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1.
Antiviral Res ; 202: 105311, 2022 06.
Article in English | MEDLINE | ID: covidwho-1773103

ABSTRACT

Nelfinavir is an HIV protease inhibitor that has been widely prescribed as a component of highly active antiretroviral therapy, and has been reported to exert in vitro antiviral activity against SARS-CoV-2. We here assessed the effect of Nelfinavir in a SARS-CoV-2 infection model in hamsters. Despite the fact that Nelfinavir, [50 mg/kg twice daily (BID) for four consecutive days], did not reduce viral RNA load and infectious virus titres in the lung of infected animals, treatment resulted in a substantial improvement of SARS-CoV-2-induced lung pathology. This was accompanied by a dense infiltration of neutrophils in the lung interstitium which was similarly observed in non-infected hamsters. Nelfinavir resulted also in a marked increase in activated neutrophils in the blood, as observed in non-infected animals. Although Nelfinavir treatment did not alter the expression of chemoattractant receptors or adhesion molecules on human neutrophils, in vitro migration of human neutrophils to the major human neutrophil attractant CXCL8 was augmented by this protease inhibitor. Nelfinavir appears to induce an immunomodulatory effect associated with increasing neutrophil number and functionality, which may be linked to the marked improvement in SARS-CoV-2 lung pathology independent of its lack of antiviral activity. Since Nelfinavir is no longer used for the treatment of HIV, we studied the effect of two other HIV protease inhibitors, namely the combination Lopinavir/Ritonavir (Kaletra™) in this model. This combination resulted in a similar protective effect as Nelfinavir against SARS-CoV2 induced lung pathology in hamsters.


Subject(s)
COVID-19 , HIV Infections , HIV Protease Inhibitors , Animals , COVID-19/drug therapy , Cricetinae , HIV Infections/drug therapy , HIV Protease Inhibitors/pharmacology , HIV Protease Inhibitors/therapeutic use , Lopinavir/pharmacology , Lopinavir/therapeutic use , Lung , Mesocricetus , Nelfinavir/pharmacology , Nelfinavir/therapeutic use , RNA, Viral , Ritonavir/therapeutic use , SARS-CoV-2
2.
Nat Commun ; 12(1): 6055, 2021 10 18.
Article in English | MEDLINE | ID: covidwho-1475294

ABSTRACT

COVID-19 caused by the SARS-CoV-2 virus has become a global pandemic. 3CL protease is a virally encoded protein that is essential across a broad spectrum of coronaviruses with no close human analogs. PF-00835231, a 3CL protease inhibitor, has exhibited potent in vitro antiviral activity against SARS-CoV-2 as a single agent. Here we report, the design and characterization of a phosphate prodrug PF-07304814 to enable the delivery and projected sustained systemic exposure in human of PF-00835231 to inhibit coronavirus family 3CL protease activity with selectivity over human host protease targets. Furthermore, we show that PF-00835231 has additive/synergistic activity in combination with remdesivir. We present the ADME, safety, in vitro, and in vivo antiviral activity data that supports the clinical evaluation of PF-07304814 as a potential COVID-19 treatment.


Subject(s)
COVID-19/drug therapy , Coronavirus 3C Proteases/antagonists & inhibitors , Coronavirus Protease Inhibitors/administration & dosage , Indoles/administration & dosage , Leucine/administration & dosage , Pyrrolidinones/administration & dosage , Adenosine Monophosphate/administration & dosage , Adenosine Monophosphate/adverse effects , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacokinetics , Alanine/administration & dosage , Alanine/adverse effects , Alanine/analogs & derivatives , Alanine/pharmacokinetics , Animals , COVID-19/virology , Chlorocebus aethiops , Coronavirus 229E, Human/drug effects , Coronavirus 229E, Human/enzymology , Coronavirus Protease Inhibitors/adverse effects , Coronavirus Protease Inhibitors/pharmacokinetics , Disease Models, Animal , Drug Design , Drug Synergism , Drug Therapy, Combination , HeLa Cells , Humans , Indoles/adverse effects , Indoles/pharmacokinetics , Infusions, Intravenous , Leucine/adverse effects , Leucine/pharmacokinetics , Mice , Pyrrolidinones/adverse effects , Pyrrolidinones/pharmacokinetics , SARS Virus/drug effects , SARS Virus/enzymology , SARS-CoV-2/drug effects , SARS-CoV-2/enzymology , Vero Cells
3.
EBioMedicine ; 72: 103595, 2021 Oct.
Article in English | MEDLINE | ID: covidwho-1433162

