Dr. L | Mar, 3 2020
At present, the prevention and treatment of coronavirus is the most urgent issue for scientific and medical research. In addition to the ACE2 host receptor target that is being highly focused on, what other targets deserve the attention of current coronavirus researchers? Herein, experts from Cyagen investigate the lesser-known targets that are pertinent to coronavirus research, aiming to provide some helpful references to guide scientific researchers.
Dipeptidyl peptidase (DPP4) belongs to the family of proline-specific serine proteases and is capable of cleaving dipeptides from the amino terminal (N-terminal). DPP4 is the most typical peptidase in this family, and it is also the third exo-peptidase that was found to be a coronavirus receptor (after ACE2 and APN). DPP4 is a multifunctional type II transmembrane glycoprotein with 766 amino acids; it exists as a homodimer on the cell surface. The dimerization of peptidase depends on the molecules’ connection between the hydrolase domain and the extension chain of b propeller blade IV. Therefore, the lateral binding of MERS-CoV RBD to DPP4 will not dysregulate the DPP4 dimerization. DPP4 is involved in a variety of biological processes, including regulation of the immune system and more specifically: T cell activation, chemotaxis regulation, cell adhesion, apoptosis and tumorigenicity. DPP4 can cleave dipeptides from N-terminal of proteins and of various chemokines. DPP4 protein showed high conservation of amino acid sequences among different species, including sequences obtained from H. pylori and bat cells, especially at the carboxyl terminal (C-terminal).
Studies have shown that the direct and specific binding of MERS-CoV S1 with human DPP4 leads to the occurrence of Middle East respiratory syndrome (MERS). The model of virus binding to host consists of E382-C585 residues of MERS-CoV RBD, S39-P766 residues of DPP4 extracellular domain and DPP4 extracellular domain of glycans linked to N85, n92, N150, n219, n229, n281, n321 and n520 residues. The cleavage site of DPP4 was far away from MERS-CoV RBD binding site, similar to the structure of ACE2 binding to SARS-CoV RBD.
Through the study of coronavirus, researchers found that β - coronaviruses may have similar core folding domains in the S protein, and take the external amino acids with different structures as the regions recognized by virus receptor. DPP4 is a clear receptor of MERS-CoV infection - learning the structural characteristics of its binding with MERS-CoV RBD is helpful for us to understand the interaction between virus and receptor and guide the treatment of MERS-CoV infection towards the development of a vaccine. It is also significant for the study of the host receptors of the new coronavirus SARS-CoV-2 outbreak in 2019.
In immunology, aminopeptidase N (APN) is usually called astrocyte antigen CD13. In 1989, it was found that APN and CD13 are the same proteins. APN is expressed in many different human cells, such as macrophages, stromal cells, smooth muscle cells, and fibroblasts. APN is located on the cell membrane and is involved in the biological processes of peptide cleaving, virus infection, endocytosis, and cell signal transduction.
In 1992, Yeager et al. found that hCoV-229E used human aminopeptidase N (hAPN) as a receptor to infect diseases. In 2017, Wong et al. explored the structure of the receptor binding domain of hAPN and hCoV-229E, which showed that the three extended RBD rings on the surface protein of the virus are fully responsible for receptor binding. In addition, they also found that these rings are the most variable regions of the whole viral genome. In the viral genome, the kind of circular RBD receptor binding domain has a different binding ability compared with specific neutralizing antibodies - these two molecules also have differing affinities to hAPN. Although immune escape provides an explanation for the emergence of these RBD, it is still unknown how they adapt to a wide range of ring changes while maintaining receptor binding. Ring changes are also expected to promote the acquisition of new receptor interactions, thereby supporting the potential for cross-species transmission.
Recently, hCoV-229E-like coronaviruses have been found in bats, camels, and alpacas - the transmission from bats to humans may involve these intermediate hosts. Experimental results show that mice that were transfected (via NIH3T3 cells) with retroviral vector containing hAPN cDNA were more likely to become infected with hCoV-229E virus than the control group. The research confirmed that hAPN host receptor combined with hCoV-229E to infect mice with virus. Cellular experiments have also shown that CoV-229E can use hAPN as its receptor. These viruses may bind to host APN in a structurally conservative way. However, it should be noted that the locus of α-coronavirus PRCoV binding pig APN is different from that of hCoV-229E.
