In January 2020, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was identified as the causative agent of a new respiratory syndrome that was later named COVID-19 (1). The virus has rapidly spread throughout the world, causing an ongoing pandemic, with millions of deaths (2). SARS-CoV-2 is a member of Coronaviridae, a family of enveloped, single-strand, positive-sense RNA viruses (3). This family is composed of both human and animal pathogens, including two other emerging human pathogens [SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV)] as well as four endemic human viruses that are the second most common cause of the common cold (HCoV-OC43, 229E, NL63, and HKU1) (4).

Upon entry into the host cell cytoplasm, the viral genome is translated into roughly 30 proteins. Of these, 16 are initially translated as two polyproteins that must be cleaved into the individual viral proteins for infection to proceed. This cleavage is mediated by two virally encoded proteases: the main viral protease, known as Mpro, 3CLpro, or nonstructural protein 5 (nsp5); and a second protease known as the papain-like protease, PLpro, a domain within nsp3 (3). There is interest in developing de novo inhibitors to target these proteases (5–10), but this is a lengthy process.

Although several vaccines received emergency use authorization from health authorities worldwide and are being deployed, it will take a long time to vaccinate the world population, and the emergence of viral escape mutants that render vaccines ineffective remains a possibility. Therefore, there is a continued need for new treatment options for COVID-19, as well as for broad-spectrum antivirals that could be used against future emerging viruses. Remdesivir, an RNA-dependent RNA-polymerase inhibitor, has been reported to shorten COVID-19 hospitalization times (11), but it failed a large clinical trial in hospitalized patients (12) and its efficacy is unclear.

Drug-repurposing screens have been used to identify safe-in-human drugs with potential anti–SARS-CoV-2 properties (9, 13, 14). Repurposed drugs that have existing clinical data on the effective dose, treatment duration, side effects, and toxicity could be rapidly translated into the treatment of patients.

We screened a library of 1900 clinically used drugs, either approved for human use or with extensive safety data in humans (phase 2 or 3 clinical trials), for their ability to inhibit infection of A549 cells by OC43. We chose OC43 because it is a human pathogen that belongs to the same clade of beta coronaviruses as SARS-CoV-2 and can be studied under “regular” biosafety conditions, as well as in an attempt to discover broad-spectrum anti-coronavirus drugs that would be beneficial against SARS-CoV-2 and future emerging coronaviruses. One day after plating, cells were infected at a multiplicity of infection (MOI) of 0.3 and incubated at 33°C for 1 hour, and drugs were added to a final concentration of 10 μM. Cells were then incubated at 33°C for 4 days, fixed, and stained for the presence of the viral nucleoprotein (Fig. 1A). We imaged the cells at day zero (after drug addition) and day four (after staining) to determine the effect of the drugs on cell growth and OC43 infection.