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1.
EuropePMC; 2022.
Preprint in English | EuropePMC | ID: ppcovidwho-335225

ABSTRACT

SUMMARY The authorized mRNA- and adenovirus-based SARS-CoV-2 vaccines are intramuscularly injected and effective in preventing COVID-19, but do not induce efficient mucosal immunity, or prevent viral transmission. We developed a bacteriophage T4-based, multicomponent, needle and adjuvant-free, mucosal vaccine by engineering spike trimers on capsid exterior and nucleocapsid protein in the interior. Intranasal administration of T4-COVID vaccine induced higher virus neutralization antibody titers against multiple variants, balanced Th1/Th2 antibody and cytokine responses, stronger CD4 + and CD8 + T cell immunity, and higher secretory IgA titers in sera and bronchoalveolar lavage with no effect on the gut microbiota, compared to vaccination of mice intramuscularly. The vaccine is stable at ambient temperature, induces apparent sterilizing immunity, and provides complete protection against original SARS-CoV-2 strain and its Delta variant with minimal lung histopathology. This mucosal vaccine is an excellent candidate for boosting immunity of immunized and/or as a second-generation vaccine for the unimmunized population.

2.
EuropePMC; 2022.
Preprint in English | EuropePMC | ID: ppcovidwho-329211

ABSTRACT

Numerous mutations in the spike protein of SARS-CoV-2 B.1.1.529 Omicron variant pose a crisis for antibody-based immunotherapies. The efficacy of emergency use authorized (EUA) antibodies that developed in early SARS-CoV-2 pandemic seems to be in flounder. In this work, we examined the Omicron variant neutralization using an early B cell antibody repertoire as well as several EUA antibodies in pseudovirus and authentic virus systems. More than half of the antibodies in the repertoire that showed good activity against WA1/2020 previously had completely lost neutralizing activity against Omicron, while antibody 8G3 from our early B cell repertoire displayed non-regressive activity. EUA antibodies Etesevimab, Casirivimab, Imdevimab and Bamlanivimab neutralized authentic WA1/2020 virus with low half maximal inhibitory concentration (IC50) values, but were entirely desensitized by Omicron. Only Sotrovimab targeting the non-ACE2 overlap epitope showed activity but with a dramatic decrease. Interestingly, antibody 8G3 efficiently neutralized Omicron in pseudovirus and authentic virus systems. 8G3 also showed excellent activity against other variants of concern (VOCs). Collectively, our results suggest that neutralizing antibodies with breadth remains broad neutralizing activity in tackling SARS-CoV-2 infection despite the universal evasion from EUA antibodies by Omicron variant.

3.
Cell Discov ; 8(1): 16, 2022 Feb 15.
Article in English | MEDLINE | ID: covidwho-1692632

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) continue to wreak havoc across the globe. Higher transmissibility and immunologic resistance of VOCs bring unprecedented challenges to epidemic extinguishment. Here we describe a monoclonal antibody, 2G1, that neutralizes all current VOCs and has surprising tolerance to mutations adjacent to or within its interaction epitope. Cryo-electron microscopy structure showed that 2G1 bound to the tip of receptor binding domain (RBD) of spike protein with small contact interface but strong hydrophobic effect, which resulted in nanomolar to sub-nanomolar affinities to spike proteins. The epitope of 2G1 on RBD partially overlaps with angiotensin converting enzyme 2 (ACE2) interface, which enables 2G1 to block interaction between RBD and ACE2. The narrow binding epitope but high affinity bestow outstanding therapeutic efficacy upon 2G1 that neutralized VOCs with sub-nanomolar half maximal inhibitory concentration in vitro. In SARS-CoV-2, Beta or Delta variant-challenged transgenic mice and rhesus macaque models, 2G1 protected animals from clinical illness and eliminated viral burden, without serious impact to animal safety. Mutagenesis experiments suggest that 2G1 is potentially capable of dealing with emerging SARS-CoV-2 variants in the future. This report characterized the therapeutic antibodies specific to the tip of spike against SARS-CoV-2 variants and highlights the potential clinical applications as well as for developing vaccine and cocktail therapy.

