COVID-19 began as an outbreak of a viral pneumonia-like illness from an unknown origin in Wuhan city, the capital of Hubei province in China. By 7 Jan 2020, Chinese scientists had isolated a novel strain of coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as the causative agent from these patients with viral pneumonia. Here, I want to start by reviewing the structure of this virus, how the virus gains entry and five potential therapeutic interventions: Chloroquine, Remdesivir, TMPRSS2 protease inhibitors, IL-6 inhibitors (Sarilumab/ Tocilizumab), and recombinant ACE2. I’ll do my best to keep this article updated as more information becomes available. Following is a comparison of COVID-19 to other members of coronavirus family by @VirusesImmunity
1. Structure and viral entry
SARS-CoV-2 is a 30 kb positive-stranded, enveloped RNA virus. To better appreciate targeted therapies, we need to first understanding the structure of SARS-CoV-2, and how the virus enters target cells. The novel coronavirus is highly homologous to the coronavirus in bats, and has significant homology with the SARS-CoV (phylogeny shown below). The main structural proteins include: Spike Protein (S), envelope protein (E), membrane protein (M), and nucleocapsid phosphoprotein. Additionally, pertinent transcribed non-structural proteins include: orf1ab, ORF3a, ORF6, ORF7a, ORF10 and ORF8. Of note, spike (S) proteins, ORF8 and ORF3a proteins in SARS-CoV-2 are significantly different from other known SARS-like coronaviruses. These differences may result in more serious virulence and disease transmission than the original SARS-CoV. In particular, ORF8 is mutating rapidly between the multiple emerging strains across different countries 1.
|Phylogenetic analysis of full-length genomes of 2019-nCoV|
The major human cell targets for coronavirus are the type II pneumocytes and enterocytes. So how does the virus enter human cells? To better understand this, we need to review Angiotensin converting enzyme 2 (ACE2), a homolog of ACE. ACE2 is a type-1 integral membrane glycoprotein, and is expressed most actively in kidney, the endothelium, the lungs, and in the heart. The major substrate for ACE2 appears to be Ang II. ACE cleaves angiotensin I to generate angiotensin II, whereas ACE2 inactivates angiotensin II (Ang II) and is a negative regulator of the renin-angiotensin-system (RAS). More specifically, ACE2 degrades Ang II, the main effector of the RAS, to the vasodilators Ang 1-7 (thus reducing vasoconstriction). Additionally, unlike ACE which is a dipeptidase, ACE2 functions as a carboxypeptidase, and therefore ACE2 activity is not antagonized by conventional ACE inhibitors.
ACE2 is relevant to our discussion because the coronavirus S (spike) protein utilizes ACE2 as a receptor for host cell entry. The S protein binds the catalytic domain of ACE2 with high affinity, and this domain is highly conserved between SARS-CoV and SARS-Cov-2. Binding of the coronavirus S protein to ACE2 triggers a conformational change in the S protein of the coronavirus, allowing for proteolytic digestion by host cell proteases (TMPRSS2). This interaction of host cell TMPRSS2 and the viral envelop is a potential therapeutic target, and we’ll return to it later.
Even though ACE2 is considered the main receptor serving as portal of entry, structural analysis of the spike (S) protein reveals that the S protein weakly binds to the ACE2 receptor compared to original SARS-CoV. Cui et al point out that ACE2 is not highly expressed in the lung, a major target organ for the virus 2. Their work presents early computational evidence for angiotensin II receptor type 2 (AGTR2) as a potential secondary receptor needed for SARS-CoV-2 entry into human cells. AGTR2 is a G-protein coupled receptor that can interact with ACE2 and is expressed densely in lung, with a high tissue specificity. Structural simulations of 3D protein-protein interaction reveal that AGTR2 shows a higher binding affinity with the S protein of 2019-nCov than ACE2. It must be noted that further confirmation with molecular experiments is necessary to support these computational claims. Even with early data, ACE2 is still considered the major receptor for viral entry.
|Current view of ACE and ACE2 functions|
- Remdesivir: Remdesivir (RDV) is an experimental prodrug; A 1′-cyano-substituted adenosine nucleotide analogue with broad spectrum antiviral activity against single-stranded RNA viruses, including SARS-CoV and MERS. Originally developed for treatment of Ebola virus, RDV is showing some promise in treatment of COVID-19. Let’s take a look at the mechanism of action. Essentially, RDV is a nucleotide analog inhibitor of RNA-dependent RNA polymerases (RdRps) 3. One preliminary study suggests intravenous administration of 10 mg/kg dose of RDV (in animal models) can result in persistent levels of its active form in the blood. The triphosphate form of RDV (RDV-TP) competes with its natural counterpart ATP. Based on molecular docking studies, it seems that RDV-TP is more efficiently incorporated than ATP by the virons. Once incorporated at position i, the inhibitor caused RNA synthesis arrest at position i+3. Hence, the likely mechanism of action is delayed RNA chain termination. The additional three nucleotides may protect the RDV-TP from excision by the viral 3’–5’ exonuclease activity.
|Structure of RDV|
TMPRSS2 inhibitor: SARS-CoV-2 uses the ACE2 receptor for viral entry and the spike (S) protein is primed by the cellular serine protease TMPRSS2. This interaction can be blocked by a TMPRSS2 protease inhibitor. We can break down the viral entry to roughly three steps:
Attachment: The viral spike protein (S) is a fusion protein that undergoes a substantial structural rearrangement to fuse the viral membrane with the host cell membrane. This process is triggered when the S1 subunit binds to a host cell receptor. Receptor binding destabilizes the spike protein, resulting in shedding of the S1 subunit and transition of the S2 subunit to a stable post-fusion conformation. At a protein level, to engage a host cell receptor, the receptor-binding domain (RBD) of S1 undergoes hinge-like conformational movements that switch between a down confirmation that corresponds to receptor-inaccessible state, and an up confirmation that corresponds to a receptor accessible state.
