Cell Entry Mechanism Research Articles

Potential use of Citrus essential oils against acute respiratory syndrome caused by coronavirus

ABSTRACT The treatments being investigated for the pandemic associated with a severe acute respiratory syndrome caused by the new coronavirus (SARS-CoV2) aim to inhibit viral replication or inhibit enzymatic elastase in the lung, responsible for the SARS-CoV cell entry mechanism and the pathogenesis of severe pneumonia caused by infection. There is also a need to improve alveolar surfactant capacity and inhibition of the exacerbated inflammatory response. Current knowledge allows us to infer that Citrus essential oils could be considered an individual or a complementary highly valuable alternative for the viral infection caused by SARS-CoV due to its antiviral activity, particularly against coronavirus, anti-elastase, anti-inflammatory, antibacterial and antibiofilm properties. Furthermore, its proven capacity of biosurfactants, antioxidants, and pain inhibitors would help to treat acute respiratory syndrome. This review aims to encourage the development of more detailed studies on Citrus essential oils as a new multi-objective strategy to treat SARS-CoV2 infection. Abbreviations: SARS-CoV: Severe Acute Respiratory Syndrome Coronavirus; GRAS: Generally Recognized as Safe; TRP: Transient Receptor Proteins; HeLa-CEACAM1a: HeLa-epithelial carcinoembryonic antigen-related cell adhesion molecule 1a; HSV: Herpes Simplex Virus; FDA: Food and Drug Administration; ANMAT: National Administration of Medicines, Food and Medical Technology, Argentina; AFSSAPS: Agence Française de Sécurité Sanitaire des Produits de Santé, France; ACE2:Angiotensin-converting enzyme-2

Understanding SARS-CoV-2 features of infectivity, aggressiveness, and transmissibility: an insect-vector theory for SARS-CoV-2 dissemination

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a ribonucleic acid–based (RNA-based) lineage B β-coronavirus characterized by 10-20 times higher infectivity and transmissibility even across species than previous coronaviruses. The significant infectivity rate of SARS-CoV-2 is due to its different host cell entry mechanisms that are mainly via angiotensin-converting enzyme 2 (ACE2) receptors contrasting earlier coronaviruses that used mainly the endosomal route. Due to the widespread distribution of ACE2 receptors throughout our body, various routes of infectivity are possible, highlighting the necessity of employing multiple forms of protection besides face masks to limit inter-human transmissibility. SARS-CoV-2 exhibits other remarkable features such as the ability to escape the immune system repeated genomic mutations that make it difficult to design a vaccine to address all viral strains and form huge host cell syncytia leading to massive tissue destruction. If we accept SARS-CoV-2 primary reservoir from bats, we should investigate the routes of viral inter-species propagation. In this article, a new theory is proposed- that the dissemination of the virus from the bats to other species and humans could have been possible via an insect vector, as insects possess significant amounts of both ACE2 receptors and a disintegrin and metalloprotease 17 (ADAM-17) enzymes that are essential for virus infectivity.
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Atomistic dynamics of a viral infection process: Release of membrane lytic peptides from a non-enveloped virus.

Molecular simulations have played an instrumental role in uncovering the structural dynamics and physical properties of virus capsids. In this work, we move beyond equilibrium physicochemical characterization of a virus system to study a stage of the infection process that is required for viral proliferation. Despite many biochemical and functional studies, the molecular mechanism of host cell entry by non-enveloped viruses remains largely unresolved. Flock House virus (FHV) is a model system for non-enveloped viruses and is the subject of the current study. FHV infects through the acid-dependent endocytic pathway, where low pH triggers externalization of membrane-disrupting (γ) peptides from the capsid interior. Using all-atom equilibrium and enhanced sampling simulations, the mechanism and energetics of γ peptide liberation and the effect of pH on this process are investigated. Our computations agree with experimental findings and reveal nanoscopic details regarding the pH control mechanism, which are not readily accessible in experiments.

Transportan Peptide Stimulates the Nanomaterial Internalization into Mammalian Cells in the Bystander Manner through Macropinocytosis.

