Homoharringtonine Inhibits CVS-11 and Clinical Isolates of Rabies Virus In Vitro: Identified via High-Throughput Screening of an FDA-Approved Drug Library

Abstract

Rabies, a viral encephalitis caused by rabies virus (RABV), is 100% fatal upon the onset of symptoms. Effective post-exposure prophylaxis (PEP) measures are available, but they are often difficult to access in low-income countries. WHO estimates about 59,000 deaths due to rabies globally, and the majority are contributed by developing countries. Hence, developing drugs for the treatment of post-symptomatic rabies is an urgent and unmet demand. It is worth noting that previous efforts regarding antiviral strategies, such as small-interfering RNA, antibodies and small-molecule inhibitors, against the rabies virus have failed to show efficacy in pre-clinical studies, especially when the virus has reached the central nervous system (CNS). Therefore, drug repurposing seems to be an alternative tool for the development of new anti-rabies drugs. We validated and used a high-throughput, FITC-conjugated antibody-based flow cytometry assay to expedite the identification of repurposable new drug candidates against the RABV. The assay was validated using ribavirin and salinomycin as reference compounds, which showed EC50 values of 10.08 µM and 0.07 µM, respectively. We screened a SelleckChem library comprising 3035 FDA-approved compounds against RABV (CVS-11) at 10 µM concentration. Five compounds (clofazimine, tiamulin, difloxacin, harringtonine and homoharringtonine) were active against RABV, with greater than 90% inhibition. Homoharringtonine (HHT) identified in the present study is active against laboratory-adapted RABV (CVS-11) and clinical isolates of RABV, with an average EC50 of 0.3 µM in both BHK-21 and Neuro-2a cell lines and exhibits post-entry inhibition.

Keywords: CVS-11; RABV; clinical isolate; flow cytometry; high-throughput assay; homoharringtonine.

Figure 1

Schematic representation of (A) optimization of anti-RABV antibody dilution and (B) optimization of MOI for flow cytometry assay development. Created with NIH BIOART (NIAID Visual & Medical Arts. (10/8/2024). 96 Well Plate and Flow Cytometer. NIAID NIH BIOART Source. bioart.niaid.nih.gov/bioart/7 accessed on 24 June 2025, and bioart.niaid.nih.gov/bioart/160 accessed on 24 June 2025).

Figure 2

Schematic representation depicting the development of a flow cytometry-based high-throughput antiviral assay and cytotoxicity study. Created with NIH BIOART (NIAID Visual & Medical Arts. (10 August 2024). 96 Well Plate and Flow Cytometer. NIAID NIH BIOART Source. bioart.niaid.nih.gov/bioart/7 and bioart.niaid.nih.gov/bioart/160).

Figure 3

Optimization of a flow cytometry-based high-throughput antiviral assay. (A) Optimization of FITC-conjugated antibody dilution and (B) optimization of RABV (CVS-11) MOI. Optimum dilution of FITC-conjugated antibody and MOI required for high-throughput antiviral assay were determined in a 96-well plate using BHK-21 cells and RABV (CVS-11). The Z’ factor was calculated to determine the efficiency of the FITC-conjugated antibody dilution assay, and it was found to be 0.631 for a 1:150 dilution and 0.538 for a 1:200 dilution. **, p-value < 0.01; and ****, p-value < 0.0001.

Figure 4

Dose-response curve (DRC) generation. (A) structure of HHT, (B) EC50 determination (n = 4) of HHT in concomitant assay (HHT was added along with infection) via flow cytometry and (C) antiviral activity of HHT by conventional DFA assay in BHK-21 cells. The cells were fixed and stained with FITC-conjugated anti-RABV antibody. The RABV appears green; the cells stained with Evans blue appear red (counterstain). Representative photomicrographs were taken after 48 h of incubation. The EC50 was determined through flow cytometry using the standard protocol. For the conventional DFA test, virus titer in the supernatant was determined by DFA staining to measure the EC50, and cells were fixed and stained to corroborate the data.

Figure 5

Time of addition assay (ToA) and cell–cell infection assay in BHK-21 cells. (A) HHT at given concentrations was added at different time points such as 24 h before infection (prophylactic, −24 h), simultaneously with infection (concomitant, 0 h) and 24 h after infection (post-infection, +24 h). The EC50 was determined via flow cytometry for each condition. (B) Cell–cell infection assay; the BHK-21 cells were infected with RABV (CVS-11) in the presence of HHT and then processed for a cluster of fluorescent foci to be counted under the microscope after DFA staining. The 50% reduction of RABV (EC50) was determined compared to the virus control.

Figure 6

Antiviral activity of HHT against RABV (CVS-11) and clinical isolates. (A) Determination of EC50 of HHT against RABV (CVS-11) in Neuro-2a cells via flow cytometry and (B) determination of inhibitory activity of HHT against RABV (clinical isolates) in BHK-21 and Neuro-2a cells at 1.0 µM by flow cytometry.

Figure 7

Antiviral activity of HHT against RABV (clinical isolates) by DFA staining. For the DFA assay, the cells (BHK-21 and Neuro-2a) were infected with RABV (clinical isolates) with or without HHT (1.0 µM) and incubated for 48 h. The cells were fixed and stained with FITC-conjugated anti-RABV antibody in the presence of counterstain Evans blue. Photomicrographs were taken; the RABV appears green, and the cells appear red (counterstain). In addition, the supernatant was collected from the wells for virus titration to corroborate the inhibitory activity of HHT.