Host Angiotensin-converting Enzyme Research Articles

5-chloro-3-(2-(2,4-dinitrophenyl) hydrazono)indolin-2-one: synthesis, characterization, biochemical and computational screening against SARS-CoV-2

Chemical prototypes with broad-spectrum antiviral activity are important toward developing new therapies that can act on both existing and emerging viruses. Binding of the SARS-CoV-2 spike protein to the host angiotensin-converting enzyme 2 (ACE2) receptor is required for cellular entry of SARS-CoV-2. Toward identifying new chemical leads that can disrupt this interaction, including in the presence of SARS-CoV-2 adaptive mutations found in variants like omicron that can circumvent vaccine, immune, and therapeutic antibody responses, we synthesized 5-chloro-3-(2-(2,4-dinitrophenyl)hydrazono)indolin-2-one (H2L) from the condensation reaction of 5-chloroisatin and 2,4-dinitrophenylhydrazine in good yield. H2L was characterised by elemental and spectral (IR, electronic, Mass) analyses. The NMR spectrum of H2L indicated a keto–enol tautomerism, with the keto form being more abundant in solution. H2L was found to selectively interfere with binding of the SARS-CoV-2 spike receptor-binding domain (RBD) to the host angiotensin-converting enzyme 2 receptor with a 50% inhibitory concentration (IC50) of 0.26 μM, compared to an unrelated PD-1/PD-L1 ligand–receptor-binding pair with an IC50 of 2.06 μM in vitro (Selectivity index = 7.9). Molecular docking studies revealed that the synthesized ligand preferentially binds within the ACE2 receptor-binding site in a region distinct from where spike mutations in SARS-CoV-2 variants occur. Consistent with these models, H2L was able to disrupt ACE2 interactions with the RBDs from beta, delta, lambda, and omicron variants with similar activities. These studies indicate that H2L-derived compounds are potential inhibitors of multiple SARS-CoV-2 variants, including those capable of circumventing vaccine and immune responses.

Predicting potential SARS-CoV-2 spillover and spillback in animals

BackgroundThe COVID-19 pandemic is spreading rapidly around the world, causing countries to impose lockdowns and efforts to develop vaccines on a global scale. However, human-to-animal and animal-to-human transmission cannot be ignored, as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can spread rapidly in farmed and wild animals. This could create a worrying cycle of SARS-CoV-2 spillover from humans to animals and spillback of new strains back into humans, rendering vaccines ineffective. MethodThis study provides a key indicator of animals that may be potential susceptible hosts for SARS-CoV-2 and coronavirus infections by analysing the phylogenetic distance between host angiotensin-converting enzyme 2 and the coronavirus spike protein. Crucially, our analysis identifies animals that are at elevated risk from a spillover and spillback incident. ResultsOne group of animals has been identified as potentially susceptible to SARS-CoV-2 by harbouring a parasitic coronavirus spike protein similar to the SARS-CoV-2 spike protein. These animals may serve as amplification hosts in spillover events from zoonotic reservoirs. This group consists of a mixture of animals infected internally and naturally: minks, dogs, cats, tigers. Additionally, no internal or natural infections have been found in masked palm civet. ConclusionTracing interspecies transmission in multi-host environments based solely on in vitro and in vivo examinations of animal susceptibility or serology is a time-consuming task. This approach allows rapid identification of high-risk animals to prioritize research and assessment of the risk of zoonotic disease transmission in the environment. It is a tool to rapidly identify zoonotic species that may cause outbreaks or participate in expansion cycles of coexistence with their hosts. This prevents the spread of coronavirus infections between species, preventing spillover and spillback incidents from occurring.

Mechanisms of SARS-CoV-2 Placental Transmission.