4.
Viruses ; 14(1)2021 12 31.
Article in English | MEDLINE | ID: covidwho-1580398

ABSTRACT

We report the discovery of several highly potent small molecules with low-nM potency against severe acute respiratory syndrome coronavirus (SARS-CoV; lowest half-maximal inhibitory concentration (IC50: 13 nM), SARS-CoV-2 (IC50: 23 nM), and Middle East respiratory syndrome coronavirus (MERS-CoV; IC50: 76 nM) in pseudovirus-based assays with excellent selectivity index (SI) values (>5000), demonstrating potential pan-coronavirus inhibitory activities. Some compounds showed 100% inhibition against the cytopathic effects (CPE; IC100) of an authentic SARS-CoV-2 (US_WA-1/2020) variant at 1.25 µM. The most active inhibitors also potently inhibited variants of concern (VOCs), including the UK (B.1.1.7) and South African (B.1.351) variants and the Delta variant (B.1.617.2) originally identified in India in pseudovirus-based assay. Surface plasmon resonance (SPR) analysis with one potent inhibitor confirmed that it binds to the prefusion SARS-CoV-2 spike protein trimer. These small-molecule inhibitors prevented virus-mediated cell-cell fusion. The absorption, distribution, metabolism, and excretion (ADME) data for one of the most active inhibitors, NBCoV1, demonstrated drug-like properties. An in vivo pharmacokinetics (PK) study of NBCoV1 in rats demonstrated an excellent half-life (t1/2) of 11.3 h, a mean resident time (MRT) of 14.2 h, and oral bioavailability. We expect these lead inhibitors to facilitate the further development of preclinical and clinical candidates.


Subject(s)
Antiviral Agents/pharmacology , SARS-CoV-2/drug effects , Virus Internalization/drug effects , Animals , Antiviral Agents/chemistry , Antiviral Agents/pharmacokinetics , Biological Availability , Cell Line , Cell Survival/drug effects , Coronavirus/classification , Coronavirus/drug effects , HIV Fusion Inhibitors/chemistry , HIV Fusion Inhibitors/pharmacokinetics , HIV Fusion Inhibitors/pharmacology , Humans , Protein Binding , Rats , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacokinetics , Small Molecule Libraries/pharmacology , Spike Glycoprotein, Coronavirus/antagonists & inhibitors
5.
2021.
Preprint in English | Other preprints | ID: ppcovidwho-294541

ABSTRACT

ABSTRACT We report here the discovery of several highly potent small molecules that showed low nM potency against SARS-CoV (IC 50 : as low as 13 nM), SARS-CoV-2 (IC 50 : as low as 23 nM), and MERS-CoV (IC 50 : as low as 76 nM) in pseudovirus based assays with excellent selectivity indices (SI: as high as > 5000) demonstrating their pancoronavirus inhibition. Some compounds also show 100% inhibition of CPE (IC 100 ) at 1.25 µM against an authentic SARS-CoV-2 (US_WA-1/2020). Furthermore, the most active inhibitors also potently inhibited variants of concerns (VOCs), such as the UK (B.1.1.7), South Africa (B.1.351), and Delta variant (B.1.617.2), originated in India. We confirmed that one of the potent inhibitors binds to the prefusion spike protein trimer of SARS-CoV-2 by SPR. Besides, we showed that they inhibit virus-mediated cell-cell fusion. The ADME data of one of the most active inhibitors, NBCoV1, show drug-like properties. In vivo PK of NBCoV1 in rats demonstrated excellent half-life (t1/2) of 11.3 h, mean resident time (MRT) of 14.2 h, and oral bioavailability. We expect the lead inhibitors to pave the way for further development to preclinical and clinical candidates.

6.
J Med Chem ; 65(4): 2956-2970, 2022 02 24.
Article in English | MEDLINE | ID: covidwho-1500413

ABSTRACT

Cathepsin L is a key host cysteine protease utilized by coronaviruses for cell entry and is a promising drug target for novel antivirals against SARS-CoV-2. The marine natural product gallinamide A and several synthetic analogues were identified as potent inhibitors of cathepsin L with IC50 values in the picomolar range. Lead molecules possessed selectivity over other cathepsins and alternative host proteases involved in viral entry. Gallinamide A directly interacted with cathepsin L in cells and, together with two lead analogues, potently inhibited SARS-CoV-2 infection in vitro, with EC50 values in the nanomolar range. Reduced antiviral activity was observed in cells overexpressing transmembrane protease, serine 2 (TMPRSS2); however, a synergistic improvement in antiviral activity was achieved when combined with a TMPRSS2 inhibitor. These data highlight the potential of cathepsin L as a COVID-19 drug target as well as the likely need to inhibit multiple routes of viral entry to achieve efficacy.


