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1.
Nat Commun ; 12(1): 3802, 2021 06 21.
Article in English | MEDLINE | ID: covidwho-1387351

ABSTRACT

SARS-CoV-2 has mutated during the global pandemic leading to viral adaptation to medications and vaccinations. Here we describe an engineered human virus receptor, ACE2, by mutagenesis and screening for binding to the receptor binding domain (RBD). Three cycles of random mutagenesis and cell sorting achieved sub-nanomolar affinity to RBD. Our structural data show that the enhanced affinity comes from better hydrophobic packing and hydrogen-bonding geometry at the interface. Additional disulfide mutations caused the fixing of a closed ACE2 conformation to avoid off-target effects of protease activity, and also improved structural stability. Our engineered ACE2 neutralized SARS-CoV-2 at a 100-fold lower concentration than wild type; we also report that no escape mutants emerged in the co-incubation after 15 passages. Therapeutic administration of engineered ACE2 protected hamsters from SARS-CoV-2 infection, decreased lung virus titers and pathology. Our results provide evidence of a therapeutic potential of engineered ACE2.


Subject(s)
Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/pharmacology , COVID-19/drug therapy , Mutation , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/metabolism , Angiotensin-Converting Enzyme 2/metabolism , Animals , COVID-19/metabolism , COVID-19/virology , Cells, Cultured , Cricetinae , Crystallography, X-Ray , Disease Models, Animal , Humans , Male , Molecular Dynamics Simulation , Protein Binding , Protein Engineering/methods , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism
3.
Int J Mol Sci ; 22(17)2021 Aug 25.
Article in English | MEDLINE | ID: covidwho-1376841

ABSTRACT

In recent years, enzymes have risen as promising therapeutic tools for different pathologies, from metabolic deficiencies, such as fibrosis conditions, ocular pathologies or joint problems, to cancer or cardiovascular diseases. Treatments based on the catalytic activity of enzymes are able to convert a wide range of target molecules to restore the correct physiological metabolism. These treatments present several advantages compared to established therapeutic approaches thanks to their affinity and specificity properties. However, enzymes present some challenges, such as short in vivo half-life, lack of targeted action and, in particular, patient immune system reaction against the enzyme. For this reason, it is important to monitor serum immune response during treatment. This can be achieved by conventional techniques (ELISA) but also by new promising tools such as microarrays. These assays have gained popularity due to their high-throughput analysis capacity, their simplicity, and their potential to monitor the immune response of patients during enzyme therapies. In this growing field, research is still ongoing to solve current health problems such as COVID-19. Currently, promising therapeutic alternatives using the angiotensin-converting enzyme 2 (ACE2) are being studied to treat COVID-19.


Subject(s)
Angiotensin-Converting Enzyme 2/therapeutic use , COVID-19/drug therapy , Enzyme Therapy/methods , Recombinant Proteins/therapeutic use , Angiotensin-Converting Enzyme 2/pharmacology , Clinical Trials, Phase II as Topic , Drug Compounding/methods , Enzyme Stability , Enzyme Therapy/history , Enzyme Therapy/trends , Half-Life , History, 20th Century , History, 21st Century , Humans , Recombinant Proteins/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/metabolism , Treatment Outcome , Virus Internalization/drug effects
4.
Antiviral Res ; 194: 105147, 2021 10.
Article in English | MEDLINE | ID: covidwho-1347484

ABSTRACT

The SARS-CoV-2 receptor angiotensin converting enzyme 2 (ACE2) was previously engineered into a high affinity tetravalent format (ACE2-Fc-TD) that is a potential decoy protein in SARS-CoV-2 infection.We report that this protein shows greatly enhanced binding to SARS-CoV-2 spike proteins of the SARS-CoV-2 variants of concern B.1.1.7 (alpha variant, originally isolated in the United Kingdom) and B.1.351 (beta variant, originally isolated in South Africa) with picomolar compared with nanomolar Kd values. In addition, ACE2-Fc-TD displays greater neutralization of SARS-CoV-2 pseudotype viruses compared to a dimeric ACE2-Fc, with enhanced activity on variant B.1.351. This tetrameric decoy protein would be a valuable addition to SARS-CoV-2 therapeutic approaches, especially where vaccination cannot be used but also should there be any future coronavirus pandemics.