The widespread occurrence of SARS-CoV-2 infections and the diverse range of symptoms have placed significant strain on healthcare systems worldwide. Pregnancy has also been affected by COVID-19, with an increased risk of complications and unfavorable outcomes for expectant mothers. Multiple studies indicate that SARS-CoV-2 can infiltrate the placenta, breach its protective barrier, and infect the fetus. Although the precise mechanisms of intrauterine transmission remain unclear, factors such as perinatal infection, macrophages, sexual intercourse, and the virus' interaction with host angiotensin-converting enzyme 2 (ACE2) and neuropilin-1 (NRP-1) proteins appear to play a role in this process. The integrity of the placental barrier fluctuates throughout pregnancy and appears to influence the likelihood of fetal transmission. The expression of placental cell receptors, like ACE2, changes during pregnancy and in response to placental damage. However, due to the consistent presence of others, such as NRP-1, SARS-CoV-2 may potentially enter the fetus at different stages of pregnancy. NRP-1 is also found in macrophages, implicating maternal macrophages and Hofbauer cells as potential routes for viral transmission. Our current understanding of SARS-CoV-2's vertical transmission pathways remains limited. Some researchers question the ACE2-associated transmission model due to the relatively low expression of ACE2 in the placenta. Existing studies investigating perinatal transmission and the impact of sexual intercourse have either involved small sample sizes or lacked statistical significance. This review aims to explore the current state of knowledge regarding the potential mechanisms of COVID-19 vertical transmission, identifying areas where further research is needed to fill the gaps in our understanding.

Enhancement of SARS-CoV-2 Infection via Crosslinking of Adjacent Spike Proteins by N-Terminal Domain-Targeting Antibodies.

The entry of SARS-CoV-2 into host cells is mediated by the interaction between the spike receptor-binding domain (RBD) and host angiotensin-converting enzyme 2 (ACE2). Certain human antibodies, which target the spike N-terminal domain (NTD) at a distant epitope from the host cell binding surface, have been found to augment ACE2 binding and enhance SARS-CoV-2 infection. Notably, these antibodies exert their effect independently of the antibody fragment crystallizable (Fc) region, distinguishing their mode of action from previously described antibody-dependent infection-enhancing (ADE) mechanisms. Building upon previous hypotheses and experimental evidence, we propose that these NTD-targeting infection-enhancing antibodies (NIEAs) achieve their effect through the crosslinking of neighboring spike proteins. In this study, we present refined structural models of NIEA fragment antigen-binding region (Fab)-NTD complexes, supported by molecular dynamics simulations and hydrogen-deuterium exchange mass spectrometry (HDX-MS). Furthermore, we provide direct evidence confirming the crosslinking of spike NTDs by NIEAs. Collectively, our findings advance our understanding of the molecular mechanisms underlying NIEAs and their impact on SARS-CoV-2 infection.

Determination of the factors responsible for the tropism of SARS-CoV-2-related bat coronaviruses to Rhinolophus bat ACE2.

The efficiency of infection receptor use is the first step in determining the species tropism of viruses. After the coronavirus disease 2019 pandemic, a number of SARS-CoV-2-related coronaviruses (SC2r-CoVs) were identified in Rhinolophus bats, and some of them can use human angiotensin converting enzyme 2 (ACE2) for the infection receptor without acquiring additional mutations. This means that the potential of certain SC2r-CoVs to cause spillover from bats to humans is "off-the-shelf." However, both SC2r-CoVs and Rhinolophus bat species are highly diversified, and the host tropism of SC2r-CoVs remains unclear. Here, we focus on two Laotian SC2r-CoVs, BANAL-20-236 and BANAL-20-52, and determine how the tropism of SC2r-CoVs to Rhinolophus bat ACE2 is determined at the amino acid resolution level.

Prime editor-mediated functional reshaping of ACE2 prevents the entry of multiple human coronaviruses, including SARS-CoV-2 variants.

The spike protein of SARS-CoV-2 hijacks the host angiotensin converting enzyme 2 (ACE2) to meditate its entry and is the primary target for vaccine development. Nevertheless, SARS-CoV-2 keeps evolving and the latest Omicron subvariants BQ.1 and XBB have gained exceptional immune evasion potential through mutations in their spike proteins, leading to sharply reduced efficacy of current spike-focused vaccines and therapeutics. Compared with the fast-evolving spike protein, targeting host ACE2 offers an alternative antiviral strategy that is more resistant to viral evolution and can even provide broad prevention against SARS-CoV and HCoV-NL63. Here, we use prime editor (PE) to precisely edit ACE2 at structurally selected sites. We demonstrated that residue changes at Q24/D30/K31 and/or K353 of ACE2 could completely ablate the binding of tested viruses while maintaining its physiological role in host angiotensin II conversion. PE-mediated ACE2 editing at these sites suppressed the entry of pseudotyped SARS-CoV-2 major variants of concern and even SARS-CoV or HCoV-NL63. Moreover, it significantly inhibited the replication of the Delta variant live virus. Our work investigated the unexplored application potential of prime editing in high-risk infectious disease control and demonstrated that such gene editing-based host factor reshaping strategy can provide broad-spectrum antiviral activity and a high barrier to viral escape or resistance.

