Identification of small-molecule inhibitors of Zika virus infection and induced neural cell death via a drug repurposing screen

Abstract

In response to the current global health emergency posed by the Zika virus (ZIKV) outbreak and its link to microcephaly and other neurological conditions, we performed a drug repurposing screen of ∼6,000 compounds that included approved drugs, clinical trial drug candidates and pharmacologically active compounds; we identified compounds that either inhibit ZIKV infection or suppress infection-induced caspase-3 activity in different neural cells. A pan-caspase inhibitor, emricasan, inhibited ZIKV-induced increases in caspase-3 activity and protected human cortical neural progenitors in both monolayer and three-dimensional organoid cultures. Ten structurally unrelated inhibitors of cyclin-dependent kinases inhibited ZIKV replication. Niclosamide, a category B anthelmintic drug approved by the US Food and Drug Administration, also inhibited ZIKV replication. Finally, combination treatments using one compound from each category (neuroprotective and antiviral) further increased protection of human neural progenitors and astrocytes from ZIKV-induced cell death. Our results demonstrate the efficacy of this screening strategy and identify lead compounds for anti-ZIKV drug development.

Figure 1

Emricasan suppresses cell death of ZIKV-infected human astrocytes and hNPCs in 2D monolayer cultures and in 3D brain organoids. (a) Top, chemical structure of Emricasan. Bottom, dose-response curves displaying the effect of Emricasan treatment on caspase activity (left) and cell viability (right) in SNB-19 glioblastoma cells exposed to 3 different ZIKV strains. Values represent mean ± s.d. (n = 3 cultures). Curves represent best fits for calculating IC₅₀, and the insets in each panel report the calculated IC₅₀ value against each strain. (b) Left, example images of 2D monolayer cultures of forebrain-specific hNPCs immunostained for ZIKV envelop protein (ZIKVE; green), cleaved-caspase-3 (Cas3; red) and DAPI (gray) 72 hours after ZIKV FSS-13025 exposure (with or without Emricasan treatment) compared to mock infections (multiplicity of infection/MOI = 0.04 – 0.08; scale bar: 20 μm, applies to all image panels in b). Right, quantification comparing the percentage of CAS3⁺ cells over DAPI in mock or ZIKV-infected hNPCs cultures treated with the indicated concentrations of Emricasan. Values represent mean ± s.e.m. (n = 3 cultures; **P < 0.01; One-way ANOVA). (c) Left, example images of 3D human forebrain-specific organoid cultures (28 days in vitro) immunostained for ZIKVE (green), Cas3 (red) and DAPI (gray), 10 days after ZIKV FSS-13025 exposure (Scale bar: 100 μm, applies to all image panels in c). Right, quantification of the percentage of CAS3⁺ cells over DAPI in the ventricular zone. Values represent mean ± s.e.m. (n = 6 organoids; ***P < 0.001; One-way ANOVA).

Figure 2

Identification of PHA-690509 and Niclosamide as antiviral compounds. (a) Top, example western blot images of titration of PHA-690509 on SNB-19 cells for anti-ZIKV activity. Glioblastoma SNB-19 cells were treated with the indicated compound for 1 hour prior to inoculation with ZIKV FSS-13025, PRVABC59, or MR766 (MOI = 1) and cells were harvested 24 hours post infection for western blot analysis. Bottom, quantification of NS1/GAPDH protein band intensities. Data were normalized to that with the DMSO treatment. Values represent mean ± s.d. (n = 3 cultures; ***P < 0.001; One-way ANOVA for comparison with the DMSO treatment). Insert shows chemical structure of PHA-690509. (b) Top, example western blot images of titration of Emricasan on SNB-19 cells for anti-ZIKV activity. Similar to (a). Values represent mean ± s.d. (n = 3 cultures; P > 0.1; One-way ANOVA for comparison with the DMSO treatment). (c) Top, chemical structure of Niclosamide and example western blot images of titration of Niclosamide on SNB-19 cells for anti-ZIKV activity using the PRVABC59 strain (MOI = 1). Bottom, quantification similar to (a). Values represent mean ±s.d. (n = 3 cultures; **P < 0.01; ***P < 0.001; One-way ANOVA for comparison with the DMSO treatment). (d) Summary of ELISA quantifications of secreted ZIKV-NS1 protein in the medium of infected cells upon treatment of different doses of Niclosamide (top) or PHA-690509 (bottom) for 24 hours. Values represent mean ± s.d. (n = 3 cultures; ***P < 0.001; One-way ANOVA for comparison with the 0 μM group). (e) Summary of titration of Niclosamide and PHA-690509 on ZIKV viral production. SNB-19 cells were treated with each indicated compound at increasing concentrations for 1 hour prior to infection with PRVABC59 at MOI = 0.5. Cell culture supernatant was collected 24 hours post infection and infectious virions were quantified for focus-forming units (FFU) using Vero cells. Data represent mean ± s.d. (n = 3 cultures). Curves represent best fits for calculating IC₅₀, and the insets report the calculated IC₅₀ value for each compound.

