Identification of In Vitro Inhibitors of Monkeypox Replication

Abstract

Monkeypox virus (MPXV) infections in humans have historically been restricted to regions of endemicity in Africa. However, in 2022, an alarming number of MPXV cases were reported globally, with evidence of person-to-person transmission. Because of this, the World Health Organization (WHO) declared the MPXV outbreak a public health emergency of international concern. The supply of MPXV vaccines is limited, and only two antivirals, tecovirimat and brincidofovir, approved by the U.S. Food and Drug Administration (FDA) for the treatment of smallpox, are currently available for the treatment of MPXV infection. Here, we evaluated 19 compounds previously shown to inhibit different RNA viruses for their ability to inhibit orthopoxvirus infections. We first used recombinant vaccinia virus (rVACV) expressing fluorescence (mScarlet or green fluorescent protein [GFP]) and luciferase (Nluc) reporter genes to identify compounds with antiorthopoxvirus activity. Seven compounds from the ReFRAME library (antimycin A, mycophenolic acid, AVN-944, pyrazofurin, mycophenolate mofetil, azaribine, and brequinar) and six compounds from the NPC library (buparvaquone, valinomycin, narasin, monensin, rotenone, and mubritinib) showed inhibitory activity against rVACV. Notably, the anti-VACV activity of some of the compounds in the ReFRAME library (antimycin A, mycophenolic acid, AVN-944, mycophenolate mofetil, and brequinar) and all the compounds from the NPC library (buparvaquone, valinomycin, narasin, monensin, rotenone, and mubritinib) were confirmed with MPXV, demonstrating their inhibitory activity in vitro against two orthopoxviruses. IMPORTANCE Despite the eradication of smallpox, some orthopoxviruses remain important human pathogens, as exemplified by the recent 2022 monkeypox virus (MPXV) outbreak. Although smallpox vaccines are effective against MPXV, access to those vaccines is limited. In addition, current antiviral treatment against MPXV infections is limited to the use of the FDA-approved drugs tecovirimat and brincidofovir. Thus, there is an urgent need to identify novel antivirals for the treatment of MPXV infection and other potentially zoonotic orthopoxvirus infections. Here, we show that 13 compounds, derived from two different libraries, previously found to inhibit several RNA viruses, also inhibit VACV. Notably, 11 compounds also displayed inhibitory activity against MPXV.

Keywords: GFP; antivirals; luciferase; mScarlet; monkeypox; orthopoxvirus; poxvirus; vaccinia virus.

FIG 1

Bireporter cell-based assay for the identification of VACV inhibitors. (A) Schematic representation of rVACV Nluc/Scarlet (left) and rVACV Nluc/GFP (right). Nluc (blue boxes) and fluorescent reporter Scarlet (red boxes) or GFP (green boxes) genes were inserted downstream of F13L (gray boxes) or A27L (yellow boxes), respectively, in the viral genome. The reporter genes are expressed by an early/late VACV synthetic promoter (white arrows). Hatched boxes indicate the recombination flanking regions for the insertion of reporter genes into the VACV genome. (B and C) Bireporter cell-based assay to identify inhibitors. Human A549 cells (2 × 10⁴ cells/well, 96-well plates, in quadruplicate) were infected with 200 PFU of rVACV Nluc/Scarlet (left) or rVACV Nluc/GFP (right) and incubated with 3-fold serial dilutions (starting concentration of 50 μM) of tecovirimat. Mock-infected cells and cells infected in the absence of drug were included as internal controls. At 24 hpi, Scarlet (rVACV Nluc/Scarlet, left) and GFP (rVACV Nluc/GFP, right) expression was visualized under a fluorescence microscope (B). Representative images are shown. Bars, 100 μm. Magnification, ×20. To quantify inhibition of viral replication, Scarlet (red squares) or GFP (green triangles) expression levels were quantified at 24 hpi using a fluorescent microplate reader (C, left) or Nluc expression (C, right) was assessed using a luminometer for rVACV Nluc/Scarlet (red squares) or rVACV Nluc/GFP (green triangles). The EC₅₀ of tecovirimat was calculated using sigmoidal dose-response curves. The CC₅₀ of tecovirimat was determined using an MTT assay kit. The SI was calculated by dividing the CC₅₀ by the EC₅₀. The percent viral inhibition was normalized to non-tecovirimat-treated controls. The dotted lines indicate 50% inhibition. Data are means and SD of viral inhibition from quadruplicate wells (n = 4). (D and E) Viral titers (D) and Nluc activity (E). Human A549 cells (2 × 10⁴ cells/well, 96-well plates, in triplicate) were infected with an MOI of 0.01 (top) or 3 (bottom) of rVACV Nluc/Scarlet, rVACV Nluc/GFP, or MPXV for 1 h and incubated with 10-fold serial dilutions (starting concentration of 100 μM) of tecovirimat. At 24, 48, and 72 hpi, tissue culture supernatants were collected, and viral titers were determined by plaque assay in CV1 cells. Additionally, Nluc activity (E) in cell culture supernatants was determined using a luminometer for rVACV Nluc/Scarlet (left) or rVACV Nluc/GFP (right). Mock-infected cells and cells infected in the absence of drug were included as internal controls. Data are means and SD of viral inhibition from triplicate wells (n = 3).