Subject(s)
Antimicrobial Cationic Peptides/pharmacology , Antiviral Agents/pharmacology , Biological Products/pharmacology , COVID-19/drug therapy , Cathepsin L/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , SARS-CoV-2/drug effects , A549 Cells , Animals , Antimicrobial Cationic Peptides/chemical synthesis , Antimicrobial Cationic Peptides/chemistry , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , Biological Products/chemical synthesis , Biological Products/chemistry , COVID-19/metabolism , Cathepsin L/metabolism , Chlorocebus aethiops , Cysteine Proteinase Inhibitors/chemical synthesis , Cysteine Proteinase Inhibitors/chemistry , Dose-Response Relationship, Drug , Humans , Microbial Sensitivity Tests , Molecular Conformation , Proteomics , Structure-Activity Relationship , Vero Cells
7.
Sci Adv ; 7(37): eabh1547, 2021 Sep 10.
Article in English | MEDLINE | ID: covidwho-1405213

ABSTRACT

A "universal" platform that can rapidly generate multiplex vaccine candidates is critically needed to control pandemics. Using the severe acute respiratory syndrome coronavirus 2 as a model, we have developed such a platform by CRISPR engineering of bacteriophage T4. A pipeline of vaccine candidates was engineered by incorporating various viral components into appropriate compartments of phage nanoparticle structure. These include expressible spike genes in genome, spike and envelope epitopes as surface decorations, and nucleocapsid proteins in packaged core. Phage decorated with spike trimers was found to be the most potent vaccine candidate in animal models. Without any adjuvant, this vaccine stimulated robust immune responses, both T helper cell 1 (TH1) and TH2 immunoglobulin G subclasses, blocked virus-receptor interactions, neutralized viral infection, and conferred complete protection against viral challenge. This new nanovaccine design framework might allow the rapid deployment of effective adjuvant-free phage-based vaccines against any emerging pathogen in the future.

8.
PLoS Pathog ; 17(9): e1009897, 2021 09.
Article in English | MEDLINE | ID: covidwho-1398941

ABSTRACT

The key to battling the COVID-19 pandemic and its potential aftermath is to develop a variety of vaccines that are efficacious and safe, elicit lasting immunity, and cover a range of SARS-CoV-2 variants. Recombinant viral receptor-binding domains (RBDs) are safe vaccine candidates but often have limited efficacy due to the lack of virus-like immunogen display pattern. Here we have developed a novel virus-like nanoparticle (VLP) vaccine that displays 120 copies of SARS-CoV-2 RBD on its surface. This VLP-RBD vaccine mimics virus-based vaccines in immunogen display, which boosts its efficacy, while maintaining the safety of protein-based subunit vaccines. Compared to the RBD vaccine, the VLP-RBD vaccine induced five times more neutralizing antibodies in mice that efficiently blocked SARS-CoV-2 from attaching to its host receptor and potently neutralized the cell entry of variant SARS-CoV-2 strains, SARS-CoV-1, and SARS-CoV-1-related bat coronavirus. These neutralizing immune responses induced by the VLP-RBD vaccine did not wane during the two-month study period. Furthermore, the VLP-RBD vaccine effectively protected mice from SARS-CoV-2 challenge, dramatically reducing the development of clinical signs and pathological changes in immunized mice. The VLP-RBD vaccine provides one potentially effective solution to controlling the spread of SARS-CoV-2.


Subject(s)
COVID-19 Vaccines/immunology , COVID-19/immunology , COVID-19/prevention & control , Immunogenicity, Vaccine , Nanoparticles/therapeutic use , Angiotensin-Converting Enzyme 2/immunology , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Disease Models, Animal , Drug Design , Female , HEK293 Cells , Humans , Lung/virology , Mice , Mice, Inbred BALB C , Protein Domains/immunology
9.
ACS Chem Biol ; 16(4): 642-650, 2021 04 16.
Article in English | MEDLINE | ID: covidwho-1387141