APN plays an important role in the survival of viruses - we can control the virus by inhibiting the expression of APN. Given that APN is the receptor of hCoV-229E and it can also act as the receptor of enteropathogenic coronavirus, these together indicate that the similarity of ligands may be related to the structure, at least to some extent. Since SARS-CoV and the new pneumonia virus are both coronaviruses, it is very important for us to study all their potential targets in order to better understand the coronavirus and deal with variants that may appear in the future. As ANP is one of the host receptors of coronavirus, if we obtain more knowledge about it, we will have more avenues to restrict viral invasion.
Argonaute 4 (AGO4) is an endonuclease that can ‘cut’ RNA, playing an indispensable role in RNA interference and short interfering RNA (siRNA) mediated gene silencing. Gene silencing is an evolutionary conservative mechanism for regulating gene expression in eukaryotes. In plants, transcriptional gene silencing (TGS) can be mediated by RNA-directed DNA methylation (RdDM). The establishment of RdDM requires the formation of dsRNA by RNA polymerase IV (POL IV) and RNA-dependent RNA polymerase 2 (rdr2); dsRNA is processed into 24-nucleotide (NT) siRNA by Dicer like 3 (dcl3). The siRNA is then loaded into AGO4 to form the core of the effector complex, which adds methyl to the exact DNA location. The AGO4 protein is one of the main catalytic components of RdDM, which plays an important role in the defense against DNA viruses (including geminiviruses) in plants. AGO4 can combine with virus-derived siRNA (vsiRNA) to mediate the methylation of virus DNA and TGS, thus inhibiting the transcription and replication of virus. Research shows that RdDM-mediated TGS is a natural antiviral defense against geminiviruses, which are the only single-stranded DNA viruses with twin particle morphology in plants.
Interferon response is the most important immune response for mammalian organisms to resist viral infection, this is closely followed in importance by the antiviral defense induced by RNA interference. Since AGO4 is the effect protein of RNAi and miRNA, researchers have explored whether AGO4 is also involved in viral defense in mammalian cells. It was found that AGO2 in the AGO family did participate in the antiviral defense of mammals, so the researchers verified the antiviral activities of AGO1-4 separately. The results showed that the AGO4-deficient macrophages were highly sensitive to influenza infection - the virus titer and RNA levels increased significantly after infection - which confirmed the important role of AGO4 in antiviral defense of mammals. Additionally, the levels of encephalomyocarditis virus (EMCV) and vesicular stomatitis virus (VSV) increased significantly in the macrophages with AGO4 deficiency. At the same time, the antiviral effect of AGO4 in bone marrow-derived dendritic cells and mouse embryonic fibroblasts was confirmed. Therefore, researchers believe that AGO4 has a unique antiviral effect on a variety of RNA viruses. Once researchers can find out how to use AGO4 protein to enhance its inherent self-defense ability in the human body, then, theoretically, we can greatly reduce the possibility of the human infection.
The protein encoded by this gene is an interferon induced membrane protein (IFITM). IFITM3 is involved in the process of cell differentiation, apoptosis, cell adhesion, and immune cell regulation. Studies show that IFITM3 is active against a variety of viruses, including influenza A virus, SARS coronavirus (SARS-CoV), Marburg virus (MARV), Ebola virus (EBOV), dengue virus (DNV), West Nile virus (WNV), influenza A H1N1 virus, HIV-1, and vesicular stomatitis virus (VSV); it can inhibit influenza virus hemagglutinin, MARV, EBOV GP, SARS-CoV S and VSV G protein-mediated virus entry into the host. IFITM3 also plays a key role in the maintaining the structural stability of vacuolar ATPase (v-ATPase). It has been reported that IFITM3 up-regulates c-myc expression through the ERK 1/2 signaling pathway and promotes liver cancer proliferation. Moreover, the IFITM 3 / STAT 3 complex, regulated by TGF-β, can promote the invasion of glioma cells to the host.