Priming: In addition to the structural changes, successive S protein cleavage at two sites by cellular proteases is required for S protein priming. Viral entry requires S protein cleavage at the proteolytic cleavage sites S1/S2, then the S2 subunit allows fusion of viral and cellular membranes.
Uptake: SARS-CoV-2 uses the endosomal cysteine proteases cathepsin B and L (CatB/L) for uptake of RNA machinery into target cells after attachment with ACE2 receptor and all the necessary conformation changes. The cathespsin B and L can also become therapeutic targets.
A clinically-studied and used serine protease inhibitor camostat is known to have activity against TMPRSS2, and partially blocked S-protein driven viral entry into Calu-3 lung cells during an in-vitro study. Complete inhibition was attained with the addition of an experimental compound called E-64d along with camostat. E-64d has been used as a lysosomal inhibitor (in laboratory settings only) as an inhibitor of CatB/L 4.
|Interaction between SARS-CoV-2 and TMPRSS2|
IL-6 blocking agents: IL-6 is a crucial cytokine underlying hyper-inflammatory responses. Tacolizumab and Sarilumab are the two agents being considered for IL-6 blockade to attenuate the overactive inflammatory responses in the lungs of those severely infected by COVID-19. Tacolizumab was tried as an experimental treatment in a RCT for cytokine release syndrome and the results showed a rapid reduction in fevers and 75% (15 out of 20) of patients experienced a reduced need for supplemental oxygen. Sarilumab trials are just not starting up at hospitals in New York. There is considerable evidence gathering that a subset of patients with severe COVID-19 develop cytokine storm and in particular acquire Secondary Hemophagocytic Lymphohistiocytosis. Secondary Hemophagocytic Lymphohistiocytosis (sHLH) is a hyper-inflammatory syndrome characterized by a fulminant and fatal hypercytokinaemia with multiorgan failure. Key features of sHLH include unremitting fever, cytopenias, and hyperferritinaemia and pulmonary involvement (including ARDS). A cytokine profile resembling sHLH is associated with severe COVID-19 disease, characterized by increased interleukin (IL)-2, IL-7, granulocyte-colony stimulating factor, interferon-γ inducible protein 10, and tumor necrosis factor-α among others. These cytokines are all acute phase inflammatory markers that remain persistently elevated in severe COVID-19 patients. As such, all patients with severe COVID-19 should be screened for hyperinflammation using laboratory trends (eg, increasing ferritin, decreasing platelet counts, or erythrocyte sedimentation rate) and the HScore. Management of cytokine storm includes treatment with steroids, intravenous immunoglobulin, selective cytokine blockade (eg, sarilumab or tocilizumab) and JAK inhibition 5.
Recombinant ACE2 The concept behind using recombinant ACE2 is to act as a neutralizing antibody. We know from structural studies that SARS-CoV-2 binds to ACE2 and uses it as an entry receptor. Supplying exogenous ACE2 can soak up the virus and prevent it from binding the canonical human target cell receptor. This would decrease the viral burden because the virus can no longer enter cells freely, and reduce severity of illness on particularly vulnerable patient populations. This strategy can take advantage of other structural components as well. For instance, the viral spike protein has a receptor binding domain (RBD), a recombinant RBD can also redirect the virus from binding human cells. In practical terms, using an immunoadhesin approach where an antibody-like molecule can neutralize the virus from entering cells would be the simplest and the most direct solution. Another potential benefit of using recombinant ACE2 is that it could be stockpiled for future outbreaks of SARS and 2019-nCoV, or any new mutated coronavirus emerging from a zoonotic reservoir that uses the ACE2 receptor for entry 6.
Lastly, let’s talk about using ACE/ARBs to reduce the viral load. This is an actively debated topic with much speculation in humans. For starters, we know that in mice models with knocked out ACE2 receptor, infection with H7N9 influenza virus lead to universally worse lung pathology and poor survival outcomes. The nephJC community provides us a great summary of the salient physiology here:
|Angiotensin receptor (AT1), ACE2 and Losartan|
- Angiotensin receptor (AT1) and ACE2 physically interact to form complexes on the cell membrane in the absence of excess Ang II (left panel). This can potentially reduce interaction of virus with ACE2 (complete speculation)
- Ang II administration decreases the physical interaction between ACE2 and the AT1 receptor. Also induces ubiquitination of ACE2 followed by internalization, and lysosomal degradation.
- AT1 receptor antagonism with losartan prevented the internalization, degradation and ubiquitination of ACE2.
- If the viral-protein interaction with ACE2 is reduced in ACE2-AT1 receptor complexes, then ARBs could prove beneficial. Administration of an ARB could (in theory) stabilize the ACE2-AT1 receptor interaction and prevent viral-protein-ACE2 interaction and internalization.
For all practical purposes of drug development, ACE2 is still considered the major receptor for viral entry. This computational pre-print argues that because ACE2 is not highly expressed in lung tissue, there should be more genes that play key roles in the entry of SARS-CoV-2 into human cells. ↩
Janus kinase (JAK) inhibition could affect both inflammatory pathways (leading to suppression of the inflammatory response) and cellular viral entry in COVID-19 ↩
Lab testing and commercialization is thought to be easier for a recombinant ACE2 antibody. ↩