Covalent coupling with cell-penetrating peptides (CPPs) has been a common strategy to facilitate the cell entry of nanomaterial and other macromolecules. Though efficient, this strategy requires chemical modifications on nanomaterials, which is not always desired for their applications. Recent studies on a few cationic CPPs have revealed that they can stimulate the cellular uptake of nanoparticles (NPs) simply via co-administration (bystander manner), which bypasses the requirement of chemical modification. In this study, we investigated the other classes of CPPs and discovered that transportan (TP) peptide, an amphiphilic CPP, also exhibited such bystander activities. When simply co-administered, TP peptide enabled the cells to engulf a variety of NPs, as well as common solute tracers, while these payloads had little or no ability to enter the cells by themselves. This result was validated in vitro and ex vivo, and TP peptide showed no physical interaction with co-administered NPs (bystander cargo). We further explored the cell entry mechanism for TP peptide and its bystander cargo, and showed that it was mediated by a receptor-dependent macropinocytosis process. Together, our findings improve the understanding of TP-assisted cell entry, and open up a new avenue to apply this peptide for nanomaterial delivery.

Reovirus directly engages integrin to recruit clathrin for entry into host cells

Reovirus infection requires the concerted action of viral and host factors to promote cell entry. After interaction of reovirus attachment protein σ1 with cell-surface carbohydrates and proteinaceous receptors, additional host factors mediate virus internalization. In particular, β1 integrin is required for endocytosis of reovirus virions following junctional adhesion molecule A (JAM-A) binding. While integrin-binding motifs in the surface-exposed region of reovirus capsid protein λ2 are thought to mediate integrin interaction, evidence for direct β1 integrin-reovirus interactions and knowledge of how integrins function to mediate reovirus entry is lacking. Here, we use single-virus force spectroscopy and confocal microscopy to discover a direct interaction between reovirus and β1 integrins. Comparison of interactions between reovirus disassembly intermediates as well as mutants and β1 integrin show that λ2 is the integrin ligand. Finally, using fluidic force microscopy, we demonstrate a functional role for β1 integrin interaction in promoting clathrin recruitment to cell-bound reovirus. Our study demonstrates a direct interaction between reovirus and β1 integrins and offers insights into the mechanism of reovirus cell entry. These results provide new perspectives for the development of efficacious antiviral therapeutics and the engineering of improved viral gene delivery and oncolytic vectors.

Soluble ACE2-mediated cell entry of SARS-CoV-2 via interaction with proteins related to the renin-angiotensin system

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can cause acute respiratory disease and multiorgan failure. Finding human host factors that are essential for SARS-CoV-2 infection could facilitate the formulation of treatment strategies. Using a human kidney cell line—HK-2—that is highly susceptible to SARS-CoV-2, we performed a genome-wide RNAi screen and identified virus dependency factors (VDFs), which play regulatory roles in biological pathways linked to clinical manifestations of SARS-CoV-2 infection. We found a role for a secretory form of SARS-CoV-2 receptor, soluble angiotensin converting enzyme 2 (sACE2), in SARS-CoV-2 infection. Further investigation revealed that SARS-CoV-2 exploits receptor-mediated endocytosis through interaction between its spike with sACE2 or sACE2-vasopressin via AT1 or AVPR1B, respectively. Our identification of VDFs and the regulatory effect of sACE2 on SARS-CoV-2 infection shed insight into pathogenesis and cell entry mechanisms of SARS-CoV-2 as well as potential treatment strategies for COVID-19.

Mechanistic and Therapeutic Implications of Extracellular Vesicles as a Potential Link Between Covid-19 and Cardiovascular Disease Manifestations.

Extracellular vesicles (EVs), which are cell released double layered membrane particles, have been found in every circulating body fluid, and provide a tool for conveying diverse information between cells, influencing both physiological and pathological conditions. Viruses can hijack the EVs secretory pathway to exit infected cells and use EVs endocytic routes to enter uninfected cells, suggesting that EVs and viruses can share common cell entry and biogenesis mechanisms. SARS-CoV-2 is responsible of the coronavirus disease 2019 (Covid-19), which may be accompanied by severe multi-organ manifestations. EVs may contribute to virus spreading via transfer of virus docking receptors such as CD9 and ACE2. Covid-19 is known to affect the renin angiotensin system (RAS), and could promote secretion of harmful EVs. In this scenario EVs might be linked to cardiovascular manifestations of the Covid-19 disease through unbalance in RAS. In contrast EVs derived from mesenchymal stem cells or cardiosphere derived cells, may promote cardiovascular function due to their beneficial effect on angiogenesis, fibrosis, contractility and immuno-modulation. In this article we assessed the potential impact of EVs in cardiovascular manifestations of Covid-19 and highlight potential strategies to control the extracellular signaling for future therapies.