In mice, IFITM3 is resistant to influenza, and I / tm3 / mouse infected with certain dose of influenza virus (not lethal in wild-type [WT] mice) will die. IFITM3 not only protects cells of the immune system from viral infections, enabling them to perform an effective immune response, but also protects other cell types, such as the respiratory epithelial cells and cardiac tissues. During influenza infection, IFITM3 was upregulated by type I IFN in lung dendritic cells, enabling them to survive and migrate to draining lymph nodes to present virus antigens. Subsequently, IFITM3 is rapidly upregulated on activated T cells in draining lymph nodes, which maintain high IFITM3 expression (due to their migration to the site of viral infection), providing a survival advantage that enables them to exert their effector functions. Interestingly, IFITM3 is also expressed in T cells in the tissues of the lungs and airways, spleen, skin, and brain, suggesting that it may promote the virus' survival at potential infection sites. Therefore, in the study, the importance of IFITM3 in immunity against virus infection was demonstrated by improving both the survival rate of the immune effector subgroups and overall viral resistance.
In 2011, some researchers demonstrated the association between IFITM and SARS-CoV. Studies have shown that IFITM protein limits the infection mediated by the entry of MARV and EBOV to the glycoprotein (GP), and also inhibits their replication. At the same time, interferon-β specifically restricts entry of filamentous viruses and influenza A virus (IAV). The researchers further demonstrated that both SARS-CoV replication and invasion (mediated by the spike protein) were limited by the IFITM protein. IFITM limits the ability of various envelope viruses to enter the host and also regulates cell tropism independently of the expression of viral receptors. The sequence homology between SARS-CoV-2 and SARS-CoV viruses leads the prediction that IFITM can be a target for studies aiming to protect against new coronavirus invasion.
The epidermal growth factor receptor (EGFR) gene encodes a transmembrane glycoprotein that binds to the extracellular protein ligand, EGF. This binding of protein and ligand results in receptor dimerization and tyrosine autophosphorylation, leading to cell proliferation. Mutations of the EGFR gene are related to lung cancer, including ependymoma and esophageal basaloid squamous cell carcinoma. EGFR is in the ErbB family of closely-related receptor tyrosine kinases, which bind to ligands to activate multiple signal cascades and transform extracellular signals into the appropriate cellular responses. Ligands already known to activate ErbB receptors include EGF，TGFA/TGF-α，amphiregulin (AREG)，epigen (EPGN)，betacellulin (BTC)，eregregulin (EREG), and heparin binding EGF-like growth factor (HBEGF).
Research has shown that EGFR mutations are the most common target gene abnormalities in patients with non-small cell lung cancer - so what role does the gene play in a model of lung infection?
After the outbreak of SARS in 2003, autopsies of patients who died from SARS-related illness showed different degrees of pulmonary fibrosis (PF), which many survivors also suffered from. Evidence shows that PF is more common in respiratory viral infections, especially as in SARS-CoV. According to some research, lung injury caused by EGFR signaling can lead to pulmonary fibrosis, and inhibition of EGFR signaling could prevent SARS and other respiratory viral infections from resulting in PF. Therefore, the role of the EGFR is worthy of our attention in the study of SARS-CoV-2.
ICAM1 is intercellular adhesion molecule 1. The gene encodes a cell surface glycoprotein, which is usually expressed in both endothelial and immune system cells. The diseases related to ICAM1 include follicular mycosis and lupus erythematosus. In the case of viral infection, the molecule acts as a receptor of the virus on the cell surface. ICAM-1 and ICAM-2 are involved in the activation of self-invasive Th1 and Th17 cells in autoimmune encephalomyelitis, and enter the central nervous system (CNS).
ICAM1 expression and NF-κB activation are important steps in inflammatory response. Professor Wu Jun, an American Chinese expert proposed an explanation for the mechanism of infection for the new coronavirus: in the process of SARS-CoV infection, excessive free radicals produced by the body’s immune response would damage the functions of other organs of the body, which would make it difficult to deal with the disease. According to Professor Wu, by scavenging free radicals, we can protect our organ functions and enhance the innate anti-viral response. Studies have shown that by both downregulating the transcription of ICAM1 and activating NF-κB, the inflammatory response can be reduced. We can try to use this mechanism to control damage to the lungs and other organs in the process of anti-viral development.
