Screening of an FDA-approved compound library identifies four small-molecule inhibitors of Middle East respiratory syndrome coronavirus replication in cell culture

Abstract

Coronaviruses can cause respiratory and enteric disease in a wide variety of human and animal hosts. The 2003 outbreak of severe acute respiratory syndrome (SARS) first demonstrated the potentially lethal consequences of zoonotic coronavirus infections in humans. In 2012, a similar previously unknown coronavirus emerged, Middle East respiratory syndrome coronavirus (MERS-CoV), thus far causing over 650 laboratory-confirmed infections, with an unexplained steep rise in the number of cases being recorded over recent months. The human MERS fatality rate of ∼ 30% is alarmingly high, even though many deaths were associated with underlying medical conditions. Registered therapeutics for the treatment of coronavirus infections are not available. Moreover, the pace of drug development and registration for human use is generally incompatible with strategies to combat emerging infectious diseases. Therefore, we have screened a library of 348 FDA-approved drugs for anti-MERS-CoV activity in cell culture. If such compounds proved sufficiently potent, their efficacy might be directly assessed in MERS patients. We identified four compounds (chloroquine, chlorpromazine, loperamide, and lopinavir) inhibiting MERS-CoV replication in the low-micromolar range (50% effective concentrations [EC(50)s], 3 to 8 μM). Moreover, these compounds also inhibit the replication of SARS coronavirus and human coronavirus 229E. Although their protective activity (alone or in combination) remains to be assessed in animal models, our findings may offer a starting point for treatment of patients infected with zoonotic coronaviruses like MERS-CoV. Although they may not necessarily reduce viral replication to very low levels, a moderate viral load reduction may create a window during which to mount a protective immune response.

FIG 1

Low-micromolar amounts of chloroquine, chlorpromazine, loperamide, and lopinavir inhibit MERS-CoV-induced cytopathology. Huh7 cells in 96-well plates were infected with MERS-CoV isolate EMC/2012 (MOI, 0.005) in the presence of 0 to 32 μM CQ (A), 0 to 16 μM CPZ (B), 0 to 8 μM LPM (C), or 0 to 20 μM LPV (D). Cells were incubated for 2 days, and cell viability was monitored using an MTS assay. In addition, the potential toxicity of compound treatment only was monitored in parallel mock-infected Huh7 cell cultures. Graphs show the results (averages and standard deviations [SD]) of a representative experiment that was performed in quadruplicate. All experiments were repeated at least twice. For each compound, the calculated EC₅₀, CC₅₀, and SI values are given.

FIG 2

Low-micromolar amounts of chloroquine, chlorpromazine, loperamide, and lopinavir inhibit SARS-CoV-induced cytopathology. Vero E6 cells in 96-well plates were infected with SARS-CoV isolate Frankfurt-1 (MOI, 0.005) in the presence of 0 to 32 μM CQ (A), 0 to 16 μM CPZ (B), 0 to 32 μM LPM (C), or 0 to 32 μM LPV (D), given at t of +1 h p.i. Cells were incubated for 3 days, and viability was monitored using an MTS assay. In parallel, potential compound cytotoxicity was monitored in mock-infected Vero E6 cells. Graphs show the results (averages and SD) of a representative experiment that was performed in quadruplicate. All experiments were repeated at least twice. For each compound, the EC₅₀, CC₅₀, and SI values are given.

FIG 3

HCoV-229E-GFP replication is inhibited by low-micromolar amounts of chloroquine, chlorpromazine, loperamide, and lopinavir. Huh7 cells in 96-well plates were infected with HCoV-229E-GFP (MOI, 5) in the presence of 0 to 50 μM CQ (A), CPZ (B), LPM (C), or LPV (D). Compounds were given at t of −1 and remained present during infection. Cells were fixed at 24 h p.i., and GFP reporter gene expression was measured and normalized to the signal in control cells (100%; black bars), which were treated with the solvent used for the various compounds. The effect of compound treatment on the viability of mock-infected Huh7 cells, compared with solvent-treated control cells, was determined by using an MTS assay (gray lines). Graphs show the results (average and SD) of a representative quadruplicate experiment. All experiments were repeated at least twice; n.d., not detected.

FIG 4

Chloroquine, chlorpromazine, loperamide, and lopinavir affect various stages of the MERS-CoV replication cycle. Vero (A, C, E, G) and Huh7 cells (B, D, F, H) were infected with MERS-CoV isolate EMC/2012 (MOI, 1). At t of −1 or +1, the indicated concentrations of CQ (A, B), CPZ (C, D), LPM (E, F), and LPV (G, H) were added, and virus titers in the culture supernatant (n = 4; averages and SDs are shown) were determined at 24 h p.i. using plaque assays; n.d., not detected.