A Screen of FDA-Approved Drugs for Inhibitors of Zika Virus Infection

Abstract

Currently there are no approved vaccines or specific therapies to prevent or treat Zika virus (ZIKV) infection. We interrogated a library of FDA-approved drugs for their ability to block infection of human HuH-7 cells by a newly isolated ZIKV strain (ZIKV MEX_I_7). More than 20 out of 774 tested compounds decreased ZIKV infection in our in vitro screening assay. Selected compounds were further validated for inhibition of ZIKV infection in human cervical, placental, and neural stem cell lines, as well as primary human amnion cells. Established anti-flaviviral drugs (e.g., bortezomib and mycophenolic acid) and others that had no previously known antiviral activity (e.g., daptomycin) were identified as inhibitors of ZIKV infection. Several drugs reduced ZIKV infection across multiple cell types. This study identifies drugs that could be tested in clinical studies of ZIKV infection and provides a resource of small molecules to study ZIKV pathogenesis.

Figure 1. A screen for inhibitors of ZIKV infection among FDA approved drugs

(A) Screen timeline. HuH-7 cells were plated 20 hours prior to treatment with drugs for one hour. Cells were then infected with ZIKV MEX_I_7 (see Figure S1) for 24 hours before fixation and staining. Final concentration of drugs was 10 µM. (B) Screen controls. Rates of infection are shown for all positive (NITD008) and negative (DMSO) controls across screening plates. (C) Screen reproducibility. Points represent the % infection for each drug in each replicate of the screen. Points colored red were selected for validation studies. (D) Infection rate versus cell count. Cell count and corresponding % infection values for each drug are plotted for replicate screen 1. (E) Representative images showing viral antigen (red) and nuclei (blue) for controls and selected drugs. Images were acquired using a 10X objective. Data for the screen are presented in Table S1.

Figure 2. Validation of selected drugs in ZIKV-infected HuH-7 cells

(A) Infection rates as a function of drug concentration are shown. (B) Cell numbers for the indicated drug treatments are shown. Data for all 30 drugs tested in follow up experiments are in Figure S2. Data are represented as mean +/− standard deviation.

Figure 3. Evaluation of selected anti-viral drugs in ZIKV-infected human cell lines of cervical and trophoblast origin

(A) Infection rates as a function of drug concentration are shown for HeLa cells. (B) Infection rates as a function of drug concentration are shown for JEG3 cells. Data are represented as mean +/− standard deviation. (C) Representative immunofluorescence images showing virus antigen (red) and nuclei (blue) are shown for the 10 µM concentration for each drug indicated. Data for all 8 drugs tested in follow up experiments are in Figure S3.

Figure 4. Effects of selected drug on infection of human neural stem cells (hNSC)

Rates of hNSC infection by ZIKV MEX_I_7 are shown for treatment conditions of 1 µM (A) and 10 µM (B). The % of cells that were analyzed for virus infection is shown for 1 µM (C) and 10 µM (D) conditions for ZIKV MEX_I_7. (E) Infection by ZIKV DAK_41525 under conditions of 1 µM drug treatment. Data are represented as mean +/− standard deviation (F) The % of cells that were analyzed for ZIKV DAK_41525. See Figure S4 for light scattering and fluorescence intensity data associated with the ZIKV MEX_I_7 infection. Asterisks indicate statistically significant reductions of virus infection or cells analyzed at a P value of <0.05 as calculated by unpaired t-test.

Figure 5. Preclinical evaluation of select therapeutics repurposed to target ZIKV infection of human amnion epithelial cells (HAECs)

Rates of HAEC infection by ZIKV MEX_I_7 are shown for the indicated drugs at concentrations between 0.0016 and 16 µM. Vehicle (DMSO) and positive control (NITD008) from the same experiment are shown in panels (A) and (B). Infection rates for ivermectin, pyrimethamine, azathioprine, and daptomycin are shown in (A), while MPA, cyclosporine A, mefloquine, and sertraline are shown in (B). Data are represented as mean +/− standard deviation Representative micrographs for vehicle (DMSO), positive control (NITD008) and MPA are shown in (C). Virus antigen (red) and nuclei (blue) are shown for the 16 µM concentration for each drug indicated. Effects on cell number for aforementioned conditions is shown in Figure S5.