Angler Peptides: Macrocyclic Conjugates Inhibit p53:MDM2/X Interactions and Activate Apoptosis in Cancer Cells.

Peptides are being developed as targeted anticancer drugs to modulate cytosolic protein-protein interactions involved in cancer progression. However, their use as therapeutics is often limited by their low cell membrane permeation and/or inability to reach cytosolic targets. Conjugation to cell penetrating peptides has been successfully used to improve the cytosolic delivery of high affinity binder peptides, but cellular uptake does not always result in modulation of the targeted pathway. To overcome this limitation, we developed "angler peptides" by conjugating KD3, a noncell permeable but potent and specific peptide inhibitor of p53:MDM2 and p53:MDMX interactions, with a set of cyclic cell-penetrating peptides. We examined their binding affinity for MDM2 and MDMX, the cell entry mechanism, and role in reactivation of the p53 pathway. We identified two angler peptides, cTAT-KD3 and cR10-KD3, able to activate the p53 pathway in cancer cells. cTAT-KD3 entered cells via endocytic pathways, escaped endosomes, and activated the p53 pathway in breast (MCF7), lung (A549), and colon (HCT116) cancer cell lines at concentrations in the range of 1-12 μM. cR10-KD3 reached the cytosol via direct membrane translocation and activated the p53 pathway at 1 μM in all the tested cell lines. Our work demonstrates that nonpermeable anticancer peptides can be delivered into the cytosol and inhibit intracellular cancer pathways when they are conjugated with stable cell penetrating peptides. The mechanistic studies suggest that direct translocation leads to less toxicity, higher cytosol delivery at lower concentrations, and lower dependencies on the membrane of the tested cell line than occurs for an endocytic pathway with endosomal escape. The angler strategy can rescue high affinity peptide binders identified from high throughput screening and convert them into targeted anticancer therapeutics, but investigation of their cellular uptake and cell death mechanisms is essential to confirming modulation of the targeted cancer pathways.

Temporin G, an amphibian antimicrobial peptide against influenza and parainfluenza respiratory viruses: Insights into biological activity and mechanism of action.

Treatment of respiratory viral infections remains a global health concern, mainly due to the inefficacy of available drugs. Therefore, the discovery of novel antiviral compounds is needed; in this context, antimicrobial peptides (AMPs) like temporins hold great promise. Here, we discovered that the harmless temporin G (TG) significantly inhibited the early life-cycle phases of influenza virus. The in vitro hemagglutinating test revealed the existence of TG interaction with the viral hemagglutinin (HA) protein. Furthermore, the hemolysis inhibition assay and the molecular docking studies confirmed a TG/HA complex formation at the level of the conserved hydrophobic stem groove of HA. Remarkably, these findings highlight the ability of TG to block the conformational rearrangements of HA2 subunit, which are essential for the viral envelope fusion with intracellular endocytic vesicles, thereby neutralizing the virus entry into the host cell. In comparison, in the case of parainfluenza virus, which penetrates host cells upon a membrane-fusion process, addition of TG to infected cells provoked~1.2 log reduction of viral titer released in the supernatant. Nevertheless, at the same condition, an immunofluorescent assay showed that the expression of viral hemagglutinin/neuraminidase protein was not significantly reduced. This suggested a peptide-mediated block of some late steps of viral replication and therefore the impairment of the extracellular release of viral particles. Overall, our results are the first demonstration of the ability of an AMP to interfere with the replication of respiratory viruses with a different mechanism of cell entry and will open a new avenue for the development of novel therapeutic approaches against a large variety of respiratory viruses, including the recent SARS-CoV2.

The Fiber Knob Protein of Human Adenovirus Type 49 Mediates Highly Efficient and Promiscuous Infection of Cancer Cell Lines Using a Novel Cell Entry Mechanism.

