, then treatment should focus on preventing the excessive activation of STAT3 that drives the release of proinflammatory cytokines and chemokines. Care must be taken to apply these therapeutic strategies in a stage-appropriate manner, because some approaches that may help in the earlier stages of the disease could be detrimental if used in its later stages. In the following sections, we discuss the potential use for COVID-19 treatment of existing drugs that enhance STAT1 or inhibit STAT3 functions.
Problems could arise from this STAT-approach to COVID-19 therapy. Chronic mucocutaneous candidiasis (CMC) was present in 98% of patients with gain of function (GOF) mutations in STAT1 in one study of 274 individuals, and the few that did not have CMC, had invasive fungal or bacterial infections. Generally, the immune profile was relatively normal, but 82% had decreased Th17 cells. STAT1 and STAT3 reciprocally regulate each other, and consistently, STAT1 GOF patients have reduced levels of STAT3. STAT3 IgE syndrome (STAT3-HIES) is a rare autosomal dominant condition caused by loss of function mutations in the STAT3 gene. Patients have eczema, recurrent skin and respiratory tract infections, and usually high levels of IgE. These examples of genetic defects are likely the most extreme consequences of increased STAT1 or decreased STAT3 activities. It is unknown if temporary treatment with STAT1 activators and STAT3 inhibitors would be detrimental, but it is promising that a high STAT1 to STAT3 ratio is advantageous in cancer treatment.
Enhancement of STAT1 function IFN-I At the very onset of infection, SARS-CoV-2 infects a minority of cells, making its invasion hard to detect. The virus acts to delay antiviral responses while it hijacks the cell’s functions for viral replication. If the infection is detected early, then an effective strategy might be to use IFN-I to activate STAT1 in neighboring uninfected cells. It is proposed that the timing of IFN treatment is critical [30]. Early induction of IFN-I signaling protects the patients, but delay in IFN administration not only fail to inhibit viral replication, but also increase proinflammatory cytokine production, leading to fatal pneumonia [30]. Since the upper respiratory tract is the primary entry site for SARS-CoV-2, mucosal treatments with IFN-I for prevention of COVID-19 is an ideal strategy. Meng et al. used IFN-I nasal drops prophylactically applied to more than 500 high-risk medical staff who were in direct contact with SARS-CoV-2-infected patients. Remarkably, the use of the IFN-I nasal drops, with thymosin-α1, an immune-stimulator, protected all of the high-risk staff from COVID-19 pneumonia.
IFN-I inducers The use of agents that induce IFN production is another option for activating STAT1 in uninfected cells. Ampligen, poly(I:C(12)U), is a synthetic dsRNA polymer that stimulates IFN production. Mice treated with Ampligen 16 h prior to lethal infection with SARS-CoV-1 were protected against death, showed reduced virus titers in the lungs, and exhibited significantly reduced lung disease scores and weight loss. Notably, because IFN-I inducers can be administered prophylactically, the innate immune system can theoretically be primed to respond to a SARS-CoV-2 attack by immediately activating STAT1. However, such use of IFN inducers must be carefully monitored, because these agents can exacerbate the disease in the later stages of infection.
Histamine receptor-2 blocker (H-2 blocker) Administration of the H-2 blocker famotidine to patients hospitalized with COVID-19, but not initially in the ICU, was associated with a twofold reduction in clinical deterioration leading to intubation or death. Computational modeling indicated that famotidine directly binds and inhibits the SARS-CoV-2 processing enzyme NSP5, but this drug lacked a direct anti-SARS-CoV-2 effect in vitro in Vero cells, and H-2-mediated antiviral effect is suggested. In vitro, histamine pretreatment of C57BL/6 mouse splenocytes enhances STAT1 phosphorylation, and an H-2 antagonist (but not an H-1 antagonist) can augment STAT1 phosphorylation to a similar extent. Famotidine may function as an H-2 receptor antagonist that promotes STAT1 activation and IFN responses. Therefore, early stage treatment with famotidine might significantly decrease the mortality rate of COVID-19 patients.
Ivermectin Ivermectin displays broad-spectrum antiparasitic and antiviral activities. Caly et al. reported that a single dose of ivermectin reduced the amount of SARS-CoV-2 RNA by 5000-fold. They speculated that ivermectin inhibits the KPNA/KPNB1-mediated nuclear import of viral proteins. Apart from its possible role in blocking nuclear transport, Ivermectin may promote a positive clinical outcome by inhibiting STAT3 and IL-6 production. Ivermectin inhibits p21 activated kinase 1 (PAK1), a serine/threonine kinase with oncogenic activity, which then compromises STAT3 activity. In this case, ivermectin suppresses Akt/mTOR signaling by promoting the ubiquitination-mediated degradation of PAK1 . In addition, PAK1 physically binds to both JAK1 and STAT3, and the resultant PAK1/STAT3 complex activates IL-6 gene transcription. When ivermectin inhibits JAK/STAT3 signaling by promoting PAK1 degradation, STAT3 activity is compromised and IL-6 production is decreased.
Inhibition of STAT3 function There are a number of approved drugs, or drugs in cancer development, that inhibit STAT3 directly. STAT3 antagonists have been described comprehensively in recent reviews by Qin et al. [164] and Bharadwaj et al. [21]. A brief description of some prominent candidates follows.
