Discovery of Clioquinol and Analogues as Novel Inhibitors of Severe Acute Respiratory Syndrome Coronavirus 2 Infection, ACE2 and ACE2 - Spike Protein Interaction In Vitro

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus first identified in December 2019. Notable features that make SARS-CoV-2 distinct from most other previously identified betacoronaviruses include a receptor binding domain and a unique insertion of 12 nucleotides or 4 amino acids (PRRA) at the S1/S2 boundary. In this study, we identified two deletion variants of SARS-CoV-2 that either directly affect the polybasic cleavage site itself (NSPRRAR) or a flanking sequence (QTQTN). These deletions were verified by multiple sequencing methods. In vitro results showed that the deletion of NSPRRAR likely does not affect virus replication in Vero and Vero-E6 cells; however, the deletion of QTQTN may restrict late-phase viral replication. The deletion of QTQTN was detected in 3 of 68 clinical samples and 12 of 24 in vitro-isolated viruses, while the deletion of NSPRRAR was identified in 3 in vitro-isolated viruses. Our data indicate that (i) there may be distinct selection pressures on SARS-CoV-2 replication or infection in vitro and in vivo; (ii) an efficient mechanism for deleting this region from the viral genome may exist, given that the deletion variant is commonly detected after two rounds of cell passage; and (iii) the PRRA insertion, which is unique to SARS-CoV-2, is not fixed during virus replication in vitro. These findings provide information to aid further investigation of SARS-CoV-2 infection mechanisms and a better understanding of the NSPRRAR deletion variant observed here. IMPORTANCE The spike protein determines the infectivity and host range of coronaviruses. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has two unique features in its spike protein, the receptor binding domain and an insertion of 12 nucleotides at the S1/S2 boundary resulting in a furin-like cleavage site. Here, we identified two deletion variants of SARS-CoV-2 that either directly affect the furin-like cleavage site itself (NSPRRAR) or a flanking sequence (QTQTN), and we investigated these deletions in cell isolates and clinical samples. The absence of the polybasic cleavage site in SARS-CoV-2 did not affect virus replication in Vero or Vero-E6 cells. Our data indicate the PRRAR sequence and the flanking QTQTN sequence are not fixed in vitro; thus, there appears to be distinct selection pressures on SARS-CoV-2 sequences in vitro and in vivo. Further investigation of the mechanism of generating these deletion variants and their infectivity in different animal models would improve our understanding of the origin and evolution of this virus.

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Neutralizing Human Antibodies against Severe Acute Respiratory Syndrome Coronavirus 2 Isolated from a Human Synthetic Fab Phage Display Library

International Journal of Molecular Sciences ◽

10.3390/ijms22041913 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1913

Author(s):

Yu Jung Kim ◽

Min Ho Lee ◽

Se-Ra Lee ◽

Hyo-Young Chung ◽

Kwangmin Kim ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Phage Display ◽

Viral Entry ◽

Phage Display Library ◽

Cross Reactivity ◽

Spike Protein ◽

Receptor Protein ◽

In Vitro Assays ◽

Global Threat

Since it was first reported in Wuhan, China, in 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic outbreak resulting in a tremendous global threat due to its unprecedented rapid spread and an absence of a prophylactic vaccine or therapeutic drugs treating the virus. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a key player in the viral entry into cells through its interaction with the angiotensin-converting enzyme 2 (ACE2) receptor protein, and the RBD has therefore been crucial as a drug target. In this study, we used phage display to develop human monoclonal antibodies (mAbs) that neutralize SARS-CoV-2. A human synthetic Fab phage display library was panned against the RBD of the SARS-CoV-2 spike protein (SARS-2 RBD), yielding ten unique Fabs with moderate apparent affinities (EC50 = 19–663 nM) for the SARS-2 RBD. All of the Fabs showed no cross-reactivity to the MERS-CoV spike protein, while three Fabs cross-reacted with the SARS-CoV spike protein. Five Fabs showed neutralizing activities in in vitro assays based on the Fabs’ activities antagonizing the interaction between the SARS-2 RBD and ACE2. Reformatting the five Fabs into immunoglobulin Gs (IgGs) greatly increased their apparent affinities (KD = 0.08–1.0 nM), presumably due to the effects of avidity, without compromising their non-aggregating properties and thermal stability. Furthermore, two of the mAbs (D12 and C2) significantly showed neutralizing activities on pseudo-typed and authentic SARS-CoV-2. Given their desirable properties and neutralizing activities, we anticipate that these human anti-SARS-CoV-2 mAbs would be suitable reagents to be further developed as antibody therapeutics to treat COVID-19, as well as for diagnostics and research tools.