Figure 3

Inhibition of ZIKV infection by Niclosamide and PHA-690509 at a post-entry step and blockade of ZIKV infection by additional CDKis. (a) Schematic illustration of time-of-addition experiment for Niclosamide and PHA-690509. (b) Top, example western blot images of SNB-19 cells treated with 10 μg/ml anti-AXL antibody, 2 μM Niclosamide, or 92 μM PHA-690509 for 1 hour prior to or 4 hours post infection with PRVABC59 (MOI = 1). Bottom, quantification of NS1/GAPDH protein band intensities. Data were normalized to that with the DMSO treatment. Values represent mean ± s.d. (n = 3 cultures; *P < 0.05; **P < 0.01; ***P < 0.001; One-way ANOVA for comparison with the DMSO treatment). (c) Inhibition of ZIKV infection in CDKi-treated cells. Left, example western blot images of SNB-19 cells treated with the indicated compound at 92 μM for one hour prior to infection with FSS13025 or PRVABC59 (MOI = 1). Right, quantification similar to (b). Values represent mean ± s.d. (n = 3 cultures; ***P < 0.01; One-way ANOVA for comparison with the DMSO treatment). (d) Effect of various CDKis on ZIKV production. Similar to Figure 2e. All data were normalized to 0 μM for each compound. Data represent mean ± s.d. (n = 3 cultures). Curves represent best fits for calculating IC₅₀ (listed on the right panel).

Figure 4

Niclosamide and PHA-690509 inhibit ZIKV infection in human astrocytes and forebrain-specific hNPCs. (a) Top, example immunostaining images of astrocytes treated with 2 μM Niclosamide, 92 μM PHA-690509, 9 μM Emricasan, or a combination of 92 μM PHA-690509 and 9 μM Emricasan for 1 hour prior to infection with PRVABC59 (MOI = 0.5). Cells were fixed 24 hours post infection and stained for ZIKVE (green) and DAPI (blue; Scale bar: 50 μm, applies to all image panels in a). Bottom, quantification of the percentage of ZIKV-infected astrocytes over DAPI. Values represent mean ± s.d. (n = 6 cultures; ***P < 0.001; One-way ANOVA for comparison with the DMSO treatment). (b) Top, example western blot images of hNPCs infected at a MOI of 0.1 and analyzed 48 hours post infection. Bottom, quantifications of NS1/GAPDH protein band intensities. Data were normalized to that with the DMSO treatment. Values represent mean ± s.d. (n = 3 cultures; ***P < 0.01; One-way ANOVA for comparison with the DMSO treatment). (c) Virus production from compound treated iPSC-derived human astrocytes. Similar to Figure 2e. Data represent mean ± s.d. (n = 3 cultures). (d–e) Summary of additive effects of a combination of Emricasan and PHA-690509 on inhibiting increased caspase-3 activity in human astrocytes infected with FSS13025-ZIKV or MR766 ZIKV (d), and on improving cell viability as measured by ATP production (e). Similar to Figure 1a. Values represent mean ± s.d. (n = 3 cultures; *P < 0.05; **P < 0.01; One-way ANOVA).