- Elevated plasmin levels are found in COVID-19 patients
- Plasmin allows the SARS-CoV-2 virus to bind with ACE2 receptors by cleaving the S protein on the virus
- Plasmin breaks down fibrin, increasing the levels of D-dimer and decreasing platelet count; this could possibly prevent inflammation in COVID-19 patients, but it could result in hemorrhaging
- Plasminogen is expressed in both endothelial and epithelial cells
- Severe and deceased COVID-19 patients had very low platelet counts
- However, plasmin causes hypertension and dehydration in the lungs due to an increase in sodium ion uptake


Plasmin and COVID-19

Spironolactone inhibits ACE2 expression on the epithelium of lung tissue, and could potentially replace ACE inhibitors. COVID-19 patients could face withdrawal from ACE inhibitors, which could be prevented by using spironolactone. 


Spironolactone as a potential replacement for ACEI

- the novel compounds under development is human recombinant soluble ACE2 (hrsACE2 [APN01; Apeiron Biologics, Vienna, Austria])
- 2 benefits: 
1. binding the viral spike protein and thereby neutralising SARS-CoV-2.
2. minimising injury to multiple organs, including the lungs, kidneys, and heart, because of unabated renin–angiotensin system hyperactivation and increased angiotensin II concentrations
- hrsACE2 can reduce SARS-CoV-2 load by a factor of 1000–5000 in in-vitro cell-culture experiments
- hrsACE2 has been tested in 89 patients. After the first injection of hrsACE2 we observed a marked reduction of angiotensin II. Concurrent with reduced angiotensin II concentrations we detected a marked reduction in the inflammatory cytokine interleukin (IL)-6 and chemokine IL-8, both of which play an important role in lung injury and cytokine storm.
- hrsACE2 can reduce the cytokine storm which is the killer for COVID-19 patients


Human recombinant soluble ACE2 (APN01) as a potential treatment for COVID-19

ACE2 inhibitors have been shown to have antiviral abilities against other CoVs, but their efficacy has yet to be tested for SARS-CoV-2.  Different ACE2 inhibitors have different functions, with some:

- targeting the ACE2 binding region so that SARS-CoV-2 S protein cannot bind there
- creating recombinant human ACE2 (rhACE2) that "tricks" the virus into binding to the recombinant ACE2 instead of somatic ACE2
- inhibiting the function of TMPRSS2, which would reduce the virus's ability to efficiently enter host cells

Targeting human proteases would be controversial, as there could be many adverse side effects.  Recently, the main SARS-CoV-2 protease structure was determined.  It is thought that an inhibitor to this viral protease could be created, and this could be a promising drug candidate.

Michigan State University

- Inhibition of the binding between the spike protein present on the surface of the virus and the ACE-2 receptor present in the membrane of the host cell. This interaction mediates the entry of the virus in the cell. 
- Because ACE 2 plays a central role in the pathogenesis of COVID-19, it is thought that it could be an efficient drug target that could either treat or prevent infection. 

ACE2 normal role:
-Critical to regulate processes such as pressure, wound healing, and inflammation. 


-It helps modulate ANGII, a protein that increases blood pressure and inflammation, increasing damage to blood vessel lining and tissue. 

COVID-19:
-In response to COVID-19, ANGII has increased inflammation and the death of alveoli cells which are critical for bringing oxygen into the body...ACE2 prevents this.

-When COVID-19 virus binds to ACE2, it inhibits its action, thus increasing the effects of ANGII
 

ACE 2 Receptor as a Pharmacological Target for Treating or Preventing COVID-19

There are existing drugs that could, in theory, based on their physiological roles, have a positive pharmacological effect on treating or preventing COVID-19.  These drugs may not have been tested yet, or they may have been proved to be ineffective.  

- ACE2 inhibitors
- serine protease inhibitors
- Mas receptor activators
- SARS-S and MERS-S inhibitors 
- soluble ACE2 receptors
- combination therapies
- N-acetylcysteine (NAC)
- antimicrobial peptides
- androgen receptors
- probiotics targeting the gut microbiota
- coronaviral protease inhibitors
- ACE inhibitors + Angiotensin Receptor Blockers (ARBs)
- JAK inhibitors
- Alpha-lipoic acid (ALA)
- Hydrogen Sulfide (H2S)

Theoretical COVID-19 Drug Targets and Candidates

Tan, H. W., Xu, Y. M., & Lau, A. T. Angiotensin‐converting enzyme 2: The old door for new severe acute respiratory syndrome coronavirus 2 infection. Reviews in Medical Virology, e2122. https://onlinelibrary.wiley.com/doi/full/10.1002/rmv.2122