ABSTRACT

Host-cell cysteine proteases play an essential role in the processing of the viral spike protein of SARS coronaviruses. K777, an irreversible, covalent inactivator of cysteine proteases that has recently completed phase 1 clinical trials, reduced SARS-CoV-2 viral infectivity in several host cells: Vero E6 (EC50< 74 nM), HeLa/ACE2 (4 nM), Caco-2 (EC90 = 4.3 µM), and A549/ACE2 (<80 nM). Infectivity of Calu-3 cells depended on the cell line assayed. If Calu-3/2B4 was used, EC50 was 7 nM, but in the ATCC Calu-3 cell line without ACE2 enrichment, EC50 was >10 µM. There was no toxicity to any of the host cell lines at 10-100 µM K777 concentration. Kinetic analysis confirmed that K777 was a potent inhibitor of human cathepsin L, whereas no inhibition of the SARS-CoV-2 cysteine proteases (papain-like and 3CL-like protease) was observed. Treatment of Vero E6 cells with a propargyl derivative of K777 as an activity-based probe identified human cathepsin B and cathepsin L as the intracellular targets of this molecule in both infected and uninfected Vero E6 cells. However, cleavage of the SARS-CoV-2 spike protein was only carried out by cathepsin L. This cleavage was blocked by K777 and occurred in the S1 domain of the SARS-CoV-2 spike protein, a different site from that previously observed for the SARS-CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of cathepsin L-mediated viral spike protein processing.


Subject(s)
Antiviral Agents/pharmacology , Cysteine Proteinase Inhibitors/pharmacology , Phenylalanine/pharmacology , Piperazines/pharmacology , SARS-CoV-2/drug effects , Tosyl Compounds/pharmacology , Animals , Cathepsin L/antagonists & inhibitors , Cathepsin L/metabolism , Cell Line, Tumor , Chlorocebus aethiops , Humans , Microbial Sensitivity Tests , Protein Domains , Proteolysis , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells , Virus Internalization/drug effects
10.
mBio ; 11(6)2020 12 11.
Article in English | MEDLINE | ID: covidwho-1388458

ABSTRACT

SARS-CoV-2 uses human angiotensin-converting enzyme 2 (ACE2) as the primary receptor to enter host cells and initiate the infection. The critical binding region of ACE2 is an ∼30-amino-acid (aa)-long helix. Here, we report the design of four stapled peptides based on the ACE2 helix, which is expected to bind to SARS-CoV-2 and prevent the binding of the virus to the ACE2 receptor and disrupt the infection. All stapled peptides showed high helical contents (50 to 94% helicity). In contrast, the linear control peptide NYBSP-C showed no helicity (19%). We have evaluated the peptides in a pseudovirus-based single-cycle assay in HT1080/ACE2 cells and human lung cell line A549/ACE2, overexpressing ACE2. Three of the four stapled peptides showed potent antiviral activity in HT1080/ACE2 (50% inhibitory concentration [IC50]: 1.9 to 4.1 µM) and A549/ACE2 (IC50: 2.2 to 2.8 µM) cells. The linear peptide NYBSP-C and the double-stapled peptide StRIP16, used as controls, showed no antiviral activity. Most significantly, none of the stapled peptides show any cytotoxicity at the highest dose tested. We also evaluated the antiviral activity of the peptides by infecting Vero E6 cells with the replication-competent authentic SARS-CoV-2 (US_WA-1/2020). NYBSP-1 was the most efficient, preventing the complete formation of cytopathic effects (CPEs) at an IC100 of 17.2 µM. NYBSP-2 and NYBSP-4 also prevented the formation of the virus-induced CPE with an IC100 of about 33 µM. We determined the proteolytic stability of one of the most active stapled peptides, NYBSP-4, in human plasma, which showed a half-life (T 1/2) of >289 min.IMPORTANCE SARS-CoV-2 is a novel virus with many unknowns. No vaccine or specific therapy is available yet to prevent and treat this deadly virus. Therefore, there is an urgent need to develop novel therapeutics. Structural studies revealed critical interactions between the binding site helix of the ACE2 receptor and SARS-CoV-2 receptor-binding domain (RBD). Therefore, targeting the entry pathway of SARS-CoV-2 is ideal for both prevention and treatment as it blocks the first step of the viral life cycle. We report the design of four double-stapled peptides, three of which showed potent antiviral activity in HT1080/ACE2 cells and human lung carcinoma cells, A549/ACE2. Most significantly, the active stapled peptides with antiviral activity against SARS-CoV-2 showed high α-helicity (60 to 94%). The most active stapled peptide, NYBSP-4, showed substantial resistance to degradation by proteolytic enzymes in human plasma. The lead stapled peptides are expected to pave the way for further optimization of a clinical candidate.