Subject(s)
Angiotensin-Converting Enzyme 2/pharmacology , Antiviral Agents/metabolism , COVID-19/prevention & control , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Antiviral Agents/chemistry , COVID-19/drug therapy , COVID-19/enzymology , COVID-19/virology , Cell Line , Humans , Kinetics , Mutation , Protein Binding , Protein Domains , SARS-CoV-2/isolation & purification , Spike Glycoprotein, Coronavirus/metabolism
5.
J Virol Methods ; 295: 114221, 2021 09.
Article in English | MEDLINE | ID: covidwho-1284316

ABSTRACT

SARS-CoV-2 is the culprit causing Coronavirus Disease 2019 (COVID-19). For the study of SARS-CoV-2 infection in a BSL-2 laboratory, a SARS-CoV-2 pseudovirus particle (SARS2pp) production and infection system was constructed by using a lentiviral vector bearing dual-reporter genes eGFP and firefly luciferase (Luc2) for easy observation and analysis. Comparison of SARS2pp different production conditions revealed that the pseudovirus titer could be greatly improved by: 1) removing the last 19 amino acids of the spike protein and replacing the signal peptide with the mouse Igk signal sequence; 2) expressing the spike protein using CMV promoter other than CAG (a hybrid promoter consisting of a CMV enhancer, beta-actin promoter, splice donor, and a beta-globin splice acceptor); 3) screening better optimized spike protein sequences for SARS2pp production; and 4) adding 1 % BSA in the SARS2pp production medium. For infection, this SARS2pp system showed a good linear relationship between MOI 2-0.0002 and then was successfully used to evaluate SARS-CoV-2 infection inhibitors including recombinant human ACE2 proteins and SARS-CoV-2 neutralizing antibodies. The kidney, liver and small intestine-derived cell lines were also found to show different susceptibility to SARSpp and SARS2pp. Given its robustness and good performance, it is believed that this pseudovirus particle production and infection system will greatly promote future research for SARS-CoV-2 entry mechanisms and inhibitors and can be easily applied to study new emerging SARS-CoV-2 variants.


Subject(s)
Neutralization Tests/methods , SARS-CoV-2/physiology , Virus Internalization , Angiotensin-Converting Enzyme 2/pharmacology , Animals , Antibodies, Neutralizing/pharmacology , Antiviral Agents/pharmacology , Cell Line , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , Lentivirus/genetics , Luciferases, Firefly/genetics , Luciferases, Firefly/metabolism , Recombinant Proteins/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Virion , Virus Internalization/drug effects
6.
Cell Rep ; 36(3): 109415, 2021 07 20.
Article in English | MEDLINE | ID: covidwho-1283976

ABSTRACT

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants threatens efforts to contain the coronavirus disease 2019 (COVID-19) pandemic. The number of COVID-19 cases and deaths in India has risen steeply, and a SARS-CoV-2 variant, B.1.617, is believed to be responsible for many of these cases. The spike protein of B.1.617 harbors two mutations in the receptor binding domain, which interacts with the angiotensin converting enzyme 2 (ACE2) receptor and constitutes the main target of neutralizing antibodies. Therefore, we analyze whether B.1.617 is more adept in entering cells and/or evades antibody responses. B.1.617 enters two of eight cell lines tested with roughly 50% increased efficiency and is equally inhibited by two entry inhibitors. In contrast, B.1.617 is resistant against bamlanivimab, an antibody used for COVID-19 treatment. B.1.617 evades antibodies induced by infection or vaccination, although less so than the B.1.351 variant. Collectively, our study reveals that antibody evasion of B.1.617 may contribute to the rapid spread of this variant.