Angiotensin‐converting enzyme 2: The old door for new severe acute respiratory syndrome coronavirus 2 infection

Because ACE and ACE2 are structurally similar, it was thought that existing ACE inhibitor (ACE-I) drugs could be used as a COVID-19 treatment by inhibiting ACE2 activity.  However, because the enzymatic binding sites of ACE and ACE2 are different, ACE-Is do not affect ACE2 activity.  Additionally, COVID-19 patients who took ACE-I drugs were more likely to experience more severe symptoms and had a higher mortality rate than those that didn't take them.  It is thought that ACE inhibition in the RAS may exacerbate the vulnerability of patients with hypertension, diabetes mellitus, or cardiovascular disease to SARS-CoV-2 infection or COVID-19 symptoms.

Using ACE Inhibitors (ACE-Is) to Treat or Prevent COVID-19

Using ACE2 Inhibitors to Treat or Prevent COVID-19

Sriram, K., Insel, E., & Loomba, R. (2020). What is the ACE2 receptor, how is it connected to coronavirus and why might it be key to treating COVID-19? The experts explain. The Conversation.


What is the ACE2 receptor, how is it connected to coronavirus and why might it be key to treating COVID-19? The experts explain 


Four potential TMPRSS2 protease inhibitors were tested:

- Chemostat

- Upamostat

- Nafmostat

- Bromhexine hydrochloride

Each of these existing, clinically proven drugs were found to bind to the catalytic pocket of TMPRSS2 with high precision and specificity.  Theoretically, these drugs could impede SARS-CoV-2 S proteins from interacting with TMPRSS2 which would consequently influence infectivity.  This could be a hopeful target for antiviral drugs or a future vaccine.  

Using Serine Protease Inhibitors to Treat or Prevent COVID-19

It is hypothesized that a targeted Mas receptor, such as AVE0091, could prevent SARS-CoV-2 from entering cells with high ACE2 expression.  If so, this could lessen the morbidity and mortality of COVID-19.  

Using Mas Receptor Activators to Treat or Prevent COVID-19

At least 100 existing pharmaceuticals can theoretically bind to COVID-19's spike protein and hinder or slow the infection process. These include Irolapride, an antidepressant, and Pirifibrate, an antilipidemic. 
These existing drugs are valuable, as they are both readily available and may already be cleared for use in humans. Their effectiveness otherwise remains unknown. 

Using Existing Pharmaceuticals to Target Binding Pockets

In this study two SARS-CoV-2  inhibitors were identified and three compounds- cepharanthine, abemaciclib and trimipramine- were discovered to be broad-spectrum spike-mediated entry inhibitors. 
- spike protein: general information
- general procedure
- cepharanthine results
- abemaciclib results
- trimipramine results
- importance/Future Directions

Using SARS-S and MERS-S Inhibitors to Prevent COVID-19

Targeting the ACE2 receptor has been a recurring motif in pharmacological research of COVID-19, but most efforts have proven to be ineffective or dangerous to those who take these drugs.  One proposal is to create soluble "decoy" ACE2 receptors.  Theoretically, if the virus were to completely target these receptors, endogenous ACE2 receptors would remain unaffected.  This proposal also does not involve changing the expression of endogenous ACE2 which could have onerous outcomes.  Cell culture studies have shown that soluble ACE2 can reduce a viral infection by a factor of 5000.  This could be a useful therapeutic or prevention method.  

Using Soluble ACE2 Receptors to Treat or Prevent COVID-19

Hou et al. proposed three different models for treating COVID-19 based on three different phenotypes which are found in different populations.  The following drugs were used in the models:

- hydroxychloroquine
- E-64D (a protease inhibitor)
- camostat mesylate (a TMPRSS2 inhibitor approved in Japan for treating chronic pancreatitis) 

These pharmacogenomic models have yet to be tested and validated.  

Hypothesized Combination Therapies to Treat COVID-19 Based on Pharmacogenomics

N-acetylcysteine (NAC) thins mucus as a mucolytic because of its its free sulfhydryl group which reduces disulfide bonds in mucus glycoproteins.  Additionally, it is a strong antioxidant.  NAC may be beneficial in treating or preventing SARS-CoV-2 infections in the following ways:

- NAC may be able to cleave SARS-CoV-2 E protein which would decrease its infectivity
- NAC has been shown to decrease angiotensin II bonds to angiotensin II type 1 receptors in vitro
- NAC has been shown to block ACE activity and limit the amount of Ang II both in vitro and clinically 
- NAC may be able to counteract the reactive oxygen species (ROS) produced by cytokine storm syndrome
- NAC has been shown to restore platelet GSH reserves and prevent protein glycosylation, a pathological mechanism of COVID-19 in diabetic patients
- NAC has been shown to inhibit NF-κB activation in an in vitro influenza (A and B) model which mediates COVID-19 pathology in the lungs by initiating the production of pro-inflammatory cytokines