Napabucasin Napabucasin was initially identified by its ability to inhibit the properties of cancer cell stemness and STAT3 activity in gel retardation assays. A Phase III clinical trial involving napabucasin in combination with other standard chemotherapeutic agents is currently underway for several advanced malignancies. In rectal cancer, napabucasin reduced not only pSTAT3 levels but also angiogenesis through a ROS-mediated effect.
Danvatirsen Another promising anti-STAT3 agent is the 16-mer antisense oligonucleotide AZD9150 (danvatirsen), which targets the 3′UTR of the STAT3 gene and inhibits its transcription. A Phase II trial of danvatirsen plus anti-PD-L1 monoclonal antibody in patients with head-and-neck squamous cell carcinoma is currently underway, with encouraging results thus far.
Metformin Metformin is a first-line oral antidiabetic drug that has been used to treat type 2 diabetic patients for over 60 years. Metformin inhibits STAT3 by specifically reducing its phosphorylation at Tyr705 and Ser727. In addition, metformin prevents both venous and arterial thrombosis by inhibiting platelet activation and extracellular mitochondrial DNA (mtDNA) release. mtDNA induces platelet activation through a DC-SIGN-dependent pathway. Despite metformin’s apparent utility in reducing STAT3 activation and thrombosis, a major concern is that the dose that elicits these activities is very high (200–400 mg/kg/day).
PIAS3 activators The regulation of endogenous inhibitors is another way to control aberrantly activated STAT3. PIAS3 is an ideal target because of its potency to inhibit the activated STAT3. Specifically, curcumin and resveratrol have been shown to suppress constitutive activation of STAT3, through upregulation of PIAS3. Although widely used for many indications, curcumin and resveratrol have not been shown to be conclusively effective in any randomized, placebo-controlled, clinical trial.
JAK inhibitors The occurrence of a cytokine storm in COVID-19 (IL-2, IL-6, IL-7, IL-10, G-CSF, IFNγ, MIP1α, and TNF-α, which triggers cytokine receptors coupled to the JAK-STAT pathway, suggests that inhibition of the JAK pathway may be an appropriate therapeutic strategy for the management of COVID-19. Although the JAK pathway affects many STATs, perhaps the inhibition of the JAK pathway may lead to the therapeutically favorable effect of quenching cytokine storm via STAT3 inhibition. The available JAK1/2 inhibitors could be studied for their effects in COVID-19. However, as JAK inhibitors also inhibit STAT1 activation, the application of JAK inhibitors in COVID-19 cases needs careful consideration.
EGFR signaling inhibitors as an IFN-I potentiator As discussed earlier and shown in Fig. 3, acute lung injury and/or the loss of functional STAT1 can lead to the upregulation of EGFR that may cause the constitutive activation of STAT3. Therefore, it is reasonable to assume that targeting EGFR signaling is an attractive strategy for COVID-19 treatment. A promising candidate is erlotinib. Lupberger et al. reported the combination of erlotinib and IFN-I resulted in a highly synergistic antiviral response against hepatitis C virus. Furthermore, erlotinib reduced IFN-I-induced STAT3 activity by induction of SOCS3 expression. Very recently, the remarkable inhibition of SARS-CoV-2 replication by several growth factor signaling inhibitors was reported. These include lonafarnib (RAS inhibitor), omipalisib (PI3K inhibitor), pictilisib (PI3K inhibitor), RO5126766 (RAF and MEK inhibitor), sorafenib (RAF inhibitor, STAT3 inhibitor), because Ras/Raf/MAPK pathway and PI3K pathway are also involved in EGFR-mediated STAT3 activation, these inhibitors can possibly synergistically potentiate IFN-I’s anti-SARS-CoV-2 activity.
PAI-1 inhibitors and TLR4 inhibitors As noted above, PAI-1 is intimately involved in the pathogenesis of COVID-19, and its inhibition may be a key point at which to treat the disease once the escalating cycle between PAI-1 and STAT3 has been established. Unfortunately, no FDA-approved PAI-1 inhibitor is currently available. PAI-1’s labile structure appears to make it a difficult target for the development of small-molecule inhibitors. Alternatively, therapeutically targeting TLR4, PAI-1’s binding partner, may be equally effective. TLR4 has already attracted keen interest as a therapeutic target for sepsis cases. Although the molecular mechanisms have yet to be clarified, it is worth investigating whether PAI-1/TLR4 binding can be inhibited by the several TLR4 antagonists in development, or by approved drugs with anti-TLR4 activity. One TLR4 inhibitor, Eritoran, is now in clinical trials to treat ARDS in COVID-19.
Concluding remarks The SARS-CoV-2 virus has evolved multiple tools to escape immune detection and destruction, and thus has become the most formidable virus in over 100 years. Our survey of the current literature reveals that severe cases of COVID-19 are commonly dependent on the over-stimulation of the STAT3/PAI-1 signaling network. We believe that this shared node may be the Achilles’ Heel of COVID-19 and a vulnerable point of the disease. We therefore urge the immediate investigation and application of STAT therapy as a treatment for this perplexing disorder, and hope that our article provides a sound foundation for doing so. A final note: during our analysis of the literature on COVID-19 and related topics, we noticed many similarities in the pathogenesis of COVID-19 and cancers. In fact, both STAT3 and PAI-1 have been separately implicated in cancer development and are the subjects of extensive clinical investigations. A shared node of STAT3 and PAI-1 activities may also function in some pSTAT3-positive cancers, creating a cascade of harmful responses comparable to those of COVID-19. Any STAT-related agents developed to treat COVID-19 may therefore eventually enjoy much wider clinical use.
Reference & source information: https://www.nature.com/
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