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Computational drug repurposing strategy predicted peptide-based drugs that can potentially inhibit the interaction of SARS-CoV-2 spike protein with its target (humanACE2)

PLoS ONE ◽

10.1371/journal.pone.0245258 ◽

2021 ◽

Vol 16 (1) ◽

pp. e0245258

Author(s):

Samuel Egieyeh ◽

Elizabeth Egieyeh ◽

Sarel Malan ◽

Alan Christofells ◽

Burtram Fielding

Keyword(s):

Angiotensin Converting Enzyme ◽

Protein Interaction ◽

Drug Repurposing ◽

Spike Protein ◽

Converting Enzyme ◽

Peptide Drugs ◽

Angiotensin Converting Enzyme 2 ◽

Protein Protein Interaction ◽

Potential Inhibitors

Drug repurposing for COVID-19 has several potential benefits including shorter development time, reduced costs and regulatory support for faster time to market for treatment that can alleviate the current pandemic. The current study used molecular docking, molecular dynamics and protein-protein interaction simulations to predict drugs from the Drug Bank that can bind to the SARS-CoV-2 spike protein interacting surface on the human angiotensin-converting enzyme 2 (hACE2) receptor. The study predicted a number of peptide-based drugs, including Sar9 Met (O2)11-Substance P and BV2, that might bind sufficiently to the hACE2 receptor to modulate the protein-protein interaction required for infection by the SARS-CoV-2 virus. Such drugs could be validated in vitro or in vivo as potential inhibitors of the interaction of SARS-CoV-2 spike protein with the human angiotensin-converting enzyme 2 (hACE2) in the airway. Exploration of the proposed and current pharmacological indications of the peptide drugs predicted as potential inhibitors of the interaction between the spike protein and hACE2 receptor revealed that some of the predicted peptide drugs have been investigated for the treatment of acute respiratory distress syndrome (ARDS), viral infection, inflammation and angioedema, and to stimulate the immune system, and potentiate antiviral agents against influenza virus. Furthermore, these predicted drug hits may be used as a basis to design new peptide or peptidomimetic drugs with better affinity and specificity for the hACE2 receptor that may prevent interaction between SARS-CoV-2 spike protein and hACE2 that is prerequisite to the infection by the SARS-CoV-2 virus.

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Discovery of Clioquinol and Analogues as Novel Inhibitors of Severe Acute Respiratory Syndrome Coronavirus 2 Infection, ACE2 and ACE2 - Spike Protein Interaction In Vitro

10.1101/2020.08.14.250480 ◽

2020 ◽

Cited By ~ 2

Author(s):

Omonike A. Olaleye ◽

Manvir Kaur ◽

Collins Onyenaka ◽

Tolu Adebusuyi

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Cytopathic Effect ◽

Molecular Target ◽

Clinical Development ◽

Spike Protein ◽

Angiotensin Converting Enzyme 2 ◽

Global Pandemic ◽

Potential Therapeutics ◽

Micromolar Range

AbstractSevere Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiological agent for coronavirus disease 2019 (COVID-19), has emerged as an ongoing global pandemic. Presently, there are no clinically approved vaccines nor drugs for COVID-19. Hence, there is an urgent need to accelerate the development of effective antivirals. Here in, we discovered Clioquinol (5-chloro-7-iodo-8-quinolinol (CLQ)), a FDA approved drug and two of its analogues (7-bromo-5-chloro-8-hydroxyquinoline (CLBQ14); and 5, 7-Dichloro-8-hydroxyquinoline (CLCQ)) as potent inhibitors of SARS-CoV-2 infection induced cytopathic effect in vitro. In addition, all three compounds showed potent anti-exopeptidase activity against recombinant human angiotensin converting enzyme 2 (rhACE2) and inhibited the binding of rhACE2 with SARS-CoV-2 Spike (RBD) protein. CLQ displayed the highest potency in the low micromolar range, with its antiviral activity showing strong correlation with inhibition of rhACE2 and rhACE2-RBD interaction. Altogether, our findings provide a new mode of action and molecular target for CLQ and validates this pharmacophore as a promising lead series for clinical development of potential therapeutics for COVID-19.

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SARS-CoV-2 invades host cells via a novel route: CD147-spike protein

10.1101/2020.03.14.988345 ◽

2020 ◽

Cited By ~ 179

Author(s):

Ke Wang ◽

Wei Chen ◽

Yu-Sen Zhou ◽

Jian-Qi Lian ◽

Zheng Zhang ◽

...