ABSTRACT

BACKGROUND: Favipiravir and Molnupiravir, orally available antivirals, have been reported to exert antiviral activity against SARS-CoV-2. First efficacy data have been recently reported in COVID-19 patients. METHODS: We here report on the combined antiviral effect of both drugs in a SARS-CoV-2 Syrian hamster infection model. The infected hamsters were treated twice daily with the vehicle (the control group) or a suboptimal dose of each compound or a combination of both compounds. FINDINGS: When animals were treated with a combination of suboptimal doses of Molnupiravir and Favipiravir at the time of infection, a marked combined potency at endpoint is observed. Infectious virus titers in the lungs of animals treated with the combination are reduced by ∼5 log10 and infectious virus are no longer detected in the lungs of >60% of treated animals. When start of treatment was delayed with one day a reduction of titers in the lungs of 2.4 log10 was achieved. Moreover, treatment of infected animals nearly completely prevented transmission to co-housed untreated sentinels. Both drugs result in an increased mutation frequency of the remaining viral RNA recovered from the lungs of treated animals. In the combo-treated hamsters, an increased frequency of C-to-T mutations in the viral RNA is observed as compared to the single treatment groups which may explain the pronounced antiviral potency of the combination. INTERPRETATION: Our findings may lay the basis for the design of clinical studies to test the efficacy of the combination of Molnupiravir/Favipiravir in the treatment of COVID-19. FUNDING: stated in the acknowledgment.


Subject(s)
Amides/therapeutic use , COVID-19/drug therapy , Cytidine/analogs & derivatives , Hydroxylamines/therapeutic use , Lung/virology , Pyrazines/therapeutic use , Amides/pharmacology , Animals , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , COVID-19/transmission , Cytidine/pharmacology , Cytidine/therapeutic use , Disease Models, Animal , Drug Therapy, Combination , Female , Hydroxylamines/pharmacology , Mesocricetus , Pyrazines/pharmacology , RNA, Viral , Treatment Outcome , Viral Load
4.
Antiviral Res ; 192: 105122, 2021 08.
Article in English | MEDLINE | ID: covidwho-1283915

ABSTRACT

There are, besides remdesivir, no approved antivirals for the treatment of SARS-CoV-2 infections. To aid in the search for antivirals against this virus, we explored the use of human tracheal airway epithelial cells (HtAEC) and human small airway epithelial cells (HsAEC) grown at the air-liquid interface (ALI). These cultures were infected at the apical side with one of two different SARS-CoV-2 isolates. Each virus was shown to replicate to high titers for extended periods of time (at least 8 days) and, in particular an isolate with the D614G in the spike (S) protein did so more efficiently at 35 °C than 37 °C. The effect of a selected panel of reference drugs that were added to the culture medium at the basolateral side of the system was explored. Remdesivir, GS-441524 (the parent nucleoside of remdesivir), EIDD-1931 (the parent nucleoside of molnupiravir) and IFN (ß1 and λ1) all resulted in dose-dependent inhibition of viral RNA and infectious virus titers collected at the apical side. However, AT-511 (the free base form of AT-527 currently in clinical testing) failed to inhibit viral replication in these in vitro primary cell models. Together, these results provide a reference for further studies aimed at selecting SARS-CoV-2 inhibitors for further preclinical and clinical development.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , SARS-CoV-2/drug effects , Virus Replication/drug effects , Animals , Cell Line , Chlorocebus aethiops , Drug Evaluation, Preclinical/methods , Epithelial Cells/virology , Humans , RNA, Viral , SARS-CoV-2/isolation & purification , Vero Cells
5.
Nat Commun ; 12(1): 3309, 2021 06 03.
Article in English | MEDLINE | ID: covidwho-1260940

ABSTRACT

The ongoing pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), necessitates strategies to identify prophylactic and therapeutic drug candidates for rapid clinical deployment. Here, we describe a screening pipeline for the discovery of efficacious SARS-CoV-2 inhibitors. We screen a best-in-class drug repurposing library, ReFRAME, against two high-throughput, high-content imaging infection assays: one using HeLa cells expressing SARS-CoV-2 receptor ACE2 and the other using lung epithelial Calu-3 cells. From nearly 12,000 compounds, we identify 49 (in HeLa-ACE2) and 41 (in Calu-3) compounds capable of selectively inhibiting SARS-CoV-2 replication. Notably, most screen hits are cell-line specific, likely due to different virus entry mechanisms or host cell-specific sensitivities to modulators. Among these promising hits, the antivirals nelfinavir and the parent of prodrug MK-4482 possess desirable in vitro activity, pharmacokinetic and human safety profiles, and both reduce SARS-CoV-2 replication in an orthogonal human differentiated primary cell model. Furthermore, MK-4482 effectively blocks SARS-CoV-2 infection in a hamster model. Overall, we identify direct-acting antivirals as the most promising compounds for drug repurposing, additional compounds that may have value in combination therapies, and tool compounds for identification of viral host cell targets.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , Drug Repositioning/methods , Pandemics , SARS-CoV-2 , Animals , COVID-19/prevention & control , COVID-19/virology , Cell Line , Cytidine/administration & dosage , Cytidine/analogs & derivatives , Cytidine/pharmacology , Databases, Pharmaceutical , Drug Discovery/methods , Drug Evaluation, Preclinical/methods , HeLa Cells , High-Throughput Screening Assays/methods , Humans , Hydroxylamines/administration & dosage , Hydroxylamines/pharmacology , Mesocricetus , Nelfinavir/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Virus Replication/drug effects
6.
Cell Rep ; 35(1): 108940, 2021 04 06.
Article in English | MEDLINE | ID: covidwho-1157178