The human adenovirus (HAdV) phylogenetic tree is diverse, divided across seven species and comprising over 100 individual types. Species D HAdV are rarely isolated with low rates of preexisting immunity, making them appealing for therapeutic applications. Several species D vectors have been developed as vaccines against infectious diseases, where they induce robust immunity in preclinical models and early phase clinical trials. However, many aspects of the basic virology of species D HAdV, including their basic receptor usage and means of cell entry, remain understudied. Here, we investigated HAdV-D49, which previously has been studied for vaccine and vascular gene transfer applications. We generated a pseudotyped HAdV-C5 presenting the HAdV-D49 fiber knob protein (HAdV-C5/D49K). This pseudotyped vector was efficient at infecting cells devoid of all known HAdV receptors, indicating HAdV-D49 uses an unidentified cellular receptor. Conversely, a pseudotyped vector presenting the fiber knob protein of the closely related HAdV-D30 (HAdV-C5/D30K), differing in four amino acids from HAdV-D49, failed to demonstrate the same tropism. These four amino acid changes resulted in a change in isoelectric point of the knob protein, with HAdV-D49K possessing a basic apical region compared to a more acidic region in HAdV-D30K. Structurally and biologically we demonstrate that HAdV-D49 knob protein is unable to engage CD46, while potential interaction with coxsackievirus and adenovirus receptor (CAR) is extremely limited by extension of the DG loop. HAdV-C5/49K efficiently transduced cancer cell lines of pancreatic, breast, lung, esophageal, and ovarian origin, indicating it may have potential for oncolytic virotherapy applications, especially for difficult to transduce tumor types.IMPORTANCE Adenoviruses are powerful tools experimentally and clinically. To maximize efficacy, the development of serotypes with low preexisting levels of immunity in the population is desirable. Consequently, attention has focused on those derived from species D, which have proven robust vaccine platforms. This widespread usage is despite limited knowledge in their basic biology and cellular tropism. We investigated the tropism of HAdV-D49, demonstrating that it uses a novel cell entry mechanism that bypasses all known HAdV receptors. We demonstrate, biologically, that a pseudotyped HAdV-C5/D49K vector efficiently transduces a wide range of cell lines, including those presenting no known adenovirus receptor. Structural investigation suggests that this broad tropism is the result of a highly basic electrostatic surface potential, since a homologous pseudotyped vector with a more acidic surface potential, HAdV-C5/D30K, does not display a similar pantropism. Therefore, HAdV-C5/D49K may form a powerful vector for therapeutic applications capable of infecting difficult to transduce cells.

In silico study of intrinsic dynamics of full-length apo-ACE2 and RBD-ACE2 complex

The key step for SARS-CoV-2 to infect human cells is the membrane fusion triggered by the binding of the viral extracellular Spike protein to the human extracellular receptor, the angiotensin-converting enzyme 2 (ACE2). Although the Cryo-electron microscopy (Cryo-EM) uncovered the static atomic details of ACE2 homodimers, there is still a lack of research on the kinetic and thermodynamic properties of these full-length structures. This information is helpful to understand and interpret the role of ACE2 in the cell entry of SARS-CoV-2. In order to obtain this information, we performed microsecond-scale conventional and accelerated molecular dynamics (MD) simulations of full-length all-atomic systems of the RBD-ACE2 complex, the normal and torsional conformations of the apo-ACE2 homodimer. The comparative analysis of these systems showed that there were differences in their allosteric signal pathways and motion trends. These results may be helpful to further explore the cell entry mechanism of SARS-CoV-2. Moreover, the binding free energy and hydrogen bond distribution analysis of RBD-ACE2 binding interface provided the binding motifs that may be critical to allosteric signal transmission and RBD binding. These multi-conformational binding motifs can be used as targets or templates for the inhibitor design of the cell entry of SARS-CoV-2.

Characterization of Structural and Energetic Differences between Conformations of the SARS-CoV-2 Spike Protein.

The novel coronavirus disease 2019 (COVID-19) pandemic has disrupted modern societies and their economies. The resurgence in COVID-19 cases as part of the second wave is observed across Europe and the Americas. The scientific response has enabled a complete structural characterization of the Severe Acute Respiratory Syndrome—novel Coronavirus 2 (SARS-CoV-2). Among the most relevant proteins required by the novel coronavirus to facilitate the cell entry mechanism is the spike protein. This protein possesses a receptor-binding domain (RBD) that binds the cellular angiotensin-converting enzyme 2 (ACE2) and then triggers the fusion of viral and host cell membranes. In this regard, a comprehensive characterization of the structural stability of the spike protein is a crucial step to find new therapeutics to interrupt the process of recognition. On the other hand, it has been suggested that the participation of more than one RBD is a possible mechanism to enhance cell entry. Here, we discuss the protein structural stability based on the computational determination of the dynamic contact map and the energetic difference of the spike protein conformations via the mapping of the hydration free energy by the Poisson–Boltzmann method. We expect our result to foster the discussion of the number of RBD involved during recognition and the repurposing of new drugs to disable the recognition by discovering new hotspots for drug targets apart from the flexible loop in the RBD that binds the ACE2.