Isolation of ACE2-dependent and -independent sarbecoviruses from Chinese horseshoe bats.

While the spike proteins from severe acute respiratory syndrome coronaviruses-1 and 2 (SARS-CoV and SARS-CoV-2) bind to host angiotensin-converting enzyme 2 (ACE2) to infect cells, the majority of bat sarbecoviruses cannot use ACE2 from any species. Despite their discovery almost 20 years ago, ACE2-independent sarbecoviruses have never been isolated from field samples, leading to the assumption these viruses pose little risk to humans. We have previously shown how spike proteins from a small group of ACE2-independent bat sarbecoviruses may possess the ability to infect human cells in the presence of exogenous trypsin. Here, we adapted our earlier findings into a virus isolation protocol and recovered two new ACE2-dependent viruses, RsYN2012 and RsYN2016A, as well as an ACE2-independent virus, RsHuB2019A. Although our stocks of RsHuB2019A rapidly acquired a tissue-culture adaption that rendered the spike protein resistant to trypsin, trypsin was still required for viral entry, suggesting limitations on the exogenous entry factors that support bat sarbecoviruses. Electron microscopy revealed that ACE2-independent sarbecoviruses have a prominent spike corona and share similar morphology to other coronaviruses. Our findings demonstrate a broader zoonotic threat posed by sarbecoviruses and shed light on the intricacies of coronavirus isolation and propagation in vitro. IMPORTANCE Several coronaviruses have been transmitted from animals to people, and 20 years of virus discovery studies have uncovered thousands of new coronavirus sequences in nature. Most of the animal-derived sarbecoviruses have never been isolated in culture due to cell incompatibilities and a poor understanding of the in vitro requirements for their propagation. Here, we built on our growing body of work characterizing viral entry mechanisms of bat sarbecoviruses in human cells and have developed a virus isolation protocol that allows for the exploration of these understudied viruses. Our protocol is robust and practical, leading to successful isolation of more sarbecoviruses than previous approaches and from field samples that had been collected over a 10-year longitudinal study.

Design of a bifunctional pan-sarbecovirus entry inhibitor targeting the cell receptor and viral fusion protein.

Development of highly effective antivirals that are robust to viral evolution is a practical strategy for combating the continuously evolved severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Inspired by viral multistep entry process, we here focus on developing a bispecific SARS-CoV-2 entry inhibitor, which acts on the cell receptor angiotensin converting enzyme 2 (ACE2) and viral S2 fusion protein. First, we identified a panel of diverse spike (S) receptor-binding domains (RBDs) and found that the RBD derived from Guangdong pangolin coronavirus (PCoV-GD) possessed the most potent antiviral potency. Next, we created a bispecific inhibitor termed RBD-IPB01 by genetically linking a peptide fusion inhibitor IPB01 to the C-terminal of PCoV-GD RBD, which exhibited greatly increased antiviral potency via cell membrane ACE2 anchoring. Promisingly, RBD-IPB01 had a uniformly bifunctional inhibition on divergent pseudo- and authentic SARS-CoV-2 variants, including multiple Omicron subvariants. RBD-IPB01 also showed consistently cross-inhibition of other sarbecoviruses, including SARS-CoV, PCoV-GD, and Guangxi pangolin coronavirus (PCoV-GX). RBD-IPB01 displayed low cytotoxicity, high trypsin resistance, and favorable metabolic stability. Combined, our studies have provided a tantalizing insight into the design of broad-spectrum and potent antiviral agent. IMPORTANCE Ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolution and spillover potential of a wide variety of sarbecovirus lineages indicate the importance of developing highly effective antivirals with broad capability. By directing host angiotensin converting enzyme 2 receptor and viral S2 fusion protein, we have created a dual-targeted virus entry inhibitor with high antiviral potency and breadth. The inhibitor receptor-binding domain (RBD)-IPB01 with the Guangdong pangolin coronavirus (PCoV-GD) spike RBD and a fusion inhibitor IPB01 displays bifunctional cross-inhibitions on pseudo- and authentic SARS-CoV-2 variants including Omicron, as well as on the sarbecoviruses SARS-CoV, PCoV-GD, and Guangxi pangolin coronavirus. RBD-IPB01 also efficiently inhibits diverse SARS-CoV-2 infection of human Calu-3 cells and blocks viral S-mediated cell-cell fusion with a dual function. Thus, the creation of such a bifunctional inhibitor with pan-sarbecovirus neutralizing capability has not only provided a potential weapon to combat future SARS-CoV-2 variants or yet-to-emerge zoonotic sarbecovirus, but also verified a viable strategy for the designing of antivirals against infection of other enveloped viruses.