Keyword(s):

Electron Microscope ◽

Severe Acute Respiratory Syndrome ◽

Research Finding ◽

Antiviral Drugs ◽

Host Cells ◽

Spike Protein ◽

Affinity Constant ◽

Humanized Antibody ◽

The World

SUMMARYCurrently, COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been widely spread around the world; nevertheless, so far there exist no specific antiviral drugs for treatment of the disease, which poses great challenge to control and contain the virus. Here, we reported a research finding that SARS-CoV-2 invaded host cells via a novel route of CD147-spike protein (SP). SP bound to CD147, a receptor on the host cells, thereby mediating the viral invasion. Our further research confirmed this finding. First, in vitro antiviral tests indicated Meplazumab, an anti-CD147 humanized antibody, significantly inhibited the viruses from invading host cells, with an EC50 of 24.86 μg/mL and IC50 of 15.16 μg/mL. Second, we validated the interaction between CD147 and SP, with an affinity constant of 1.85×10−7M. Co-Immunoprecipitation and ELISA also confirmed the binding of the two proteins. Finally, the localization of CD147 and SP was observed in SARS-CoV-2 infected Vero E6 cells by immuno-electron microscope. Therefore, the discovery of the new route CD147-SP for SARS-CoV-2 invading host cells provides a critical target for development of specific antiviral drugs.

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The SARS-CoV-2-Inactivating Activity of Hydroxytyrosol-Rich Aqueous Olive Pulp Extract (HIDROX®) and Its Use as a Virucidal Cream for Topical Application

Viruses ◽

10.3390/v13020232 ◽

2021 ◽

Vol 13 (2) ◽

pp. 232

Author(s):

Yohei Takeda ◽

Dulamjav Jamsransuren ◽

Sachiko Matsuda ◽

Roberto Crea ◽

Haruko Ogawa

Keyword(s):

Molecular Weight ◽

Severe Acute Respiratory Syndrome ◽

Viral Genome ◽

Structural Changes ◽

Control Measures ◽

Spike Protein ◽

Virucidal Activity ◽

Olive Pulp ◽

Hand Cream

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally. Although measures to control SARS-CoV-2, namely, vaccination, medication, and chemical disinfectants are being investigated, there is an increase in the demand for auxiliary antiviral approaches using natural compounds. Here we have focused on hydroxytyrosol (HT)-rich aqueous olive pulp extract (HIDROX®) and evaluated its SARS-CoV-2-inactivating activity in vitro. We showed that the HIDROX solution exhibits time- and concentration-dependent SARS-CoV-2-inactivating activities, and that HIDROX has more potent virucidal activity than pure HT. The evaluation of the mechanism of action suggested that both HIDROX and HT induced structural changes in SARS-CoV-2, which changed the molecular weight of the spike proteins. Even though the spike protein is highly glycosylated, this change was induced regardless of the glycosylation status. In addition, HIDROX or HT treatment disrupted the viral genome. Moreover, the HIDROX-containing cream applied on film showed time- and concentration-dependent SARS-CoV-2-inactivating activities. Thus, the HIDROX-containing cream can be applied topically as an antiviral hand cream. Our findings suggest that HIDROX contributes to improving SARS-CoV-2 control measures.

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Antibody cocktail to SARS-CoV-2 spike protein prevents rapid mutational escape seen with individual antibodies

Science ◽

10.1126/science.abd0831 ◽

2020 ◽

Vol 369 (6506) ◽

pp. 1014-1018 ◽

Cited By ~ 137

Author(s):

Alina Baum ◽

Benjamin O. Fulton ◽

Elzbieta Wloga ◽

Richard Copin ◽

Kristen E. Pascal ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Human Population ◽

Spike Protein ◽

Development Of Resistance ◽

Escape Mutants ◽

Antibody Resistance ◽

Antibody Cocktail

Antibodies targeting the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) present a promising approach to combat the coronavirus disease 2019 (COVID-19) pandemic; however, concerns remain that mutations can yield antibody resistance. We investigated the development of resistance against four antibodies to the spike protein that potently neutralize SARS-CoV-2, individually as well as when combined into cocktails. These antibodies remain effective against spike variants that have arisen in the human population. However, novel spike mutants rapidly appeared after in vitro passaging in the presence of individual antibodies, resulting in loss of neutralization; such escape also occurred with combinations of antibodies binding diverse but overlapping regions of the spike protein. Escape mutants were not generated after treatment with a noncompeting antibody cocktail.