Subject(s)
Angiotensin-Converting Enzyme 2/chemistry , Peptides/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Virus Attachment/drug effects , A549 Cells , Animals , Binding Sites , Chlorocebus aethiops , Humans , Inhibitory Concentration 50 , Peptides/chemical synthesis , Protein Binding , Vero Cells
11.
J Med Chem ; 64(15): 11267-11287, 2021 08 12.
Article in English | MEDLINE | ID: covidwho-1319012

ABSTRACT

Cysteine proteases comprise an important class of drug targets, especially for infectious diseases such as Chagas disease (cruzain) and COVID-19 (3CL protease, cathepsin L). Peptide aldehydes have proven to be potent inhibitors for all of these proteases. However, the intrinsic, high electrophilicity of the aldehyde group is associated with safety concerns and metabolic instability, limiting the use of aldehyde inhibitors as drugs. We have developed a novel class of self-masked aldehyde inhibitors (SMAIs) for cruzain, the major cysteine protease of the causative agent of Chagas disease-Trypanosoma cruzi. These SMAIs exerted potent, reversible inhibition of cruzain (Ki* = 18-350 nM) while apparently protecting the free aldehyde in cell-based assays. We synthesized prodrugs of the SMAIs that could potentially improve their pharmacokinetic properties. We also elucidated the kinetic and chemical mechanism of SMAIs and applied this strategy to the design of anti-SARS-CoV-2 inhibitors.


Subject(s)
Aldehydes/chemistry , COVID-19/drug therapy , Chagas Disease/drug therapy , Cysteine Proteinase Inhibitors/therapeutic use , SARS-CoV-2/enzymology , Trypanosoma cruzi/enzymology , Aldehydes/metabolism , Aldehydes/pharmacology , Cathepsin L/antagonists & inhibitors , Cathepsin L/metabolism , Cysteine Endopeptidases/metabolism , Cysteine Proteases/metabolism , Cysteine Proteinase Inhibitors/chemistry , Drug Design , Humans , Kinetics , Models, Molecular , Molecular Structure , Protozoan Proteins/antagonists & inhibitors , Protozoan Proteins/metabolism , SARS-CoV-2/drug effects , Structure-Activity Relationship , Trypanosoma cruzi/drug effects
12.
ACS Chem Biol ; 16(4): 642-650, 2021 04 16.
Article in English | MEDLINE | ID: covidwho-1160225

ABSTRACT

Host-cell cysteine proteases play an essential role in the processing of the viral spike protein of SARS coronaviruses. K777, an irreversible, covalent inactivator of cysteine proteases that has recently completed phase 1 clinical trials, reduced SARS-CoV-2 viral infectivity in several host cells: Vero E6 (EC50< 74 nM), HeLa/ACE2 (4 nM), Caco-2 (EC90 = 4.3 µM), and A549/ACE2 (<80 nM). Infectivity of Calu-3 cells depended on the cell line assayed. If Calu-3/2B4 was used, EC50 was 7 nM, but in the ATCC Calu-3 cell line without ACE2 enrichment, EC50 was >10 µM. There was no toxicity to any of the host cell lines at 10-100 µM K777 concentration. Kinetic analysis confirmed that K777 was a potent inhibitor of human cathepsin L, whereas no inhibition of the SARS-CoV-2 cysteine proteases (papain-like and 3CL-like protease) was observed. Treatment of Vero E6 cells with a propargyl derivative of K777 as an activity-based probe identified human cathepsin B and cathepsin L as the intracellular targets of this molecule in both infected and uninfected Vero E6 cells. However, cleavage of the SARS-CoV-2 spike protein was only carried out by cathepsin L. This cleavage was blocked by K777 and occurred in the S1 domain of the SARS-CoV-2 spike protein, a different site from that previously observed for the SARS-CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of cathepsin L-mediated viral spike protein processing.


Subject(s)
Antiviral Agents/pharmacology , Cysteine Proteinase Inhibitors/pharmacology , Phenylalanine/pharmacology , Piperazines/pharmacology , SARS-CoV-2/drug effects , Tosyl Compounds/pharmacology , Animals , Cathepsin L/antagonists & inhibitors , Cathepsin L/metabolism , Cell Line, Tumor , Chlorocebus aethiops , Humans , Microbial Sensitivity Tests , Protein Domains , Proteolysis , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells , Virus Internalization/drug effects
13.
Proc Natl Acad Sci U S A ; 118(10)2021 03 09.
Article in English | MEDLINE | ID: covidwho-1087557

