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1.
Nat Commun ; 13(1): 1891, 2022 Apr 07.
Article in English | MEDLINE | ID: covidwho-1783979

ABSTRACT

The SARS-CoV-2 3CL protease is a critical drug target for small molecule COVID-19 therapy, given its likely druggability and essentiality in the viral maturation and replication cycle. Based on the conservation of 3CL protease substrate binding pockets across coronaviruses and using screening, we identified four structurally distinct lead compounds that inhibit SARS-CoV-2 3CL protease. After evaluation of their binding specificity, cellular antiviral potency, metabolic stability, and water solubility, we prioritized the GC376 scaffold as being optimal for optimization. We identified multiple drug-like compounds with <10 nM potency for inhibiting SARS-CoV-2 3CL and the ability to block SARS-CoV-2 replication in human cells, obtained co-crystal structures of the 3CL protease in complex with these compounds, and determined that they have pan-coronavirus activity. We selected one compound, termed coronastat, as an optimized lead and characterized it in pharmacokinetic and safety studies in vivo. Coronastat represents a new candidate for a small molecule protease inhibitor for the treatment of SARS-CoV-2 infection for eliminating pandemics involving coronaviruses.


Subject(s)
COVID-19 , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , COVID-19/drug therapy , Cysteine Endopeptidases/chemistry , Humans , Molecular Docking Simulation , Pandemics , Peptide Hydrolases , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Protease Inhibitors/therapeutic use , SARS-CoV-2
2.
Microbiology & infectious diseases (Wilmington, Del.) ; 5(2), 2021.
Article in English | EuropePMC | ID: covidwho-1738196

ABSTRACT

SARS-CoV-2, the novel coronavirus responsible for the COVID-19 pandemic, caused >26 million cases in the United States and >437,000 deaths as of Jan 30, 2020. Worldwide by that date, there had been 102 million cases of infections, and deaths had climbed to 2.21 million. Mutated variants of SARS-CoV-2 that have emerged from the United Kingdom, Brazil, and South Africa are associated with higher transmission rates and associated deaths. Therefore, novel therapeutic and prophylactic methods against SARS-CoV-2 are in urgent need. While some antiviral drugs, such as Remdesivir, provide relief to certain patient populations, other existing antiviral drugs or combinations of FDA approved pharmaceuticals have yet to show clinical efficacy against COVID-19. Compounds that possess strong and broad antiviral properties with different mechanisms of action against respiratory viruses may provide novel approaches to combat SARS-CoV-2 and its variants, especially if the compounds are classified as generally recognized as safe (GRAS). A large body of evidence indicates a promising potential for the use of epigallocatechin-3-gallate (EGCG) and its derivatives as effective agents against infections from a wide range of pathogenic viruses. However, EGCG or its derivatives have not been tested directly against SARS-CoV-2. The current study was designed to evaluate the potential antiviral activity of EGCG against SARS-CoV-2 infection in primate epithelial cells. Methods applied in the study include cytopathic effect (CPE) assay and virus yield reduction (VYR) assays using Vero 76 (green monkey epithelial cells) and Caco-2 (human epithelial cells) cell lines, respectively. The results demonstrated that EGCG at 0.27 μg/ml (0.59 μM) inhibited SARS-CoV-2 infection in Vero 76 cells by 50% (i.e., EC50=0.27 μg/ml). EGCG also inhibited SARS-CoV-2 infection in Caco-2 cells with EC90=28 μg/ml (61 μM). These results, to the best of our knowledge, are the first observations on the antiviral activities of EGCG against SARS-CoV-2, and suggest that EGCG and its derivatives could be used to combat COVID-19 and other respiratory viral infection-induced illness, pending in vivo and clinical studies.

3.
Science ; 374(6575): 1586-1593, 2021 Dec 24.
Article in English | MEDLINE | ID: covidwho-1666355

ABSTRACT

The worldwide outbreak of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic. Alongside vaccines, antiviral therapeutics are an important part of the healthcare response to countering the ongoing threat presented by COVID-19. Here, we report the discovery and characterization of PF-07321332, an orally bioavailable SARS-CoV-2 main protease inhibitor with in vitro pan-human coronavirus antiviral activity and excellent off-target selectivity and in vivo safety profiles. PF-07321332 has demonstrated oral activity in a mouse-adapted SARS-CoV-2 model and has achieved oral plasma concentrations exceeding the in vitro antiviral cell potency in a phase 1 clinical trial in healthy human participants.


