- 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

- HCoV‐NL63

- SARS‐CoV

The receptor binding domain (RBD) of the S protein of these CoVs differ from each other, so it is thought that they utilize different mechanisms to enter host cells.  However, it has been observed that the S protein of SARS-CoV-2 is much more similar to that of SARS-CoV than HCoV‐NL63.  

Other CoVs that Utilize the Human ACE2 Receptor for Entry

The receptor binding domain (RBD) is the part the viral S protein that interacts with human ACE2.  It can exist in two conformations: open (B) or closed (A).  Molecular docking assays have shown that SARS-CoV-2 S protein can interact with the  human ACE2 in both of its conformations (D - open; C - closed).  However, the open conformation interacts protease domain (PD) at a more favorable binding energy than the closed domain which interacts with a different region of ACE2.  This confirms the importance of the open conformation of S protein in SARS-CoV-2. infection. 

SARS-CoV-2 S Protein Conformation and ACE2 Receptor Interaction

SARS-CoV-2 S protein has two relevant subunits involved in the pathogenesis of COVID-19: S1 and S2.  S1 has a high binding affinity with the receptor binding domain (RBD) of ACE2.  S2 contains amino acid sequences that are required for the virus to enter host cells.  

ACE2 Interaction with SARS-CoV-2 S Protein by Subunit

A549 are a cell line that do not express ACE2 receptors.  Partridge et al. observed that SARS-CoV-2 S protein interact with these cells even though they do not express ACE2.  Further studies were conducted using two types of 293T cells: cells that express low amounts of ACE2 and cells that over-express ACE2.  S protein interacted with both cell types, but the binding affinity for those expressing low amounts of ACE2 was nearly undetectable.  This suggests that the S protein, specifically the S1 and S2 subunits, may initially interact with host cells via  a different receptor.  However, binding affinity studies suggest that the viral receptor binding domain (RBD) requires higher expression levels of ACE2 to interact with cells.

SARS-CoV-2 S Protein May Interact with Human Cells Independently of ACE2

Five potential binding sites between HCQ/CQ and ACE2 were identified using computerized molecular docking assays.  Reported values are calculated Glide Standard Precision (SP) Glide Extra Precision (XP) measurements which quantify protein/ligand interactions.  These findings support the observation that HCQ is a more effective anti-SARS-CoV-2 drug than CQ in vitro because of HCQ's stronger interaction with the ACE2 allosteric site, which is involved with regulation of the receptor.

ACE2 as a Target for Hydroxychloroquine/Chloroquine in COVID-19 Treatment

Allelic variants in the ACE2 gene has been observed in different ethinic populations, as well as in case-control studies within ethnic groups. However, these are present in relatively low frequencies. Functional studies are encouraged to characterize the relationship between ACE2 variants and COVID-19 severity, but no causal relationship can be made to date.

ACE2 variants and COVID-19

ADAM17 has been demonstrated to upregulate ACE2 expression in cells via a process known as shedding, where it cleaves the extracellular portion of the receptor off of the cell.  Increased ACE2 receptors increases the number of SARS-CoV-2 viral receptors on the cellular surface, and this may exacerbate the infection or COVID-19 symptoms.  

ADAM17 Induced ACE2 Upregulation May Contribute to SARS-CoV-2 Infections

SARS-CoV-2 relies on its spike (S) protein to bind to cellular receptors to enter host cells. SARS-S, the SARS spike protein, uses ACE2 as an entry receptor into cells. Then the virus utilizes the serine protease TMPRSS2 found within the cell to enhance entry and prime future S proteins. SARS‐CoV‐2 is thought to be more transmissible than others CoVs because its S proteins have a greater affinity for ACE2.

Michigan State University

University of Michigan - Ann Arbor

Smith College

Three primary observations have been made about ACE2 and its relation to SARS/COVID-19:

- ACE2 acts as a functional receptor for SARS-CoV-2 to enter host cells
- Increased ACE2 expression may elevate the risk of a SARS-CoV-2 infection as well as the development of COVID-19
- Reduced ACE2 expression may lead to acute respiratory distress syndrome

SARS‐CoV‐2 may be able to infect other tissues that express ACE2 using viral RNA that circulates in the blood.  

ACE2 in the Pathogenesis of COVID-19

The spike glycoprotein (S) is responsible for attachment between the virus and a cell-surface receptor, leading to the fusion of virus to the cell membrane. This membrane-fusion activity can only occur with the proteolytic cleavage of the S glycoprotein into S1 and S2 subunits, which continue to remain associated with one another after cleavage. 


Spike glycoprotein (S)

Hoffmann, M., Kleine-Weber, H., Krüger, N., Mueller, M. A., Drosten, C., & Pöhlmann, S. (2020). The novel coronavirus 2019 (2019-nCoV) uses the SARS-coronavirus receptor ACE2 and the cellular protease TMPRSS2 for entry into target cells. BioRxiv. https://www.biorxiv.org/content/10.1101/2020.01.31.929042v1
doi: 10.1101/2020.01.31.929042

The novel coronavirus 2019 (2019-nCoV) uses the SARS-coronavirus receptor ACE2 and the cellular protease TMPRSS2 for entry into target cells.