Natural selection shapes the evolution of SARS-CoV-2 Omicron in Bangladesh.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved to give rise to a highly transmissive and immune-escaping variant of concern, known as Omicron. Many aspects of the evolution of SARS-CoV-2 and the driving forces behind the ongoing Omicron outbreaks remain unclear. Substitution at the receptor-binding domain (RBD) in the spike protein is one of the primary strategies of SARS-CoV-2 Omicron to hinder recognition by the host angiotensin-converting enzyme 2 (ACE2) receptor and avoid antibody-dependent defense activation. Here, we scanned for adaptive evolution within the SARS-CoV-2 Omicron genomes reported from Bangladesh in the public database GISAID (www.gisaid.org; dated 2 April 2023). The ratio of the non-synonymous (Ka) to synonymous (Ks) nucleotide substitution rate, denoted as ω, is an indicator of the selection pressure acting on protein-coding genes. A higher proportion of non-synonymous to synonymous substitutions (Ka/Ks or ω > 1) indicates positive selection, while Ka/Ks or ω near zero indicates purifying selection. An equal amount of non-synonymous and synonymous substitutions (Ka/Ks or ω = 1) refers to neutrally evolving sites. We found evidence of adaptive evolution within the spike (S) gene of SARS-CoV-2 Omicron isolated from Bangladesh. In total, 22 codon sites of the S gene displayed a signature of positive selection. The data also highlighted that the receptor-binding motif within the RBD of the spike glycoprotein is a hotspot of adaptive evolution, where many of the codons had ω > 1. Some of these adaptive sites at the RBD of the spike protein are known to be associated with increased viral fitness. The M gene and ORF6 have also experienced positive selection. These results suggest that although purifying selection is the dominant evolutionary force, positive Darwinian selection also plays a vital role in shaping the evolution of SARS-CoV-2 Omicron in Bangladesh.

SARS-CoV-2 spike ectodomain targets α7 nicotinic acetylcholine receptors

Virus entry into animal cells is initiated by attachment to target macromolecules located on host cells. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) trimeric spike glycoprotein targets host angiotensin converting enzyme 2 to gain cellular access. The SARS-CoV-2 glycoprotein contains a neurotoxin-like region that has sequence similarities to the rabies virus and the HIV glycoproteins, as well as to snake neurotoxins, which interact with nicotinic acetylcholine receptor (nAChR) subtypes via this region. Using a peptide of the neurotoxin-like region of SARS-CoV-2 (SARS-CoV-2 glycoprotein peptide [SCoV2P]), we identified that this area moderately inhibits α3β2, α3β4, and α4β2 subtypes, while potentiating and inhibiting α7 nAChRs. These nAChR subtypes are found in target tissues including the nose, lung, central nervous system, and immune cells. Importantly, SCoV2P potentiates and inhibits ACh-induced α7 nAChR responses by an allosteric mechanism, with nicotine enhancing these effects. Live-cell confocal microscopy was used to confirm that SCoV2P interacts with α7 nAChRs in transfected neuronal-like N2a and human embryonic kidney 293cells. The SARS-CoV-2 ectodomain functionally potentiates and inhibits the α7 subtype with nanomolar potency. Our functional findings identify that the α7 nAChR is a target for the SARS-CoV-2 glycoprotein, providing a new aspect to our understanding of SARS-CoV-2 and host cell interactions, in addition to disease pathogenesis.

Activation of the Interferon Pathway in Trophoblast Cells Productively Infected with SARS-CoV-2.