Subject(s)
COVID-19/drug therapy , Lactams/pharmacology , Lactams/therapeutic use , Leucine/pharmacology , Leucine/therapeutic use , Nitriles/pharmacology , Nitriles/therapeutic use , Proline/pharmacology , Proline/therapeutic use , SARS-CoV-2/drug effects , Viral Protease Inhibitors/pharmacology , Viral Protease Inhibitors/therapeutic use , Administration, Oral , Animals , COVID-19/virology , Clinical Trials, Phase I as Topic , Coronavirus/drug effects , Disease Models, Animal , Drug Therapy, Combination , Humans , Lactams/administration & dosage , Lactams/pharmacokinetics , Leucine/administration & dosage , Leucine/pharmacokinetics , Mice , Mice, Inbred BALB C , Microbial Sensitivity Tests , Nitriles/administration & dosage , Nitriles/pharmacokinetics , Proline/administration & dosage , Proline/pharmacokinetics , Randomized Controlled Trials as Topic , Ritonavir/administration & dosage , Ritonavir/therapeutic use , SARS-CoV-2/physiology , Viral Protease Inhibitors/administration & dosage , Viral Protease Inhibitors/pharmacokinetics , Virus Replication/drug effects
4.
Arch Pharmacol Ther ; 3(2): 52-65, 2021.
Article in English | MEDLINE | ID: covidwho-1515741

ABSTRACT

BACKGROUND: Inhaled budesonide benefits patients with COVID-19. ProLung™-budesonide enables the sustained, low dose administration of budesonide within a delivery vehicle similar to lung surfactant. ProLung™-budesonide may offer anti-inflammatory and protective effects to the lung in COVID-19, yet it's effect on SARS-CoV-2 replication is unknown. OBJECTIVE: To determine the efficacy of ProLung™-budesonide against SARS-CoV-2-infection in vitro, evaluate its ability to decrease inflammation, and airway hyperresponsiveness in an animal model of lung inflammation. METHODS: SARS-CoV-2-infected Vero 76 cells were treated with ProLung™-budesonide ([0.03-100 µg/ml]) for 3 days, and virus yield in the supernatant was measured. Ovalbumin-sensitized C57BL/6 mice received aerosolized (a) ProLung™-budesonide weekly, (b) only budesonide, either daily or weekly, or (c) weekly empty ProLung™ carrier (without budesonide). All treatment groups were compared to sensitized untreated, or normal mice using histopathologic examination, electron microscopy (EM), airway hyperresponsiveness (AHR) to Methacholine (Mch) challenge, and eosinophil peroxidase activity (EPO) measurements in bronchioalveolar lavage (BAL). RESULTS: ProLung™-budesonide showed significant inhibition of viral replication of SARS-CoV-2-infected cells with the selectivity index (SI) value >24. Weekly ProLung™-budesonide and daily budesonide therapy significantly decreased lung inflammation and EPO in BAL. ProLung™-budesonide localized in type II pneumocytes, and was the only group to significantly decrease AHR, and EPO in BAL with Mch challenge. CONCLUSIONS: ProLung™-budesonide significantly inhibited viral replication in SARS-CoV-2-infected cells. It localized into type II pneumocytes, decreased lung inflammation, AHR and EPO activity with Mch challenge. This novel drug formulation may offer a potential inhalational treatment for COVID-19.

5.
Sci Rep ; 11(1): 20595, 2021 10 18.
Article in English | MEDLINE | ID: covidwho-1475487

ABSTRACT

The delivery of safe, visible wavelengths of light can be an effective, pathogen-agnostic, countermeasure that would expand the current portfolio of SARS-CoV-2 intervention strategies beyond the conventional approaches of vaccine, antibody, and antiviral therapeutics. Employing custom biological light units, that incorporate optically engineered light-emitting diode (LED) arrays, we harnessed monochromatic wavelengths of light for uniform delivery across biological surfaces. We demonstrated that primary 3D human tracheal/bronchial-derived epithelial tissues tolerated high doses of a narrow spectral band of visible light centered at a peak wavelength of 425 nm. We extended these studies to Vero E6 cells to understand how light may influence the viability of a mammalian cell line conventionally used for assaying SARS-CoV-2. The exposure of single-cell monolayers of Vero E6 cells to similar doses of 425 nm blue light resulted in viabilities that were dependent on dose and cell density. Doses of 425 nm blue light that are well-tolerated by Vero E6 cells also inhibited infection and replication of cell-associated SARS-CoV-2 by > 99% 24 h post-infection after a single five-minute light exposure. Moreover, the 425 nm blue light inactivated cell-free betacoronaviruses including SARS-CoV-1, MERS-CoV, and SARS-CoV-2 up to 99.99% in a dose-dependent manner. Importantly, clinically applicable doses of 425 nm blue light dramatically inhibited SARS-CoV-2 infection and replication in primary human 3D tracheal/bronchial tissue. Safe doses of visible light should be considered part of the strategic portfolio for the development of SARS-CoV-2 therapeutic countermeasures to mitigate coronavirus disease 2019 (COVID-19).