ABSTRACT

Guided by a computational docking analysis, about 30 Food and Drug Administration/European Medicines Agency (FDA/EMA)-approved small-molecule medicines were characterized on their inhibition of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) main protease (Mp ro). Of these small molecules tested, six displayed a concentration that inhibits response by 50% (IC50) value below 100 µM in inhibiting Mp ro, and, importantly, three, that is, pimozide, ebastine, and bepridil, are basic molecules that potentiate dual functions by both raising endosomal pH to interfere with SARS-CoV-2 entry into the human cell host and inhibiting Mp ro in infected cells. A live virus-based modified microneutralization assay revealed that bepridil possesses significant anti-SARS-CoV-2 activity in both Vero E6 and A459/ACE2 cells in a dose-dependent manner with low micromolar effective concentration, 50% (EC50) values. Therefore, the current study urges serious considerations of using bepridil in COVID-19 clinical tests.


Subject(s)
Antiviral Agents/pharmacology , Bepridil/pharmacology , Drug Discovery , SARS-CoV-2/drug effects , A549 Cells , Animals , Chlorocebus aethiops , Humans , Molecular Docking Simulation , Molecular Structure , Small Molecule Libraries , Vero Cells
14.
ChemMedChem ; 16(6): 942-948, 2021 03 18.
Article in English | MEDLINE | ID: covidwho-959133

ABSTRACT

The COVID-19 pathogen, SARS-CoV-2, requires its main protease (SC2MPro ) to digest two of its translated long polypeptides to form a number of mature proteins that are essential for viral replication and pathogenesis. Inhibition of this vital proteolytic process is effective in preventing the virus from replicating in infected cells and therefore provides a potential COVID-19 treatment option. Guided by previous medicinal chemistry studies about SARS-CoV-1 main protease (SC1MPro ), we have designed and synthesized a series of SC2MPro inhibitors that contain ß-(S-2-oxopyrrolidin-3-yl)-alaninal (Opal) for the formation of a reversible covalent bond with the SC2MPro active-site cysteine C145. All inhibitors display high potency with Ki values at or below 100 nM. The most potent compound, MPI3, has as a Ki value of 8.3 nM. Crystallographic analyses of SC2MPro bound to seven inhibitors indicated both formation of a covalent bond with C145 and structural rearrangement from the apoenzyme to accommodate the inhibitors. Virus inhibition assays revealed that several inhibitors have high potency in inhibiting the SARS-CoV-2-induced cytopathogenic effect in both Vero E6 and A549/ACE2 cells. Two inhibitors, MPI5 and MPI8, completely prevented the SARS-CoV-2-induced cytopathogenic effect in Vero E6 cells at 2.5-5 µM and A549/ACE2 cells at 0.16-0.31 µM. Their virus inhibition potency is much higher than that of some existing molecules that are under preclinical and clinical investigations for the treatment of COVID-19. Our study indicates that there is a large chemical space that needs to be explored for the development of SC2MPro inhibitors with ultra-high antiviral potency.


Subject(s)
Antiviral Agents/pharmacology , Coronavirus 3C Proteases/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , SARS-CoV-2/drug effects , A549 Cells , Alanine/analogs & derivatives , Alanine/metabolism , Alanine/pharmacology , Animals , Antiviral Agents/chemical synthesis , Antiviral Agents/metabolism , Catalytic Domain , Chlorocebus aethiops , Coronavirus 3C Proteases/chemistry , Coronavirus 3C Proteases/metabolism , Cysteine/chemistry , Cysteine Proteinase Inhibitors/chemical synthesis , Cysteine Proteinase Inhibitors/metabolism , Humans , Microbial Sensitivity Tests , Protein Binding , Pyrrolidinones/chemical synthesis , Pyrrolidinones/metabolism , Pyrrolidinones/pharmacology , SARS-CoV-2/enzymology , Vero Cells
15.
bioRxiv ; 2020 Oct 30.
Article in English | MEDLINE | ID: covidwho-915969