SARS-CoV-2 infection during pregnancy has been associated with poor maternal and neonatal outcomes and placental defects. The placenta, which acts as a physical and immunological barrier at the maternal-fetal interface, is not established until the end of the first trimester. Therefore, localized viral infection of the trophoblast compartment early in gestation could trigger an inflammatory response resulting in altered placental function and consequent suboptimal conditions for fetal growth and development. In this study, we investigated the effect of SARS-CoV-2 infection in early gestation placentae using placenta-derived human trophoblast stem cells (TSCs), a novel in vitro model, and their extravillous trophoblast (EVT) and syncytiotrophoblast (STB) derivatives. SARS-CoV-2 was able to productively replicate in TSC-derived STB and EVT, but not undifferentiated TSCs, which is consistent with the expression of SARS-CoV-2 entry host factors, ACE2 (angiotensin-converting enzyme 2) and TMPRSS2 (transmembrane cellular serine protease) in these cells. In addition, both TSC-derived EVT and STB infected with SARS-CoV-2 elicited an interferon-mediated innate immune response. Combined, these results suggest that placenta-derived TSCs are a robust in vitro model to investigate the effect of SARS-CoV-2 infection in the trophoblast compartment of the early placenta and that SARS-CoV-2 infection in early gestation activates the innate immune response and inflammation pathways. Therefore, placental development could be adversely affected by early SARS-CoV-2 infection by directly infecting the developing differentiated trophoblast compartment, posing a higher risk for poor pregnancy outcomes.

Investigating the binding affinity of andrographolide against human SARS-CoV-2 spike receptor-binding domain through docking and molecular dynamics simulations

SARS-CoV-2 is a positive-sense single-stranded RNA virus that causes a deadly coronavirus disease (COVID-19) in humans. The infection of SARS-CoV-2 in humans involves a viral surface spike glycoprotein containing the receptor-binding domain (RBD). The interactions of SARS-CoV-2 with the host angiotensin-converting enzyme 2 (ACE2) receptor are mediated by RBD. It binds to the host ACE2 and influences viral replication and disease pathogenesis. Therefore, targeting the RBD to prevent SARS-CoV-2 infections is of utmost importance. In this study, we used docking and molecular dynamics simulations to understand the binding effect of andrographolide on the SARS-CoV-2 spike protein. During docking, a strong binding affinity was observed between the ligand and the target receptor protein. MD results demonstrated higher conformational fluctuations in the ligand-free protein compared to the bound form. Several residues in the active sites make conformational rearrangements for the S protein to interact with the ligand. While RBD experiences conformational transition to gain more stability upon binding with the ligand. This binding is strengthened via several non-covalent interactions that make the complex structure more stable with higher binding affinity. Overall findings of the study may shed some valuable insights concerning the development of potential therapeutics in the strategies for COVID-19 prevention.

Rational approaches to discover SARS-CoV-2/ACE2 interaction inhibitors: Pharmacophore-based virtual screening, molecular docking, molecular dynamics and binding free energy studies

The lack of effective treatment remains a bottleneck in combating the current coronavirus family pandemic, particularly coronavirus 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The infection of host cells by SARS-CoV-2 is mediated by the binding of its receptor-binding domain (RBD) on the spike (S) glycoprotein to the host angiotensin-converting enzyme (ACE2) receptor. As all developed and available vaccines against COVID-19 do not provide long-term immunity, the creation of an effective drug for the treatment of COVID-19 is necessary and cannot be ignored. Therefore, the aim of this study is to present a computational screening method to identify potential inhibitor candidates with a high probability of blocking the binding of RBD to the ACE2 receptor. Pharmacophore mapping, molecular docking, molecular dynamics (MD) simulations, and binding free-energy analyses were performed to identify potential inhibitor candidates against ACE2/SARS-CoV-2. In conclusion, we propose the compound PubChem-84280085 as a potential inhibitor of protein–protein interactions to disrupt the binding of the SARS-CoV-2-RBD to the ACE2 receptor.

De novo design of a stapled peptide targeting SARS-CoV-2 spike protein receptor-binding domain.