Subject(s)
COVID-19/drug therapy , COVID-19/prevention & control , Light , SARS-CoV-2 , Trachea/radiation effects , Virus Replication/radiation effects , Adult , Animals , Antiviral Agents/pharmacology , Bronchi , Calibration , Cell-Free System , Chlorocebus aethiops , Epithelium/pathology , Female , Humans , Respiratory Mucosa/radiation effects , Trachea/virology , Vero Cells
6.
Vaccine ; 39(38): 5410-5421, 2021 09 07.
Article in English | MEDLINE | ID: covidwho-1351060

ABSTRACT

Traditional bolus vaccine administration leads to rapid clearance of vaccine from lymphoid tissue. However, there is increasing evidence suggesting that the kinetics of antigen delivery can impact immune responses to vaccines, particularly when tailored to mimic natural infections. Here, we present the specific enhancements sustained release immunization confers to seasonal influenza vaccine, including the magnitude, durability, and breadth of humoral responses. To achieve sustained vaccine delivery kinetics, we have developed a microneedle array patch (MIMIX), with silk fibroin-formulated vaccine tips designed to embed in the dermis after a short application to the skin and release antigen over 1-2 weeks, mimicking the time course of a natural influenza infection. In a preclinical murine model, a single influenza vaccine administration via MIMIX led to faster seroconversion, response-equivalence to prime-boost bolus immunization, higher HAI titers against drifted influenza strains, and improved protective efficacy upon lethal influenza challenge when compared with intramuscular injection. These results highlight infection mimicry, achieved through sustained release silk microneedles, as a powerful approach to improve existing seasonal influenza vaccines, while also suggesting the broader potential of this platform technology to enable more efficacious next-generation vaccines and vaccine combinations.


Subject(s)
Influenza Vaccines , Influenza, Human , Animals , Humans , Immunogenicity, Vaccine , Influenza, Human/prevention & control , Mice , Needles , Silk
7.
J Am Chem Soc ; 143(36): 14748-14765, 2021 09 15.
Article in English | MEDLINE | ID: covidwho-1397838

ABSTRACT

The COVID-19 pandemic highlights the need for platform technologies enabling rapid development of vaccines for emerging viral diseases. The current vaccines target the SARS-CoV-2 spike (S) protein and thus far have shown tremendous efficacy. However, the need for cold-chain distribution, a prime-boost administration schedule, and the emergence of variants of concern (VOCs) call for diligence in novel SARS-CoV-2 vaccine approaches. We studied 13 peptide epitopes from SARS-CoV-2 and identified three neutralizing epitopes that are highly conserved among the VOCs. Monovalent and trivalent COVID-19 vaccine candidates were formulated by chemical conjugation of the peptide epitopes to cowpea mosaic virus (CPMV) nanoparticles and virus-like particles (VLPs) derived from bacteriophage Qß. Efficacy of this approach was validated first using soluble vaccine candidates as solo or trivalent mixtures and subcutaneous prime-boost injection. The high thermal stability of our vaccine candidates allowed for formulation into single-dose injectable slow-release polymer implants, manufactured by melt extrusion, as well as microneedle (MN) patches, obtained through casting into micromolds, for prime-boost self-administration. Immunization of mice yielded high titers of antibodies against the target epitope and S protein, and data confirms that antibodies block receptor binding and neutralize SARS-CoV and SARS-CoV-2 against infection of human cells. We present a nanotechnology vaccine platform that is stable outside the cold-chain and can be formulated into delivery devices enabling single administration or self-administration. CPMV or Qß VLPs could be stockpiled, and epitopes exchanged to target new mutants or emergent diseases as the need arises.