Using N-acetylcysteine to Treat or Prevent COVID-19

Computational screening and molecular docking are two methods that are being used to discover new drugs to combat SARS-CoV-2 infections and COVID-19.  Many of these efforts to discover new drugs use SARS-CoV-2 S protein as a pharmacological target.  The concept of antimicrobial peptides is to introduce peptides to SARS-CoV-2 S protein that would change the viral protein's conformation so that it cannot properly interact with ACE2 receptors.  This would consequently stop the virus from infecting a cell.  These synthetic peptides would be more clinically favorable than other antivirals because they have no side effects, cause no hemolytic activity, and are not toxic to human cells.  The following peptides were tested by Souza et al.:

- Mo-CBP3-PepI
- Mo-CBP3-PepII
- Mo-CBP3-PepIII [22,23]
- RcAlb-PepI
- RcAlb-PepII
- RcAlb-PepIII [24]
- PepGAT
- PepKAA

Using Antimicrobial Peptides to Treat or Prevent COVID-19

TMPRSS2 is a serine protease involved in the cellular entry of SARS-CoV-2.  Its gene is regulated in part by upstream androgen receptors and has consequently been shown to be androgen-sensitive.  It may be possible to treat a SARS-CoV-2 infection using androgen deprivation therapy (ADT) or -anti-androgens which are currently used as prostate cancer therapies.  

Using Androgen Receptor Inhibitors to Treat or Prevent COVID-19

The gut microbiota is thought to be  an important regulator of antiviral immune responses.  Studies have shown that modulating the gut microbiota has been able to reduce viral-related enteritis, severe pneumonia cases requiring ventilator assistance, and the spread of influenza in lung epithelial cells.  It is thought that probiotic treatment in COVID-19 patients could promote a healthy gut microecology, which could mitigate GI symptoms, as well as protect the respiratory system from other infections via the bi-directional communication network that is thought to exist between the gut and lung microbiotas.  

Using Probiotics to Treat or Prevent COVID-19

Figure 1
- People who develop ALI/ARDS along with COVID-19 end up with an infected lung
- Eventually, inflammation in organ tissues will develop due to elevated IL-6 levels and decreased white blood cells; oxidation stress occurs
- Administering melatonin into the system could lead to recovery, as it can lower IL-6 levels and the effect of oxidative enzymes is decreased
- If melatonin does not work, the body’s immune system will go out of control (cytokine storm)
- Administering melatonin once again can help prevent problems associated with ALI/ARDS


Using Melatonin to Treat or Prevent COVID-19

JAK inhibitors are usually used to help people who may be affected by rheumatism, which is an autoimmune disease. They are known as DMARDs. People who have rheumatism, their medications usually consist of immunosuppressants which cause a higher infectious risk, leading to concerns of several diseases. Interestingly, people who are taking medications with JAK inhibitors are not experiencing this issue with COVID-19. No data suggests that patients with rheumatic diseases are at an increased risk for COVID-19 compared to the general population. Medications such as Baricitinib and Xeljanz (generic name Tofacitinib) which have JAK inhibitors in them are, predicted to block SARS-CoV-2 receptor-mediated endocytosis into pulmonary epithelial cells. An open-label study with 12 patients with oral baricitinib added to ritonavir/lopinavir therapy showed promising results as all patients improved at 2 weeks and required no ICU admission.


Using JAK Inibitors to Treat or Prevent COVID-19

Alpha-lipoic acid (ALA) is a drug that is currently used to treat diabetic polyneuropathy.  It increases insulin signalling sensitivity, functions as an antioxidant, and inhibits NF-kB activity.  Additionally, ALA has been demonstrated to have anti-viral properties.  Its ability to reduce NF-kB activity, increase intracellular glutathione concentrations, chelating metal ions, and restoring antioxidant levels have all been shown to have protective effects against different viruses.  ALA may be specifically useful in treating or preventing COVID-19 because of its ability to prevent ACE2 upregulation by inhibiting ADAM17, increasing intracellular pH by activating sodium/potassium pumps, and increasing the amount of intracellular antioxidants to bolster the host cell's defense mechanism.  

Using Alpha-Lipoic Acid (ALA) to Treat or Prevent COVID-19

Coronavirus 3C-like proteases are the enzymes on the virus that play a key role in viral replication. Coronaviral protease inhibitors could theoretically prevent the virus from initially infecting the host cell, and appear to be promising drug targets. 

Types:
- GC376
- C-Phycoyanin
- peptidomimetic α-ketoamides 

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