Although effective vaccines have been developed against SARS-CoV-2, many regions in the world still have low rates of vaccination and new variants with mutations in the viral spike protein have reduced the effectiveness of most available vaccines and treatments. There is an urgent need for a drug to cure this disease and prevent infection. The SARS-CoV-2 virus enters the host cell through protein-protein interaction between the virus's spike protein and the host's angiotensin converting enzyme (ACE2). Using protein design software and molecular dynamics simulations, we have designed a 17-residue peptide (pep39), that binds to the spike protein receptor-binding domain (RBD) and blocks interaction of spike protein with ACE2. We have confirmed the binding activity of the designed peptide for the original spike protein and the delta variant spike protein using micro-cantilever and bio-layer interferometry (BLI) based methods. We also confirmed that pep39 strongly inhibits SARS-CoV-2 virus replication in Vero E6 cells. Taken together these data suggest that a newly designed spike protein RBD blocking peptide pep39 has a potential as a SARS-CoV-2 virus inhibitor.

Hepatic Injury in COVID-19 Patients

The practical studies have already proved that the laboratory liver tests are highly useful in the evaluation and treatment of patients with hepatic dysfunction. It has been found that some of the enzymes and the end products of the metabolic pathway such as serum bilirubin, alanine amino transferase, aspartate amino transferase, ratio of aminotransferases, alkaline phosphatase, gamma glutamyl transferase, 5’ nucleotidase, ceruloplasmin that are very sensitive for the abnormality occurred may be considered as an outstanding biochemical marker of liver dysfunction. It is noticed that the novel coronavirus Severe Acute Respiratory Syndrome Corona Virus-2 (SARS-CoV-2) infection mostly leads to respiratory distress syndrome, at the same time liver injury is also documented. As a matter of fact, the mechanism of liver injury is limited and poorly understood. Therefore, the hepatic injury might be due to a consequence of systemic inflammatory response, viral infection of hepatocytes, or it comes as a result of intensive care treatment or drug toxicity. The host angiotensin-converting enzyme 2 (ACE2) receptors, which are widely distributed in type 2 alveolar cells, are the proposed route of viral entrance. It is interesting to note that ACE2 receptors are found in the liver's cholangiocytes, vascular endothelium, and gastrointestinal tract. 
 Histological pictures compatible with vascular alterations are observed, characterized by the increase in number of portal vein branches associated with lumen massive dilatation, partial or complete luminal thrombosis of portal and sinusoidal vessels, fibrosis of portal tract, focally markedly enlarged and fibrotic.

Unveiling Acacia farnesiana phytochemicals as Angiotensin Converting Enzyme (ACE) inhibitors via in-silico drug design, molecular docking, and bioavailability predictions: An illustration against SARS-CoV-2 spike protein

Angiotensin-converting enzyme 2 (ACE2) is a transmembrane protein that functions as a receptor for coronavirus spike protein. When spike protein fragments as the ligand binds with ACE2 protein, this ACE2 protein functions as a virus receptor, participating in the biological process known as the viral particle entry in the host cell. Hence, an in-silico study was carried out since it is faster and less expensive than trial and error methods based on experimental investigations. To study the effect of Acacia farnesiana phytochemicals on spike protein, molecular docking analyses were carried out. In this study, twelve phytochemicals from Acacia farnesiana have been selected as small molecules based on their ACEI and anti- inflammatory nature to evaluate molecular interaction between spike protein of SARS-CoV2 with ACE2 of the human complex molecule. Gallic acid, methyl gallate, kaempferol, Rhamnocitrin, naringenin, apigenin, ellagic acid, ferulic acid, myricetin, Diosmetin, Caffeic acid, and Quercetin were chosen as competent natural compounds from Acacia farnesiana as potent small molecules against COVID-19 and further ADME analysis were carried out. The result indicated that due to the presence of ACEIs and anti-inflammatory phytochemicals in Acacia farnesiana, the bound structure of ACE2 and spike protein becomes unstable. Therefore, these natural compounds can show antiviral activity by destabilizing spike protein binding with the human host ACE2 receptor.