Subject(s)
Angiotensin-Converting Enzyme 2/pharmacology , Antibodies, Monoclonal, Humanized/pharmacology , Antibodies, Viral/pharmacology , COVID-19/drug therapy , Esters/pharmacology , Guanidines/pharmacology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Antibodies, Monoclonal, Humanized/immunology , Antibodies, Viral/immunology , COVID-19/immunology , Cell Line , Humans , Protease Inhibitors/pharmacology , Protein Binding , SARS-CoV-2/genetics , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Vaccination
7.
Pharmacol Rep ; 73(6): 1539-1550, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1281363

ABSTRACT

Angiotensin-converting enzyme (ACE) and its homologue, ACE2, are commonly allied with hypertension, renin-angiotensin-aldosterone system pathway, and other cardiovascular system disorders. The recent pandemic of COVID-19 has attracted the attention of numerous researchers on ACE2 receptors, where the causative viral particle, SARS-CoV-2, is established to exploit these receptors for permitting their entry into the human cells. Therefore, studies on the molecular origin and pathophysiology of the cell response in correlation to the role of ACE2 receptors to these viruses are bringing novel theories. The varying level of manifestation and importance of ACE proteins, underlying irregularities and disorders, intake of specific medications, and persistence of assured genomic variants at the ACE genes are potential questions raising nowadays while observing the marked alteration in response to the SARS-CoV-2-infected patients. Therefore, the present review has focused on several raised opinions associated with the role of the ACE2 receptor and its impact on COVID-19 pathogenesis.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Angiotensin-Converting Enzyme 2/pharmacology , COVID-19/drug therapy , SARS-CoV-2/pathogenicity , Acute Lung Injury , Angiotensin-Converting Enzyme 2/deficiency , Angiotensin-Converting Enzyme 2/therapeutic use , Humans , Hypertension/drug therapy , Spike Glycoprotein, Coronavirus/metabolism
8.
Sci Rep ; 11(1): 10617, 2021 05 19.
Article in English | MEDLINE | ID: covidwho-1236094

ABSTRACT

Approaches are needed for therapy of the severe acute respiratory syndrome from SARS-CoV-2 coronavirus (COVID-19). Interfering with the interaction of viral antigens with the angiotensin converting enzyme 2 (ACE-2) receptor is a promising strategy by blocking the infection of the coronaviruses into human cells. We have implemented a novel protein engineering technology to produce a super-potent tetravalent form of ACE2, coupled to the human immunoglobulin γ1 Fc region, using a self-assembling, tetramerization domain from p53 protein. This high molecular weight Quad protein (ACE2-Fc-TD) retains binding to the SARS-CoV-2 receptor binding spike protein and can form a complex with the spike protein plus anti-viral antibodies. The ACE2-Fc-TD acts as a powerful decoy protein that out-performs soluble monomeric and dimeric ACE2 proteins and blocks both SARS-CoV-2 pseudovirus and SARS-CoV-2 virus infection with greatly enhanced efficacy. The ACE2 tetrameric protein complex promise to be important for development as decoy therapeutic proteins against COVID-19. In contrast to monoclonal antibodies, ACE2 decoy is unlikely to be affected by mutations in SARS-CoV-2 that are beginning to appear in variant forms. In addition, ACE2 multimeric proteins will be available as therapeutic proteins should new coronaviruses appear in the future because these are likely to interact with ACE2 receptor.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/pharmacology , Antiviral Agents/metabolism , COVID-19/prevention & control , Protein Engineering/methods , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antiviral Agents/chemistry , COVID-19/drug therapy , COVID-19/enzymology , COVID-19/virology , Cell Line , Drug Design , Haplorhini , Humans , Protein Binding , Protein Structural Elements , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/metabolism
9.
Int J Mol Med ; 47(4)2021 04.
Article in English | MEDLINE | ID: covidwho-1080914

ABSTRACT

Coronavirus disease 2019 (COVID­19) is an acute infectious pneumonia caused by a novel type of coronavirus infection. There are currently no clinically available specific drugs for the treatment of this virus. The process of host invasion is the key to viral infection, and it is a mechanism that needs to be considered when exploring antiviral drugs. At present, studies have confirmed that angiotensin­converting enzyme II (ACE2) is the main functional receptor through which severe acute respiratory syndrome coronavirus (SARS­CoV­2) invades host cells. Therefore, a number of studies have focused on this field. However, as ACE2 may play a dual role in mediating susceptibility and immunity to SARS­CoV­2 infection, the role of ACE2 in viral infection is controversial. Beginning with the physiological function of ACE2, the present review article summarizes the influence of the ACE2 content on the susceptibility to the virus and acute lung injury. Drug mechanisms were taken as the starting point, combined with the results of clinical trials, specifically elaborating upon and analyzing the efficacy of several ACE2­centered therapeutic drugs and their potential effects. In addition, the current status of ACE2 as a targeted therapy for COVID­19 is discussed in order to provide new insight into the clinical prevention and treatment of COVID­19.