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Potent Inhibition of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) by photosensitizers

10.21203/rs.3.rs-152985/v1 ◽

2021 ◽

Author(s):

Shujuan Yu ◽

Gaohui Sun ◽

Yaqun Sui ◽

Hanlin Li ◽

Ning Zhang ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Severe Acute Respiratory Syndrome ◽

Antiviral Treatment ◽

Spike Protein ◽

Converting Enzyme ◽

Viral Inactivation ◽

Angiotensin Converting Enzyme 2 ◽

Potent Inhibition

Abstract The pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has exploded since December 2019, and causes more than 2 million death with more than 95 million people infected as of Jan. 21th, 2021 globally1,2. Angiotensin-converting enzyme 2 (ACE2), expressed in the lungs, arteries, heart, kidney, intestines, and nasal epithelium3, has been shown to be the primary entry point targeted by the surface spike protein of SARS-CoV-2. Currently, no proven antiviral treatment for SARS-CoV-2 infection is available. In this study, we screened a number of photosensitizers for photodynamic viral inactivation, and found compounds pentalysine β-carbonylphthalocyanine zinc (ZnPc5K) and chlorin e6 (ce6) potently inhibited the viral infection and replication in vitro with half-maximal effective concentrations (EC50) values at nanomolar level. Such viral inactivation strategy is implementable, and has unique advantages, including resistance to virus mutations, affordability compared to the monoclonal antibodies, and lack of long-term toxicity.

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Interferons and viruses induce a novel primate-specific isoform dACE2 and not the SARS-CoV-2 receptor ACE2

10.1101/2020.07.19.210955 ◽

2020 ◽

Cited By ~ 7

Author(s):

Olusegun O. Onabajo ◽

A. Rouf Banday ◽

Wusheng Yan ◽

Adeola Obajemu ◽

Megan L. Stanifer ◽

...

Keyword(s):

Amino Acids ◽

Tumor Microenvironment ◽

Severe Acute Respiratory Syndrome ◽

Current Knowledge ◽

Spike Protein ◽

Target Cells ◽

Converting Enzyme ◽

Angiotensin Converting Enzyme 2 ◽

Terminal Amino

ABSTRACTSevere acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which causes COVID-19, utilizes angiotensin-converting enzyme 2 (ACE2) for entry into target cells. ACE2 has been proposed as an interferon-stimulated gene (ISG). Thus, interferon-induced variability in ACE2 expression levels could be important for susceptibility to COVID-19 or its outcomes. Here, we report the discovery of a novel, primate-specific isoform of ACE2, which we designate as deltaACE2 (dACE2). We demonstrate that dACE2, but not ACE2, is an ISG. In vitro, dACE2, which lacks 356 N-terminal amino acids, was non-functional in binding the SARS-CoV-2 spike protein and as a carboxypeptidase. Our results reconcile current knowledge on ACE2 expression and suggest that the ISG-type induction of dACE2 in IFN-high conditions created by treatments, inflammatory tumor microenvironment, or viral co-infections is unlikely to affect the cellular entry of SARS-CoV-2 and promote infection.

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An Exposed Domain in the Severe Acute Respiratory Syndrome Coronavirus Spike Protein Induces Neutralizing Antibodies

Journal of Virology ◽

10.1128/jvi.78.13.7217-7226.2004 ◽

2004 ◽

Vol 78 (13) ◽

pp. 7217-7226 ◽

Cited By ~ 59

Author(s):

Tong Zhou ◽

Hong Wang ◽

Danlin Luo ◽

Thomas Rowe ◽

Zheng Wang ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Monoclonal Antibodies ◽

Severe Acute Respiratory Syndrome ◽

Neutralizing Antibodies ◽

Vaccine Development ◽

Vero Cells ◽

Spike Protein ◽

Protein Fragment

ABSTRACT Exposed epitopes of the spike protein may be recognized by neutralizing antibodies against severe acute respiratory syndrome (SARS) coronavirus (CoV). A protein fragment (S-II) containing predicted epitopes of the spike protein was expressed in Escherichia coli. The properly refolded protein fragment specifically bound to the surface of Vero cells. Monoclonal antibodies raised against this fragment recognized the native spike protein of SARS CoV in both monomeric and trimeric forms. These monoclonal antibodies were capable of blocking S-II attachment to Vero cells and exhibited in vitro antiviral activity. These neutralizing antibodies mapped to epitopes in two peptides, each comprising 20 amino acids. Thus, this region of the spike protein might be a target for generation of therapeutic neutralizing antibodies against SARS CoV and for vaccine development to elicit protective humoral immunity.

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