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

ACE2 as a Receptor for SARS-CoV-2

ACE2 is known to be expressed in a variety of different tissues, including:

- Heart, kidneys, gastrointestinal (GI) tract, lungs, brain, testes, bladder, adipose tissue, vascular system, cholangiocytes (epithelial cells of the oral and nasal mucosa)

ACE2 expression may explain the SARS-CoV-2 organ localization.

Expression of ACE2 in Tissue and COVID-19

Some believe that the body may initiate an autoimmune response when SARS-CoV-2 is attached to an ACE2 receptor.  The body may consider this complex antigenic, meaning it produces antibodies.  However, these antibodies would target both the virus and ACE2.  This theory is substantiated by:

- the patterns of lung damage in patients with Pulmonary Hypertension secondary to Scleroderma, which is hallmarked by high levels of anti-ACE2 antibodies
- the severity of COVID-19 immune responses in those with cardiovascular disease and hypertension, who would be likely to have increased levels of ACE2 receptors in their blood serum

Measuring ACE2 levels in serum could be a way of predicting at-risk groups for ARDS (acute respiratory distress syndrome) and other hyperimmune responses.  

Autoimmunity to ACE2 Theory in the Pathogenesis of COVID-19

COVID-19 results in the down-regulation of ACE2 receptors by the virus binding to transcription factors at ACE2 regulatory regions on the gene.  This results in increased concentrations of Ang II, which would normally be converted to Ang 1-7 by ACE2.  This may contribute to the hyper-inflammation seen in COVID-19 patients.  

SARS-CoV-2 Down-Regulates ACE2 Receptors

- Neuronal ACE2 Expression as a Risk Factor for COVID-19
- Neuronal ACE2 Internalization in COVID-19
- ACE2 Expression in BBB
- ACE2 Expression in neuroglial cells
- ACE2 Expression in the ENS and Choroid Plexas

ACE2 and its Implications in SARS-CoV2 infection of the CNS

- Increased Concentrations of Circulating ACE2 is Associated with Mild COVID-19 Symptoms 
- Circulating ACE2 expression compared to circulating ACE

Expression of Circulating ACE2 and COVID-19

Using cry-electron microscopy (cryo-EM), the structure of a furin protease-cleaved SARS-CoV-2 S glycoprotein was visualized. These particles were in three different types of conformations: closed, intermediate, and open with an upright receptor-binding domain (RBD). 

Structure of protease-cleaved SARS-CoV-2 S glycoprotein

The presence of a furin-cleavage is favored for infection of SARS-CoV-2 as the cleavage allows the open conformation, exposing the receptor-binding domain (RBD) for easier binding access to a human receptor. The study found that the lower number of S proteins are in a closed conformation for protease-cleaved. There is also an increasing percentage of intermediate form, which is possibly transient, (39%, compared to 34% being closed and 27% being open) demonstrating a high probability that it will shift to an open form and allow binding to a receptor. Therefore, protease cleavage is favored not only for membrane fusion but also for better binding to a receptor by increasing the proportion of S protein on the virus surface. 

Furin protease-cleaved S protein is preferred for SARS-CoV-2

- Cry-electron microscopy (cryo-EM) structures of S glycoprotein of bat virus RaTG13 and uncleaved SARS-CoV-2
- S glycoprotein sequences of RaTG13 and SARS-CoV-2
- S glycoprotein stability 
- Bat virus RaTG13 and SARS-CoV-2 S glycoprotein affinities to human receptor ACE2

Comparison of S glycoprotein between RaTG13 and uncleaved SARS-CoV-2

Partridge et al. have observed multiple heparin binding sites on the SARS-CoV-2 S protein, including:

- one at the furin cleavage site between the S1 and S2 subunits
- others in the S2 domain 
- others in the receptor binding domain (RBD)

SARS-CoV-2 S Protein May Have Multiple Heparin Binding Sites

Prefusion stabilization of the S glycoprotein increases the recombinant expression of viral fusion glycoproteins. Prefusion-stabilized proteins are superior immunogens and can be used in subunit vaccine development. Four substitutions were designed and tested for the SARS-CoV-2 S glycoprotein to produce a more stable construct based on previous knowledge of class I viral fusion protein function.

Prefusion Stabilized SARS-CoV-2 Spike Proteins

- overall low homology between SARS-CoV-2 S Protein and SARS-CoV S Protein
- amino acid sequence identity of the spike proteins is 76.47%
- patches of sequences in RBD show high homology
- amino acid residues at positions 442, 472, 479, 487, and 491 are key to human transmission of the virus
- only Tyr491 is conserved in SARS-CoV-2 S protein; the remaining 4 residues are replaced by other residues of similar properties
- therefore, because the structure of the RBD of SARS-CoV-2 S protein remained the same as SARS-CoV S protein, ACE2 binding is possible and SARS-CoV-2 S protein actually binds with higher binding affinity than SARS-CoV S protein

SARS-CoV-2 S Protein Homology to SARS-CoV S Protein

- mutation sites are distributed over the whole length of the SARS-CoV-2 spike protein sequence
- maximum mutation density was near the protease cleavage site
- D614G mutation is the only most common mutation in spike protein so far among various regions
- some mutations at the interface of the spike protein and ACE2 receptor could affect the efficacy of vaccines and other drugs


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