ABSTRACT

K777 is a di-peptide analog that contains an electrophilic vinyl-sulfone moiety and is a potent, covalent inactivator of cathepsins. Vero E6, HeLa/ACE2, Caco-2, A549/ACE2, and Calu-3, cells were exposed to SARS-CoV-2, and then treated with K777. K777 reduced viral infectivity with EC50 values of inhibition of viral infection of: 74 nM for Vero E6, <80 nM for A549/ACE2, and 4 nM for HeLa/ACE2 cells. In contrast, Calu-3 and Caco-2 cells had EC50 values in the low micromolar range. No toxicity of K777 was observed for any of the host cells at 10-100 µM inhibitor. K777 did not inhibit activity of the papain-like cysteine protease and 3CL cysteine protease, encoded by SARS-CoV-2 at concentrations of ≤ 100 µM. These results suggested that K777 exerts its potent anti-viral activity by inactivation of mammalian cysteine proteases which are essential to viral infectivity. Using a propargyl derivative of K777 as an activity-based probe, K777 selectively targeted cathepsin B and cathepsin L in Vero E6 cells. However only cathepsin L cleaved the SARS-CoV-2 spike protein and K777 blocked this proteolysis. The site of spike protein cleavage by cathepsin L was in the S1 domain of SARS-CoV-2 , differing from the cleavage site observed in the SARS CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of viral spike protein processing.

16.
Proc Natl Acad Sci U S A ; 117(47): 29832-29838, 2020 11 24.
Article in English | MEDLINE | ID: covidwho-900111

ABSTRACT

Effective therapies are urgently needed for the SARS-CoV-2/COVID-19 pandemic. We identified panels of fully human monoclonal antibodies (mAbs) from large phage-displayed Fab, scFv, and VH libraries by panning against the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) glycoprotein. A high-affinity Fab was selected from one of the libraries and converted to a full-size antibody, IgG1 ab1, which competed with human ACE2 for binding to RBD. It potently neutralized replication-competent SARS-CoV-2 but not SARS-CoV, as measured by two different tissue culture assays, as well as a replication-competent mouse ACE2-adapted SARS-CoV-2 in BALB/c mice and native virus in hACE2-expressing transgenic mice showing activity at the lowest tested dose of 2 mg/kg. IgG1 ab1 also exhibited high prophylactic and therapeutic efficacy in a hamster model of SARS-CoV-2 infection. The mechanism of neutralization is by competition with ACE2 but could involve antibody-dependent cellular cytotoxicity (ADCC) as IgG1 ab1 had ADCC activity in vitro. The ab1 sequence has a relatively low number of somatic mutations, indicating that ab1-like antibodies could be quickly elicited during natural SARS-CoV-2 infection or by RBD-based vaccines. IgG1 ab1 did not aggregate, did not exhibit other developability liabilities, and did not bind to any of the 5,300 human membrane-associated proteins tested. These results suggest that IgG1 ab1 has potential for therapy and prophylaxis of SARS-CoV-2 infections. The rapid identification (within 6 d of availability of antigen for panning) of potent mAbs shows the value of large antibody libraries for response to public health threats from emerging microbes.


Subject(s)
COVID-19 Serological Testing/methods , COVID-19 Vaccines/immunology , COVID-19/therapy , Angiotensin-Converting Enzyme 2/metabolism , Animals , Antibodies, Viral/blood , Antibodies, Viral/immunology , Antibody-Dependent Cell Cytotoxicity , COVID-19 Serological Testing/standards , COVID-19 Vaccines/standards , Chlorocebus aethiops , Cricetinae , Female , Humans , Immunization, Passive/methods , Immunization, Passive/standards , Immunogenicity, Vaccine , Immunoglobulin G/blood , Immunoglobulin G/immunology , Mice , Mice, Inbred BALB C , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology , Vero Cells
17.
Cell ; 183(2): 429-441.e16, 2020 10 15.
Article in English | MEDLINE | ID: covidwho-878393

ABSTRACT

Novel COVID-19 therapeutics are urgently needed. We generated a phage-displayed human antibody VH domain library from which we identified a high-affinity VH binder ab8. Bivalent VH, VH-Fc ab8, bound with high avidity to membrane-associated S glycoprotein and to mutants found in patients. It potently neutralized mouse-adapted SARS-CoV-2 in wild-type mice at a dose as low as 2 mg/kg and exhibited high prophylactic and therapeutic efficacy in a hamster model of SARS-CoV-2 infection, possibly enhanced by its relatively small size. Electron microscopy combined with scanning mutagenesis identified ab8 interactions with all three S protomers and showed how ab8 neutralized the virus by directly interfering with ACE2 binding. VH-Fc ab8 did not aggregate and did not bind to 5,300 human membrane-associated proteins. The potent neutralization activity of VH-Fc ab8 combined with good developability properties and cross-reactivity to SARS-CoV-2 mutants provide a strong rationale for its evaluation as a COVID-19 therapeutic.