What is the significance of the conjunctiva as apotential transmission route for SARS-CoV-2 infections?

Recent studies have described conjunctivitis in approximately 1% of COVID-19 patients and speculated that SARS-CoV‑2 can be transmitted via the conjunctiva. In this article we recapitulate the molecular mechanisms of host cell entry of SARS-CoV‑2 and discuss the current evidence for a potential conjunctival transmission of SARS-CoV‑2. The current body of evidence indicates that SARS-CoV‑2 requires the membrane-bound angiotensin-converting enzyme 2 (ACE2) and the membrane-bound serine protease TMPRSS2 to enter cells. Recent studies suggest that COVID-19 patients rarely exhibit viral RNA in tear film and conjunctival smears and that, ACE2 and TMPRSS2 are only expressed in small amounts in the conjunctiva, making conjunctival infection with SARS-CoV‑2 via these mediators unlikely. Nevertheless, we consider the current evidence to be still too limited to provide a conclusive statement and recommend appropriate protective measures for healthcare personnel who are in close contact with suspected and confirmed COVID-19 patients.

Cell-penetrating peptide-mediated cell entry of H5N1 highly pathogenic avian influenza virus

H5N1 highly pathogenic avian influenza virus (HPAIV) poses a huge threat to public health and the global economy. These viruses cause systemic infection in poultry and accidental human infection leads to severe pneumonia, associated with high mortality rates. The hemagglutinin (HA) of H5N1 HPAIV possesses multiple basic amino acids, as in the sequence RERRRKKR at the cleavage site; however, the role of this motif is not fully understood. Here, we showed that a 33-amino acid long peptide derived from HA of H5N1 HPAIV (HA314-46) has the potential to penetrate various cells and lung tissue through a sialic acid-independent endocytotic pathway. Mutant peptide analyses revealed that the cysteine residue at position 318 and multiple basic amino acids were essential for the cell-penetrating activity. Moreover, reassortant viruses possessing H5 HA could enter sialic acid-deficient cells, and virus internalisation was facilitated by cleavage with recombinant furin. Thus, our findings demonstrate that the HA314-46 motif exhibits cell-penetrating activity through a sialic acid-independent cell entry mechanism.

Defusing SARS-CoV-2: Emergency Brakes in a Vaccine Failure Scenario.

Deactivation of primed SARS-CoV-2 prior to cell entry constitutes an emergency brake in COVID-19 infection if vaccine-induced antibodies fail to block recognition of the human angiotensin-converting enzyme 2 (hACE2) receptor. The timing and locus for the therapeutic intervention are dictated by the cell entry mechanism and by the selective advantage of the dominant D614G mutation.

Pharmacotherapeutic modifications in cardiopulmonary patients during COVID-19 outbreak

Severe acute respiratory syndrome coronavirus (SARS-CoV-2), has a well-established host cell entry mechanism by the angiotensin-converting enzyme2 (ACE2 receptor) most commonly found in epithelial cells of blood vessels, respiratory tract including lungs, intestines and kidney. Cardiopulmonary disorders were the most important comorbidities for COVID-19 patients. Recent studies conducted in Wuhan, China (the epicenter of the COVID19 outbreak) showed that an alarming 1,099 patients with polymerase chain reaction (PCR) confirmed COVID19 had serious illnesses such as hypertension, diabetes mellitus both types, diseases coronary heart disease (CAD), kidney and cerebrovascular disease. Chronic inflammation and respiratory tract infections are usually treated with fluoroquinolones and corticosteroids. Fluoroquinolones increase the QT interval and threaten the evolution of arrhythmias and overuse of chloroquine or hydroxychloroquine in COVID-19;expanding the threat of developing arrhythmias;on the other hand, corticosteroids mask the immune response resulting in worsening of the condition. While inhibitors cause respiratory depression, it increases the severity in COVID-19 patients. Based on available pharmacological data, there are no studies showing that calcium channel blocker increases ACE2 expression. Therefore, calcium channel blockers may be a suitable alternative treatment in these patients. Macrolides (azithromycin) may be a better choice than fluoroquinolones, as they are reported to be more effective in reducing secondary infection in COVID-19, paracetamol can be used than ibuprofen and diclofenac in COVID-19 patients. It is suggested that by modifying pharmacotherapy, the severity in cardiopulmonary patients during this COVID-19 outbreak can be reduced.