Subject(s)
Angiotensin-Converting Enzyme 2/physiology , Antiviral Agents/pharmacology , COVID-19/prevention & control , COVID-19/therapy , Host-Pathogen Interactions/physiology , Angiotensin-Converting Enzyme 2/antagonists & inhibitors , Angiotensin-Converting Enzyme 2/pharmacology , COVID-19/virology , Cardiovascular Diseases/etiology , Chloroquine/analogs & derivatives , Chloroquine/pharmacology , Host-Pathogen Interactions/drug effects , Humans , Indoles/pharmacology , Molecular Targeted Therapy , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/metabolism , Virus Internalization
10.
J Cell Mol Med ; 25(3): 1342-1349, 2021 02.
Article in English | MEDLINE | ID: covidwho-1030565

ABSTRACT

SARS-CoV-2, the virus responsible for the global coronavirus disease (COVID-19) pandemic, attacks multiple organs of the human body by binding to angiotensin-converting enzyme 2 (ACE2) to enter cells. More than 20 million people have already been infected by the virus. ACE2 is not only a functional receptor of COVID-19 but also an important endogenous antagonist of the renin-angiotensin system (RAS). A large number of studies have shown that ACE2 can reverse myocardial injury in various cardiovascular diseases (CVDs) as well as is exert anti-inflammatory, antioxidant, anti-apoptotic and anticardiomyocyte fibrosis effects by regulating transforming growth factor beta, mitogen-activated protein kinases, calcium ions in cells and other major pathways. The ACE2/angiotensin-(1-7)/Mas receptor axis plays a decisive role in the cardiovascular system to combat the negative effects of the ACE/angiotensin II/angiotensin II type 1 receptor axis. However, the underlying mechanism of ACE2 in cardiac protection remains unclear. Some approaches for enhancing ACE2 expression in CVDs have been suggested, which may provide targets for the development of novel clinical therapies. In this review, we aimed to identify and summarize the role of ACE2 in CVDs.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , COVID-19/complications , Cardiovascular Diseases/metabolism , Angiotensin-Converting Enzyme 2/pharmacology , Animals , Arrhythmias, Cardiac/metabolism , Arrhythmias, Cardiac/physiopathology , COVID-19/drug therapy , COVID-19/metabolism , Cardiovascular Diseases/complications , Cardiovascular Diseases/physiopathology , Diminazene/pharmacology , Heart Failure/etiology , Humans , Hypertension/metabolism , Hypertension/physiopathology , Myocardial Infarction/drug therapy , Myocardial Infarction/metabolism , Myocardial Infarction/physiopathology , Recombinant Proteins/pharmacology
11.
EMBO Mol Med ; 13(1): e13426, 2021 01 11.
Article in English | MEDLINE | ID: covidwho-1024813

ABSTRACT

There is a critical need for safe and effective drugs for COVID-19. Only remdesivir has received authorization for COVID-19 and has been shown to improve outcomes but not decrease mortality. However, the dose of remdesivir is limited by hepatic and kidney toxicity. ACE2 is the critical cell surface receptor for SARS-CoV-2. Here, we investigated additive effect of combination therapy using remdesivir with recombinant soluble ACE2 (high/low dose) on Vero E6 and kidney organoids, targeting two different modalities of SARS-CoV-2 life cycle: cell entry via its receptor ACE2 and intracellular viral RNA replication. This combination treatment markedly improved their therapeutic windows against SARS-CoV-2 in both models. By using single amino-acid resolution screening in haploid ES cells, we report a singular critical pathway required for remdesivir toxicity, namely, Adenylate Kinase 2. The data provided here demonstrate that combining two therapeutic modalities with different targets, common strategy in HIV treatment, exhibit strong additive effects at sub-toxic concentrations. Our data lay the groundwork for the study of combinatorial regimens in future COVID-19 clinical trials.


Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Angiotensin-Converting Enzyme 2/pharmacology , Antiviral Agents/pharmacology , COVID-19/drug therapy , SARS-CoV-2/drug effects , Adenosine Monophosphate/pharmacology , Alanine/pharmacology , Animals , Cells, Cultured , Chlorocebus aethiops , Drug Synergism , Humans , Models, Molecular , Recombinant Proteins/pharmacology , SARS-CoV-2/physiology , Vero Cells , Virus Internalization/drug effects , Virus Replication/drug effects
13.
Int J Biol Macromol ; 165(Pt B): 1626-1633, 2020 Dec 15.
Article in English | MEDLINE | ID: covidwho-866724

ABSTRACT

Angiotensin-converting enzyme 2 (ACE2) is the entry receptor for SARS-CoV-2, and recombinant ACE2 decoys are being evaluated as new antiviral therapies. We designed and tested an antibody-like ACE2-Fc fusion protein, which has the benefit of long pharmacological half-life and the potential to facilitate immune clearance of the virus. Out of a concern that the intrinsic catalytic activity of ACE2 may unintentionally alter the balance of its hormonal substrates and cause adverse cardiovascular effects in treatment, we performed a mutagenesis screening for inactivating the enzyme. Three mutants, R273A, H378A and E402A, completely lost their enzymatic activity for either surrogate or physiological substrates. All of them remained capable of binding SARS-CoV-2 and could suppress the transduction of a pseudotyped virus in cell culture. This study established new ACE2-Fc candidates as antiviral treatment for SARS-CoV-2 without potentially harmful side effects from ACE2's catalytic actions toward its vasoactive substrates.


Subject(s)
Angiotensin-Converting Enzyme 2 , COVID-19 , Immunoglobulin Fc Fragments , Recombinant Fusion Proteins , SARS-CoV-2/metabolism , Amino Acid Substitution , Angiotensin-Converting Enzyme 2/chemistry , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/pharmacology , Animals , COVID-19/drug therapy , COVID-19/metabolism , COVID-19/pathology , Cell Line , Female , Humans , Immunoglobulin Fc Fragments/chemistry , Immunoglobulin Fc Fragments/genetics , Immunoglobulin Fc Fragments/pharmacology , Mice , Mice, Inbred BALB C , Mutation, Missense , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/pharmacology
15.
J Biomol Struct Dyn ; 39(10): 3605-3614, 2021 07.
Article in English | MEDLINE | ID: covidwho-245040

ABSTRACT

The current pandemic of Covid-19 caused by SARS-CoV-2 is continued to spread globally and no potential drug or vaccine against it is available. Spike (S) glycoprotein is the structural protein of SARS-CoV-2 located on the envelope surface, involve in interaction with angiotensin converting enzyme 2 (ACE2), a cell surface receptor, followed by entry into the host cell. Thereby, blocking the S glycoprotein through potential inhibitor may interfere its interaction with ACE2 and impede its entry into the host cell. Here, we present a truncated version of human ACE2 (tACE2), comprising the N terminus region of the intact ACE2 from amino acid position 21-119, involved in binding with receptor binding domain (RBD) of SARS-CoV-2. We analyzed the in-silico potential of tACE2 to compete with intact ACE2 for binding with RBD. The protein-protein docking and molecular dynamic simulation showed that tACE2 has higher binding affinity for RBD and form more stabilized complex with RBD than the intact ACE2. Furthermore, prediction of tACE2 soluble expression in E. coli makes it a suitable candidate to be targeted for Covid-19 therapeutics. This is the first MD simulation based findings to provide a high affinity protein inhibitor for SARS-CoV-2 S glycoprotein, an important target for drug designing against this unprecedented challenge.Communicated by Ramaswamy H. Sarma.


Subject(s)
Angiotensin-Converting Enzyme 2/pharmacology , COVID-19 , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Angiotensin-Converting Enzyme 2/chemistry , Binding Sites , COVID-19/therapy , Escherichia coli , Humans , Molecular Dynamics Simulation , Protein Binding , SARS-CoV-2
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