SARS-CoV-2 spike protein variant binding affinity to an angiotensin-converting enzyme 2 fusion glycoproteins.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of the Coronavirus disease 2019 (Covid-19) pandemic, continues to evolve and circulate globally. Current prophylactic and therapeutic countermeasures against Covid-19 infection include vaccines, small molecule drugs, and neutralizing monoclonal antibodies. SARS-CoV-2 infection is mainly mediated by the viral spike glycoprotein binding to angiotensin converting enzyme 2 (ACE2) on host cells for viral entry. As emerging mutations in the spike protein evade efficacy of spike-targeted countermeasures, a potential strategy to counter SARS-CoV-2 infection is to competitively block the spike protein from binding to the host ACE2 using a soluble recombinant fusion protein that contains a human ACE2 and an IgG1-Fc domain (ACE2-Fc). Here, we have established Chinese Hamster Ovary (CHO) cell lines that stably express ACE2-Fc proteins in which the ACE2 domain either has or has no catalytic activity. The fusion proteins were produced and purified to partially characterize physicochemical properties and spike protein binding. Our results demonstrate the ACE2-Fc fusion proteins are heavily N-glycosylated, sensitive to thermal stress, and actively bind to five spike protein variants (parental, alpha, beta, delta, and omicron) with different affinity. Our data demonstrates a proof-of-concept production strategy for ACE2-Fc fusion glycoproteins that can bind to different spike protein variants to support the manufacture of potential alternative countermeasures for emerging SARS-CoV-2 variants.

Sequence difference of angiotensin-converting enzyme 2 between nonhuman primates affects its binding-affinity with SARS-CoV-2 S receptor binding domain

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused many deaths and contributed to a tremendous public health concern worldwide since 2020. Angiotensin-converting enzyme 2 (ACE2) binds to the SARS-CoV-2 virus as a receptor. The challenge of different nonhuman primate (NHP) species by SARS-CoV-2 virus demonstrated different effects on virus replication and disease pathology. This study characterizes differences between host ACE2 sequences of three NHP species: Macaca mulatta, Macaca fascicularis, and Chlorocebus sabaeus. In addition, the binding affinity between the ACE2 ectodomain and the SARS-CoV-2 S receptor-binding domain (RBD) was analyzed. Variation of ACE2 sequence among NHP species and the binding affinity may account for different susceptibility and responses to SARS-CoV-2 infection.

Exploring the Binding Affinity and Mechanism between ACE2 and the Trimers of Delta and Omicron Spike Proteins by Molecular Dynamics Simulation and Bioassay.

Five major variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged and posed challenges in controlling the pandemic. Among them, the current dominant variant, viz., Omicron, has raised serious concerns about its infectiousness and antibody neutralization. However, few studies pay attention to the effect of the mutations on the dynamic interaction network of Omicron S protein trimers binding to the host angiotensin-converting enzyme 2 (ACE2). In this study, we conducted molecular dynamics (MD) simulations and enzyme linked immunosorbent assay (ELISA) to explore the binding strength and mechanism of wild type (WT), Delta, and Omicron S protein trimers to ACE2. The results showed that the binding capacities of both the two variants' S protein trimers to ACE2 are enhanced in varying degrees, indicating possibly higher cell infectiousness. Energy decomposition and protein-protein interaction network analysis suggested that both the mutational and conserved sites make effects on the increase in the overall affinity through a variety of interactions. The experimentally determined KD values by biolayer interferometry (BLI) and the predicted binding free energies of the RBDs of Delta and Omicron to mAb HLX70 revealed that the two variants may have the high risk of immune evasion from the mAb. These results are not only helpful in understanding the binding strength and mechanism of S protein trimer-ACE2 but also beneficial for drug, especially for antibody development.

Putative role of mitochondria in SARS-CoV-2 mediated brain dysfunctions: a prospect

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the COVID-19 pandemic. Though the virus primarily damages the respiratory and cardiovascular systems after binding to the host angiotensin-converting enzyme 2 (ACE2) receptors, it has the potential to affect all major organ systems, including the human nervous system. There are multiple clinical reports of anosmia, dizziness, headache, nausea, ageusia, encephalitis, demyelination, neuropathy, memory loss, and neurological complications in SARS-CoV-2 infected individuals. Though the molecular mechanism of these brain dysfunctions during SARS-CoV-2 infection is elusive, the mitochondria seem to be an integral part of this pathogenesis. Emerging research findings suggest that the dysfunctional mitochondria and associated altered bioenergetics in the infected host cells lead to altered energy metabolism in the brain of Covid-19 patients. The interactome between viral proteins and mitochondrial proteins during Covid-19 pathogenesis also provides evidence for the involvement of mitochondria in SARS-CoV-2-induced brain dysfunctions. The present review discusses the possible role of mitochondria in disturbing the SARS-CoV-2 mediated brain functions, with the potential to use this information to prevent and treat these impairments.