FIG 2

Inhibitory activity of the ReFRAME compounds against VACV: Human A549 cells (2 × 10⁴ cells/well, 96-well plates, in quadruplicate) were infected with 200 PFU of rVACV Nluc/Scarlet (red squares) or rVACV Nluc/GFP (green triangles) and incubated with 3-fold serial dilutions of antimycin A (A), Osu-03012 (B), mycophenolic acid (C), AVN-944 (D), azauridine (E), pyrazofurin (F), mycophenolate mofetil (G), azaribine (H), or brequinar (I). Benzimidazole (J) and tecovirimat (K) were included as negative and positive controls, respectively. Mock-infected cells and cells infected in the absence of drug were included as internal controls. At 24 hpi, inhibition of rVACV Nluc/Scarlet and rVACV Nluc/GFP viral replication was evaluated by quantifying fluorescent Scarlet or GFP (top graphs) and Nluc (bottom graphs) expression using a fluorescent microplate reader and a luminometer, respectively. The EC₅₀ for each compound was calculated using sigmoidal dose-response curves with GraphPad Prism. The dotted lines indicate 50% inhibition. Data are means and SD of viral inhibition from quadruplicate wells (n = 4). At the same time point (hpi), Scarlet (rVACV Nluc/Scarlet, top) and GFP (rVACV Nluc/GFP, bottom) fluorescent expression in infected cells in the absence (0 μM) or in the presence of the indicated concentrations of the compounds (maximum concentration and EC₅₀) was visualized using a fluorescence microscope. Representative images are shown. Bars, 100 μm. Magnification, ×20.

FIG 3

Inhibitory activity of the NPC compounds against VACV. Human A549 cells (2 × 10⁴ cells/well, 96-well plates, in quadruplicate) were infected with 200 PFU of rVACV Nluc/Scarlet (red squares) or rVACV Nluc/GFP (green triangles) and incubated with 3-fold serial dilutions of azoxystrobin (A), buparvaquone (B), valinomycin (C), narasin (D), amuvatinib (E), monensin (F), spautin-1 (G), tryptanthrin (H), rotenone (I), or mubritinib (J). Benzimidazole (K) and tecovirimat (L) were included as negative and positive controls, respectively. Mock-infected cells and cells infected in the absence of drug were included as internal controls. Inhibition of rVACV Nluc/Scarlet or rVACV Nluc/GFP viral replication was evaluated by quantifying Scarlet or GFP (top graphs) and Nluc (bottom graphs) expression at 24 hpi using fluorescent and luciferase microplate readers, respectively. The EC₅₀ for each compound was calculated using sigmoidal dose-response curves with GraphPad Prism. The dotted lines indicate 50% inhibition. Data are means and SD of viral inhibition from quadruplicate wells (n = 4). At the same time point (hpi), Scarlet (rVACV Nluc/Scarlet; top) and GFP (rVACV Nluc/GFP; bottom) fluorescent expression in infected cells in the absence (0 μM) or presence of the indicated concentrations of the compounds (maximum concentration and EC₅₀) was visualized using a fluorescence microscope. Representative images are shown. Bars, 100 μm. Magnification, ×20.

FIG 4

Inhibitory activity of the ReFRAME and NPC compounds against MPXV. Human A549 cells (2 × 10⁴ cells/well, 96-well plates, in quadruplicate) were infected with 200 PFU of MPXV and incubated with 3-fold serial dilutions (starting concentration of 50 μM) of the ReFRAME library compound antimycin A, Osu-03012, mycophenolic acid, AVN-944, azauridine, pyrazofurin, mycophenolate mofetil, azaribine, or brequinar (A) or the NPC library compound azoxystrobin, buparvaquone, valinomycin, narasin, amuvatinib, monensin, spautin-1, tryptanthrin, rotenone, or mubritinib (B). Benzimidazole and tecovirimat were included as negative and positive controls, respectively (A and B). Mock-infected cells and cells infected in the absence of drug were included as an internal control. Inhibition of viral replication was evaluated by quantifying the number of plaques at 24 hpi using a cross-reactive anti-VACV A33R polyclonal antibody and developed with an anti-rabbit Vectastain and DAB reagent. The EC₅₀ of each compound was calculated using sigmoidal dose-response curves with GraphPad Prism. The dotted line indicates 50% inhibition. Data are means and SD of viral inhibition from quadruplicate wells (n = 4).