ABSTRACT

SARS-CoV-2 has currently precipitated the COVID-19 global health crisis. We developed a medium-throughput drug-screening system and identified a small-molecule library of 34 of 430 protein kinase inhibitors that were capable of inhibiting the SARS-CoV-2 cytopathic effect in human epithelial cells. These drug inhibitors are in various stages of clinical trials. We detected key proteins involved in cellular signaling pathways mTOR-PI3K-AKT, ABL-BCR/MAPK, and DNA-damage response that are critical for SARS-CoV-2 infection. A drug-protein interaction-based secondary screen confirmed compounds, such as the ATR kinase inhibitor berzosertib and torin2 with anti-SARS-CoV-2 activity. Berzosertib exhibited potent antiviral activity against SARS-CoV-2 in multiple cell types and blocked replication at the post-entry step. Berzosertib inhibited replication of SARS-CoV-1 and the Middle East respiratory syndrome coronavirus (MERS-CoV) as well. Our study highlights key promising kinase inhibitors to constrain coronavirus replication as a host-directed therapy in the treatment of COVID-19 and beyond as well as provides an important mechanism of host-pathogen interactions.


Subject(s)
Antiviral Agents/pharmacology , COVID-19/drug therapy , DNA Damage , Isoxazoles/pharmacology , Pyrazines/pharmacology , SARS-CoV-2/physiology , Virus Replication/drug effects , A549 Cells , Animals , COVID-19/metabolism , COVID-19/pathology , Chlorocebus aethiops , Drug Evaluation, Preclinical , HEK293 Cells , HeLa Cells , Humans , MAP Kinase Signaling System/drug effects , Middle East Respiratory Syndrome Coronavirus/metabolism , Vero Cells
7.
Nature ; 586(7827): 113-119, 2020 10.
Article in English | MEDLINE | ID: covidwho-672174

ABSTRACT

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019 has triggered an ongoing global pandemic of the severe pneumonia-like disease coronavirus disease 2019 (COVID-19)1. The development of a vaccine is likely to take at least 12-18 months, and the typical timeline for approval of a new antiviral therapeutic agent can exceed 10 years. Thus, repurposing of known drugs could substantially accelerate the deployment of new therapies for COVID-19. Here we profiled a library of drugs encompassing approximately 12,000 clinical-stage or Food and Drug Administration (FDA)-approved small molecules to identify candidate therapeutic drugs for COVID-19. We report the identification of 100 molecules that inhibit viral replication of SARS-CoV-2, including 21 drugs that exhibit dose-response relationships. Of these, thirteen were found to harbour effective concentrations commensurate with probable achievable therapeutic doses in patients, including the PIKfyve kinase inhibitor apilimod2-4 and the cysteine protease inhibitors MDL-28170, Z LVG CHN2, VBY-825 and ONO 5334. Notably, MDL-28170, ONO 5334 and apilimod were found to antagonize viral replication in human pneumocyte-like cells derived from induced pluripotent stem cells, and apilimod also demonstrated antiviral efficacy in a primary human lung explant model. Since most of the molecules identified in this study have already advanced into the clinic, their known pharmacological and human safety profiles will enable accelerated preclinical and clinical evaluation of these drugs for the treatment of COVID-19.


Subject(s)
Antiviral Agents/analysis , Antiviral Agents/pharmacology , Betacoronavirus/drug effects , Coronavirus Infections/drug therapy , Coronavirus Infections/virology , Drug Evaluation, Preclinical , Drug Repositioning , Pneumonia, Viral/drug therapy , Pneumonia, Viral/virology , Adenosine Monophosphate/analogs & derivatives , Adenosine Monophosphate/pharmacology , Alanine/analogs & derivatives , Alanine/pharmacology , Alveolar Epithelial Cells/cytology , Alveolar Epithelial Cells/drug effects , Betacoronavirus/growth & development , COVID-19 , Cell Line , Cysteine Proteinase Inhibitors/analysis , Cysteine Proteinase Inhibitors/pharmacology , Dose-Response Relationship, Drug , Drug Synergism , Gene Expression Regulation/drug effects , Humans , Hydrazones , Induced Pluripotent Stem Cells/cytology , Models, Biological , Morpholines/analysis , Morpholines/pharmacology , Pandemics , Pyrimidines , Reproducibility of Results , SARS-CoV-2 , Small Molecule Libraries/analysis , Small Molecule Libraries/pharmacology , Triazines/analysis , Triazines/pharmacology , Virus Internalization/drug effects , Virus Replication/drug effects
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