In Silico Structure-Based Repositioning of Approved Drugs for Spike Glycoprotein S2 Domain Fusion Peptide of SARS-CoV-2: Rationale from Molecular Dynamics and Binding Free Energy Calculations.

The membrane-anchored spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a pivotal role in directing the fusion of the virus particle mediated by the host cell receptor angiotensin-converting enzyme 2 (ACE-2). The fusion peptide region of the S protein S2 domain provides SARS-CoV-2 with the biological machinery needed for direct fusion to the host lipid membrane. In our present study, computer-aided drug design strategies were used for the identification of FDA-approved small molecules using the optimal structure of the S2 domain, which exhibits optimal interaction ratios, structural features, and energy variables, which were evaluated based on their performances in molecular docking, molecular dynamics simulations, molecular mechanics/generalized Born model and solvent accessibility binding free energy calculations of molecular dynamics trajectories, and statistical inferences. Among the 2,625 FDA-approved small molecules, chloramphenicol succinate, imipenem, and imidurea turned out to be the molecules that bound the best at the fusion peptide hydrophobic pocket. The principal interactions of the selected molecules suggest that the potential binding site at the fusion peptide region is centralized amid the Lys790, Thr791, Lys795, Asp808, and Gln872 residues.IMPORTANCE The present study provides the structural identification of the viable binding residues of the SARS-CoV-2 S2 fusion peptide region, which holds prime importance in the virus's host cell fusion and entry mechanism. The classical molecular mechanics simulations were set on values that mimic physiological standards for a good approximation of the dynamic behavior of selected drugs in biological systems. The drug molecules screened and analyzed here have relevant antiviral properties, which are reported here and which might hint toward their utilization in the coronavirus disease 2019 (COVID-19) pandemic owing to their attributes of binding to the fusion protein binding region shown in this study.

Human Pluripotent Stem Cell-Derived Neural Cells and Brain Organoids Reveal SARS-CoV-2 Neurotropism Predominates in Choroid Plexus Epithelium.

Neurological complications are common in patients with COVID-19. Although severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causal pathogen of COVID-19, has been detected in some patient brains, its ability to infect brain cells and impact their function is not well understood. Here, we investigated the susceptibility of human induced pluripotent stem cell (hiPSC)-derived monolayer brain cells and region-specific brain organoids to SARS-CoV-2 infection. We found that neurons and astrocytes were sparsely infected, but choroid plexus epithelial cells underwent robust infection. We optimized a protocol to generate choroid plexus organoids from hiPSCs and showed that productive SARS-CoV-2 infection of these organoids is associated with increased cell death and transcriptional dysregulation indicative of an inflammatory response and cellular function deficits. Together, our findings provide evidence for selective SARS-CoV-2 neurotropism and support the use of hiPSC-derived brain organoids as a platform to investigate SARS-CoV-2 infection susceptibility of brain cells, mechanisms of virus-induced brain dysfunction, and treatment strategies.

Insights on SARS-CoV-2 Molecular Interactions With the Renin-Angiotensin System.

