Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of Coronavirus Disease 2019 (COVID-19). There is a dire need for novel effective antivirals to treat COVID-19, as the only approved direct-acting antiviral to date is remdesivir, targeting the viral polymerase complex. A potential alternate target in the viral life cycle is the main SARS-CoV-2 protease 3CLpro (Mpro). The drug candidate PF-00835231 is the active compound of the first anti-3CLpro regimen in clinical trials. Here, we perform a comparative analysis of PF-00835231, the pre-clinical 3CLpro inhibitor GC-376, and the polymerase inhibitor remdesivir, in alveolar basal epithelial cells modified to express ACE2 (A549+ACE2 cells). We find PF-00835231 with at least similar or higher potency than remdesivir or GC-376. A time-of-drug-addition approach delineates the timing of early SARS-CoV-2 life cycle steps in A549+ACE2 cells and validates PF-00835231's early time of action. In a model of the human polarized airway epithelium, both PF-00835231 and remdesivir potently inhibit SARS-CoV-2 at low micromolar concentrations. Finally, we show that the efflux transporter P-glycoprotein, which was previously suggested to diminish PF-00835231's efficacy based on experiments in monkey kidney Vero E6 cells, does not negatively impact PF-00835231 efficacy in either A549+ACE2 cells or human polarized airway epithelial cultures. Thus, our study provides in vitro evidence for the potential of PF-00835231 as an effective SARS-CoV-2 antiviral and addresses concerns that emerged based on prior studies in non-human in vitro models.Importance:The arsenal of SARS-CoV-2 specific antiviral drugs is extremely limited. Only one direct-acting antiviral drug is currently approved, the viral polymerase inhibitor remdesivir, and it has limited efficacy. Thus, there is a substantial need to develop additional antiviral compounds with minimal side effects and alternate viral targets. One such alternate target is its main protease, 3CLpro (Mpro), an essential component of the SARS-CoV-2 life cycle processing the viral polyprotein into the components of the viral polymerase complex. In this study, we characterize a novel antiviral drug, PF-00835231, which is the active component of the first-in-class 3CLpro-targeting regimen in clinical trials. Using 3D in vitro models of the human airway epithelium, we demonstrate the antiviral potential of PF-00835231 for inhibition of SARS-CoV-2.

Highlights

  • Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of coronavirus disease 2019 (COVID-19)

  • Using immunofluorescence staining for SARS-CoV-2 nucleocapsid protein (N) and high-content microscopy, we found A5491ACE2 cells permissive to SARS-CoV-2 infection, whereas parent A549 cells were not (Fig. 1f)

  • We studied and compared three compounds in regard to SARS-CoV-2 antiviral activity and cytotoxicity, as follows: (i) PF-00835231, (ii) the preclinical 3CLpro inhibitor GC-376, which is licensed for veterinary use in feline coronavirus infections [29] and was recently shown to inhibit SARS-CoV-2 in Vero E6 cells [30], and (iii) the polymerase inhibitor remdesivir, which is currently the only direct-acting antiviral approved in the United States to treat SARS-CoV-2 infections, and is the standard of care

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Summary

Introduction

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of coronavirus disease 2019 (COVID-19). Remdesivir is incorporated into viral RNA by the RdRp, resulting in chain termination of both viral transcripts and de novo synthesized viral genomes [3] Given this considerably limited arsenal of directacting antivirals for COVID-19, it remains a strategic priority to develop novel compounds with minimal side effects and that are directed against alternate viral targets. One such alternate SARS-CoV-2 target is its main protease, 3CLpro (Mpro), which plays an essential role in the viral life cycle. There have been intense efforts to develop 3CLpro inhibitors specific for SARS-CoV-2 [6,7,8,9,10,11,12,13], only one inhibitor has been brought to the clinic, PF-07304814, which is the first anti-3CLpro compound in clinical trials

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