In the experimental autoimmune encephalomyelitis (EAE) animal model of multiple sclerosis (MS), myelin sheath-specific T cells are activated in the peripheral and differentiated into the central nervous system (CNS) upon passing through the blood-brain barrier (BBB) effector cells Th1 and Th17, causing neuroinflammation. Endothelial ICAM-1 and ICAM-2 mediate the migration of Th1 and Th17 cells across the BBB. ICAM1- and ICAM2-deficient mice improved the typical and atypical EAE, respectively, caused by Th1 and Th17 cell metastasis,.
Although the ICAM gene has been extensively studied in the BBB model, it is recommended that researchers should pay attention to it since it functions as a cellular receptor for the virus in the case of viral infection.
HSPA1B is a heat shock 70 kDa protein 1B. HSPA1A, HSPA1B and HSPA1L are three protein encoding genes belonging to the 70 kilodalton heat shock protein (HSP70) family. HSPA1A and HSPA1B may be involved in the immune response against specific infections, including Epstein-Barr virus (EBV), Legionella, Toxoplasma, measles, and influenza A. HSPA1B-related diseases include polygenic mutations, acute altitude sickness, male infertility, glaucoma and more. Studies have found that HSPA1B gene polymorphisms are related to the risk of infection and prognosis of lung cancer. Transgenic animal models and pretreatment experiments have demonstrated that overexpression of HSP70 in neurons and glial cells can exert neuroprotective effects against ischemic injury and excitotoxicity mediated by alginic acid and glutamic acid. In animal models of neurodegenerative diseases, Hsp70 (protein) has been shown to be a powerful inhibitor of neurodegeneration.
Research has shown that pulmonary endothelial injury is related to the expression of HSP70 protein - increased levels of Hsp70 has a protective effect on hyperoxia-induced destruction of the lung endothelium barrier. The combination of Hsp70 and AIF can prevent nuclear translocation of AIF, which helps protect endothelial cells against hyperoxia-induced apoptosis.
A majority of patients with SARS-CoV-2 infection have suffered from lung injury. Further learning the damage function causing by HSPA1B to the lung endothelial barrier, we can elucidate the mechanism of viral infection.
The ITGB6 gene is a member of the integrin superfamily of transmembrane receptors which play a role in transferring signals from the extracellular matrix into the cell, facilitating cell-extracellular matrix (ECM) adhesion. Integrins are heterodimeric integral membrane proteins composed of α and β chains. ITGB6 is a key subunit that controls the expression of the entire heterodimer αVβ6. Integrin αVβ6 is also a receptor for fibronectin and bladder actin, which additionally recognizes the arginylglycylaspartic acid (RGD) peptide sequence in its ligand. It has also been shown that αVβ6 promotes cancer cell invasion through clathrin-mediated endocytosis.
In the experimental model, pulmonary fibrosis (PF) depends on transforming growth factor beta (TGF-β) signal transduction. TGF-β is latent in the complex with its propeptide region, and TGF-β activators release TGF-β from the complex. Given that integrin αVβ6 is the main activator of TGF-β in the lungs, inhibiting αVβ6-mediated activation of TGF-β is a logical strategy for the treatment and prevention of pulmonary fibrosis. Therefore, it can be said that ITGB6 is expressed in pulmonary epithelial cells, and may exhibit control over the occurrence of specialized pulmonary fibrosis.
It was also found that agonists of protease-activated receptor 1 (PAR1) promoted αVβ6-dependent activation of TGF-β may be one mechanism behind the activation of the coagulation cascade that contributes to developing acute lung injuries. Therefore, we believe that ITGB6 may be a potential target for the treatment of viral pneumonia caused by SARS-CoV-2.
WW domain-containing transcription coactivator 1 (WWTR1), also known as TAZ, was originally identified as a 14–3–3 binding protein. WWTR1 not only acts as a downstream regulatory target in the Hippo signaling pathway, but also regulates cell proliferation and apoptosis. Additionally, WWTR1 is involved in organ size control, stem cell differentiation, and human cancer development. Diseases associated with WWTR1 include epithelioid hemangioendothelioma and Barth syndrome.