Subject(s)
COVID-19 Vaccines/metabolism , COVID-19/epidemiology , COVID-19/prevention & control , Delayed-Action Preparations/chemistry , SARS-CoV-2/metabolism , Vaccines, Subunit/metabolism , Animals , Comovirus , Computer Simulation , Drug Compounding , Epitopes/chemistry , Hot Temperature , Humans , Male , Mice, Inbred BALB C , Nanoparticles/chemistry , Peptides/chemistry , Vaccination , Vaccines, Virus-Like Particle/chemistry
8.
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
9.
PLoS One ; 16(4): e0250019, 2021.
Article in English | MEDLINE | ID: covidwho-1197380

ABSTRACT

SARS-CoV-2 has caused a global pandemic, and has taken over 1.7 million lives as of mid-December, 2020. Although great progress has been made in the development of effective countermeasures, with several pharmaceutical companies approved or poised to deliver vaccines to market, there is still an unmet need of essential antiviral drugs with therapeutic impact for the treatment of moderate-to-severe COVID-19. Towards this goal, a high-throughput assay was used to screen SARS-CoV-2 nsp15 uracil-dependent endonuclease (endoU) function against 13 thousand compounds from drug and lead repurposing compound libraries. While over 80% of initial hit compounds were pan-assay inhibitory compounds, three hits were confirmed as nsp15 endoU inhibitors in the 1-20 µM range in vitro. Furthermore, Exebryl-1, a ß-amyloid anti-aggregation molecule for Alzheimer's therapy, was shown to have antiviral activity between 10 to 66 µM, in Vero 76, Caco-2, and Calu-3 cells. Although the inhibitory concentrations determined for Exebryl-1 exceed those recommended for therapeutic intervention, our findings show great promise for further optimization of Exebryl-1 as an nsp15 endoU inhibitor and as a SARS-CoV-2 antiviral.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Drug Repositioning , Endoribonucleases/antagonists & inhibitors , SARS-CoV-2/drug effects , Small Molecule Libraries/pharmacology , Viral Nonstructural Proteins/antagonists & inhibitors , Animals , Antiviral Agents/chemistry , COVID-19/virology , Caco-2 Cells , Chlorocebus aethiops , Drug Repositioning/methods , Endoribonucleases/metabolism , High-Throughput Screening Assays/methods , Humans , Molecular Docking Simulation , SARS-CoV-2/metabolism , Small Molecule Libraries/chemistry , Vero Cells , Viral Nonstructural Proteins/metabolism
10.
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
11.
ACS Omega ; 6(11): 7454-7468, 2021 Mar 23.
Article in English | MEDLINE | ID: covidwho-1155692

ABSTRACT

Severe acute respiratory coronavirus 2 (SARS-CoV-2) is a newly identified virus that has resulted in over 2.5 million deaths globally and over 116 million cases globally in March, 2021. Small-molecule inhibitors that reverse disease severity have proven difficult to discover. One of the key approaches that has been widely applied in an effort to speed up the translation of drugs is drug repurposing. A few drugs have shown in vitro activity against Ebola viruses and demonstrated activity against SARS-CoV-2 in vivo. Most notably, the RNA polymerase targeting remdesivir demonstrated activity in vitro and efficacy in the early stage of the disease in humans. Testing other small-molecule drugs that are active against Ebola viruses (EBOVs) would appear a reasonable strategy to evaluate their potential for SARS-CoV-2. We have previously repurposed pyronaridine, tilorone, and quinacrine (from malaria, influenza, and antiprotozoal uses, respectively) as inhibitors of Ebola and Marburg viruses in vitro in HeLa cells and mouse-adapted EBOV in mice in vivo. We have now tested these three drugs in various cell lines (VeroE6, Vero76, Caco-2, Calu-3, A549-ACE2, HUH-7, and monocytes) infected with SARS-CoV-2 as well as other viruses (including MHV and HCoV 229E). The compilation of these results indicated considerable variability in antiviral activity observed across cell lines. We found that tilorone and pyronaridine inhibited the virus replication in A549-ACE2 cells with IC50 values of 180 nM and IC50 198 nM, respectively. We used microscale thermophoresis to test the binding of these molecules to the spike protein, and tilorone and pyronaridine bind to the spike receptor binding domain protein with K d values of 339 and 647 nM, respectively. Human Cmax for pyronaridine and quinacrine is greater than the IC50 observed in A549-ACE2 cells. We also provide novel insights into the mechanism of these compounds which is likely lysosomotropic.

12.
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.

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