Subject(s)
Coronavirus Infections/drug therapy , Immunoglobulin Heavy Chains/administration & dosage , Immunoglobulin Variable Region/administration & dosage , Peptide Library , Pneumonia, Viral/drug therapy , Angiotensin-Converting Enzyme 2 , Animals , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/ultrastructure , Antibodies, Viral/administration & dosage , Antibodies, Viral/chemistry , Antibodies, Viral/immunology , Antibodies, Viral/ultrastructure , Antibody Affinity , COVID-19 , Cricetinae , Female , Humans , Immunoglobulin Fc Fragments/immunology , Immunoglobulin Heavy Chains/chemistry , Immunoglobulin Heavy Chains/immunology , Immunoglobulin Heavy Chains/ultrastructure , Immunoglobulin Variable Region/chemistry , Immunoglobulin Variable Region/immunology , Immunoglobulin Variable Region/ultrastructure , Mice , Mice, Inbred BALB C , Mutation , Pandemics , Peptidyl-Dipeptidase A/metabolism , Protein Domains , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Spike Glycoprotein, Coronavirus/ultrastructure
18.
Vaccine ; 38(46): 7205-7212, 2020 10 27.
Article in English | MEDLINE | ID: covidwho-779733

ABSTRACT

The development of an effective vaccine against SARS-CoV-2 is urgently needed. We generated SARS-CoV-2 RBD-Fc fusion protein and evaluated its potency to elicit neutralizing antibody response in mice. RBD-Fc elicited a higher neutralizing antibodies titer than RBD as evaluated by a pseudovirus neutralization assay and a live virus based microneutralization assay. Furthermore, RBD-Fc immunized sera better inhibited cell-cell fusion, as evaluated by a quantitative cell-cell fusion assay. The cell-cell fusion assay results correlated well with the virus neutralization potency and could be used for high-throughput screening of large panels of anti-SARS-CoV-2 antibodies and vaccines without the requirement of live virus infection in BSL3 containment. Moreover, the anti-RBD sera did not enhance the pseudotyped SARS-CoV-2 infection of K562 cells. These results demonstrate that Fc fusion can significantly improve the humoral immune response to recombinant RBD immunogen, and suggest that RBD-Fc could serve as a useful component of effective vaccines against SARS-CoV-2.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Coronavirus Infections/prevention & control , Immunoglobulin Fc Fragments/immunology , Pandemics/prevention & control , Pneumonia, Viral/prevention & control , Spike Glycoprotein, Coronavirus/immunology , Viral Vaccines/immunology , Angiotensin-Converting Enzyme 2 , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Betacoronavirus/immunology , COVID-19 , COVID-19 Vaccines , Cell Fusion , Cell Line , Coronavirus Infections/immunology , Enzyme-Linked Immunosorbent Assay , Female , HEK293 Cells , High-Throughput Screening Assays/methods , Humans , Immunity, Humoral/immunology , Immunoglobulin Fc Fragments/genetics , Mice , Mice, Inbred BALB C , Neutralization Tests , Peptidyl-Dipeptidase A/genetics , Protein Domains/immunology , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , SARS-CoV-2 , Spike Glycoprotein, Coronavirus/genetics , Vaccines, Subunit/immunology
20.
MAbs ; 12(1): 1778435, 2020 01 01.
Article in English | MEDLINE | ID: covidwho-601168

ABSTRACT

Effective therapies are urgently needed for COVID-19. Here we describe the identification of a new stable human immunoglobulin G1 heavy-chain variable (VH) domain scaffold that was used for the construction of a large library, lCAT6, of engineered human VHs. This library was panned against the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) glycoprotein. Two VH domains (VH ab6 and VH m397) were selected and fused to Fc for increased half-life in circulation. The VH-Fc ab6 and m397 specifically neutralized SARS-CoV-2 with high potencies (50% neutralization at 0.35 µg/ml and 1.5 µg/ml, respectively) as measured by two independent replication-competent virus neutralization assays. Ab6 and m397 competed with ACE2 for binding to RBD, suggesting a competitive mechanism of virus neutralization. These VH domains may have potential applications for prophylaxis and therapy of COVID-19 alone or in combination, as well as for diagnosis and as tools for research.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Betacoronavirus/immunology , Coronavirus Infections , Pandemics , Pneumonia, Viral , Single-Domain Antibodies/immunology , Antibodies, Monoclonal , COVID-19 , Humans , Immunoglobulin Heavy Chains/immunology , Peptide Library , SARS-CoV-2
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