The emergence of SARS-CoV-2/human/Wuhan/X1/2019, a virus belonging to the species Severe acute respiratory syndrome-related coronavirus, and the recognition of Coronavirus Disease 2019 (COVID-19) as a pandemic have highly increased the scientific research regarding the pathogenesis of COVID-19. The Renin Angiotensin System (RAS) seems to be involved in COVID-19 natural course, since studies suggest the membrane-bound Angiotensin-converting enzyme 2 (ACE2) works as SARS-CoV-2 cellular receptor. Besides the efforts of the scientific community to understand the virus’ molecular interactions with human cells, few studies summarize what has been so far discovered about SARS-CoV-2 signaling mechanisms and its interactions with RAS molecules. This review aims to discuss possible SARS-CoV-2 intracellular signaling pathways, cell entry mechanism and the possible consequences of the interaction with RAS components, including Angiotensin II (Ang II), Angiotensin-(1-7) [Ang-(1-7)], Angiotensin-converting enzyme (ACE), ACE2, Angiotensin II receptor type-1 (AT1), and Mas Receptor. We also discuss ongoing clinical trials and treatment based on RAS cascade intervention. Data were obtained independently by the two authors who carried out a search in the PubMed, Embase, LILACS, Cochrane, Scopus, SciELO and the National Institute of Health databases using Medical Subject Heading terms as “SARS-CoV-2,” “COVID-19,” “Renin Angiotensin System,” “ACE2,” “Angiotensin II,” “Angiotensin-(1-7),” and “AT1 receptor.” Similarly to other members of Coronaviridae family, the molecular interactions between the pathogen and the membrane-bound ACE2 are based on the cleavage of the spike glycoprotein (S) in two subunits. Following the binding of the S1 receptor-binding domain (RBD) to ACE2, transmembrane protease/serine subfamily 2 (TMPRSS2) cleaves the S2 domain to facilitate membrane fusion. It is very likely that SARS-CoV-2 cell entry results in downregulation of membrane-bound ACE2, an enzyme that converts Ang II into Ang-(1-7). This mechanism can result in lung injury and vasoconstriction. In addition, Ang II activates pro-inflammatory cascades when binding to the AT1 Receptor. On the other hand, Ang-(1-7) promotes anti-inflammatory effects through its interactions with the Mas Receptor. These molecules might be possible therapeutic targets for treating COVID-19. Thus, the understanding of SARS-CoV-2 intracellular pathways and interactions with the RAS may clarify COVID-19 physiopathology and open perspectives for new treatments and strategies.

Targeted therapy strategies against SARS-CoV-2 cell entry mechanisms: A systematic review of in vitro and in vivo studies.

Due to the rapidly spreading of novel coronavirus disease (COVID-19) worldwide, there is an urgent need to develop efficient vaccines and specific antiviral treatments. Pathways of the viral entry into cells are interesting subjects for targeted therapy of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The present study aims to provide a systematic evaluation of the most recent in vitro and in vivo investigations targeting SARS-CoV-2 cell entry. A systematic search was carried out in major medical sources, including MEDLINE (through PubMed), Web of Science, Scopus, and EMBASE. Combinations of the following search terms were used: SARS-CoV-2, in vitro, in vivo, preclinical, targeted therapy, and cell entry. A modified version of the Consolidated Standards of Reporting Trials and Systematic Review Centre for Laboratory Animal Experimentation assessment tools were applied for evaluating the risk of bias of in vitro and in vivo studies, respectively. A narrative synthesis was performed as a qualitative method for the data synthesis of each outcome measure. A total of 2,649 articles were identified through searching PubMed, Web of Science, Scopus, EMBASE, Google Scholar, and Biorxiv. Finally, 22 studies (one in vivo study and 21 in vitro studies) were included. The spike (S) glycoprotein of the SARS-CoV-2 was the main target of investigation in 19 studies. SARS-CoV-2 can enter into the host cells through endocytosis or independently. SARS-CoV-2 S protein utilizes angiotensin-converting enzyme 2 or CD147 as its cell-surface receptor to attach host cells. It consists of S1 and S2 subunits. The S1 subunit mediates viral attachment to the host cells, while the S2 subunit facilitates virus-host membrane fusion. The cleavage of the S1-S2 protein, which is required for the conformational changes of the S2 subunit and processing of viral fusion, is regulated by the host proteases, including cathepsin L (during endocytosis) and type II membrane serine protease (independently). Targeted therapy strategies against SARS-CoV-2 cell entry mechanisms fall into four main categories: strategies targeting virus receptors on the host, strategies neutralizing SARS-CoV-2 spike protein, strategies targeting virus fusion to host cells, and strategies targeting endosomal and non-endosomal dependent pathways of virus entry. Inhibition of the viral entry by targeting host or virus-related components remains the most potent strategy to prevent and treat COVID-19. Further high-quality investigations are needed to assess the efficacy of the proposed targets and develop specific antivirals against SARS-CoV-2.