The lungs have significant repair capabilities after injury, including the regeneration of alveolar epithelial type I cells (AEC1s) - which mediate gas exchange in the lungs. After injury, AEC1s are differentiated from progenitor-alveolar epithelial type II cells (AEC2s), which also secrete surfactants to maintain surface tension and alveolar patency. Studies have shown that WWTR1 (TAZ) plays a crucial role in AEC differentiation. Using in vitro organoid culture systems, WWTR1/TAZ has been shown to effectively prevent AEC2-AEC1 differentiation. WWTR1/TAZ is a key factor in the differentiation of AEC2 into AEC1 and an important factor in protecting the integrity of alveolar cells after injury. At the same time, WWTR1/TAZ has also been shown to have a pathophysiological role in pulmonary fibrosis (PF), and plays a significant role in developing new therapies for non-small cell lung cancer.
Therefore, we believe that WWTR1/TAZ could be a potential therapeutic target for COVID-19 induced pneumonia.
Aldehyde dehydrogenase 1A1 (ALDH1A1) is a marker of cancer stem-like cells (CSCs). It is the second enzyme in the main oxidative pathway of alcohol metabolism, following alcohol dehydrogenase. There are two major aldehyde dehydrogenase isoenzymes in the liver, cytoplasm, and mitochondria. Studies in mice have shown that the ALDH1A1 gene may also be involved in regulating metabolism in high-fat diet reactions through its role in retinol metabolism. Studies have also shown that ALDH1A1 maintains the characteristics of esophageal squamous cell carcinoma stem cells by activating the AKT signaling pathway and interacting with β-catenin. ALDH1A1 can reverse cisplatin resistance of the human lung adenocarcinoma cell A549/DDP. In both cancerous and healthy human liver tissue, ALDH1A1 is highly expressed and active, and ALDH1A1 phosphorylation at T267 site is increased - which suggest that its regulation may be critical in both normal and diseased states.
ALDH1A1, just like VEGFA, is a landmark target molecule of β-catenin, which is involved in the pathogenesis of myofibroblast activation and pulmonary fibrosis. The phenotypic feature of SARS-CoV-2-induced viral pneumonia (COVID-19) is respiratory failure caused by lung injury. Therefore, ALDH1A1 can be used as a target for research on a variety of disease models, having many therapeutic applications beyond its potential to mitigate the severity of COVID-19.
RUNX3 (runt-related transcription factor 3) is a protein-coding gene that functions as tumor suppressor. RUNX3-related diseases include myeloid leukemia and esophageal squamous cell carcinoma – in cancer cases, the gene is often deleted or transcriptionally silenced. It can activate or inhibit transcription and also interact with other transcription factors.
RUNX3 is associated with a variety of immune regulators, including LAG3, CTLA-4, PD-1, and TIGIT. More importantly, RUNX3 is involved in immune-related pathways, especially those related to immune cell migration. Researchers have demonstrated the antitumor effect of RUNX3 in knockout (KO) mice and found a causal relationship between RUNX3 silencing and human gastric cancer.
Previous studies have shown that the expression of RUNX3, CCL3, and CCL20 recruits CD8+ T lymphocytes in human lung cancer tissues to participate in the process of immune regulation. Additionally, miR-661 directly targets RUNX3 to promote progression of non-small cell lung cancer and, similarly, miR-301a promotes the occurrence of lung cancer by inhibiting RUNX3. RUNX3 can protect against acute lung injury in rats with severe acute pancreatitis by inhibiting the JAK2/STAT3 pathway. RUNX3 is a key regulator in the process of epithelial-mesenchymal transition in animal model alveolar type II cells.
Therefore, we believe that RUNX3 could be a potential therapeutic target for the treatment of SARS-CoV-2-induced COVID-19.
Virus research is very difficult, especially considering the quickly-changing variability among viral strains and discrete differences among hosts. This guide aims to serve as a useful tool to help understand our viral opponents and fight the current outbreak of COVID-19, while addressing additional therapeutic applications, so we can lead the way into a brighter tomorrow.
For the research of new coronaviruses, accurate animal models are necessary for verifying the pathogenesis and immune mechanisms of the illness to accelerate research across vaccine development, new drug development, gene therapy, and more. Since the outbreak, the R&D team at Cyagen has made every effort to develop animal models catered to the global SARS-CoV-2 research initiative. As our way of contributing to the international epidemic prevention effort, we are opening service reservations on models for ACE2 and DPP4 receptor targets effective immediately.
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