Respiratory Syncytial Virus Two-Step Infection Screen Reveals Inhibitors of Early and Late Life Cycle Stages

Abstract

Human respiratory syncytial virus (hRSV) infection is a leading cause of severe respiratory tract infections. Effective, directly acting antivirals against hRSV are not available. We aimed to discover new and chemically diverse candidates to enrich the hRSV drug development pipeline. We used a two-step screen that interrogates compound efficacy after primary infection and a consecutive virus passaging. We resynthesized selected hit molecules and profiled their activities with hRSV lentiviral pseudotype cell entry, replicon, and time-of-addition assays. The breadth of antiviral activity was tested against recent RSV clinical strains and human coronavirus (hCoV-229E), and in pseudotype-based entry assays with non-RSV viruses. Screening 6,048 molecules, we identified 23 primary candidates, of which 13 preferentially scored in the first and 10 in the second rounds of infection, respectively. Two of these molecules inhibited hRSV cell entry and selected for F protein resistance within the fusion peptide. One molecule inhibited transcription/replication in hRSV replicon assays, did not select for phenotypic hRSV resistance and was active against non-hRSV viruses, including hCoV-229E. One compound, identified in the second round of infection, did not measurably inhibit hRSV cell entry or replication/transcription. It selected for two coding mutations in the G protein and was highly active in differentiated BCi-NS1.1 lung cells. In conclusion, we identified four new hRSV inhibitor candidates with different modes of action. Our findings build an interesting platform for medicinal chemistry-guided derivatization approaches followed by deeper phenotypical characterization in vitro and in vivo with the aim of developing highly potent hRSV drugs.

Keywords: RSV; antivirals; drug discovery; drug screen.

FIG 1

Two-step screening of a small molecule compound library against human respiratory syncytial virus (hRSV). (a) Schematic representation of two-step infection screen of hRSV B05 enhanced green fluorescent protein (eGFP) in HEp-2 cells. (b) Primary hit candidates from the first round of infection. Each circle represents the mean value of four technical replicates. Black circles represent hit compounds according to hit criteria of MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] value (cell viability) ≥ 85% and eGFP fluorescence (infection efficiency) ≤ 12%. Synagis (2 μg/mL) was used as a positive control (empty triangles). Cell viability and infection efficiency are plotted relative to the dimethyl sulfoxide (DMSO)-treated control infections, which were set to 100%. (c) Hit compounds in second-round infection assay prioritizing for late viral replication stages (of compounds showing >60% eGFP signal in first round). Cutoff criteria for hit compounds were set at MTT ≥ 85% (round 1) and eGFP fluorescence ≤ 12% (round 2). (d) Dose-response curves of selected hit candidates against a firefly luciferase-expressing hRSV A strain Long reporter virus (rHRSV-A-Luc) (15). Black circles represent infection round 1, gray squares represent infection round 2. For both round 1 and 2 infection assays, luciferase activity was assessed 24 h postinoculation and normalized to control infections conducted in the presence of DMSO. An MTT assay was performed on uninfected cells treated with the given compound doses for 24 h (filled triangles). Means and standard deviations (SD) from three independent experiments are shown. (e) Chemical structure of primary candidate molecules. Compound 9: no stereo information available. (f) Dose-response activity of given resynthesized compounds. Assay setup as in panel d.

FIG 2

Primary hit candidates target cell entry or late stages in hRSV infection. (a) Inhibition of hRSV-F mediated cell entry in the lentiviral pseudotype asssay. Values were normalized to DMSO-treated infected wells. Synagis (10 μg/mL) was used as control for hRSV-F mediated cell entry inhibition. The compound concentrations used are summarized in Table S1. Means and SD of three independent experiments are shown. (b) Activity of hit compounds against hRSV replication/transcription as determined by an hRSV replicon assay in BSR T7/5 cells. Cells were transfected with the RSV replicon and helper plasmids or with a firefly luciferase-expressing control plasmid (pWpi F-luc). Normalized means and SD of three independent replicates are shown. (c) Schematic representation of the time-of-addition infection assay conducted as outlined above (a, b, c, d). Cells were infected with the recombinant hRSV B05 eGFP (29) reporter virus for 1 h. (d) Results from the time-of-addition infection assay. The number of eGFP-positive, infected cells was assessed by flow cytometry 24 h after inoculation and is normalized to the DMSO control infections. Mean values and SD from three independent replicates are shown. (e and f) Limiting dilution infection assay. HRSV was incubated with 100 μm of given compounds or Synagis (100 μg/mL) at 1 h before serial dilution and infection of HEp-2 cells. Cells were fixed and stained for hRSV phosphoprotein. The 50% tissue culture infective dose (TCID₅₀)/mL was calculated from the number of infected wells. Mean and SD from two to three independent replicates are shown. (g, h, i) Impact of hRSV-F protein resistance to Synagis (K272E) (h) or fusion inhibitors (K394R) (i) on the antiviral potency of selected compounds. HEp-2 cells were transduced with lentiviral pseudoviruses harboring WT F protein (g) (RSV F wt) or given resistance mutations (h, i). Increasing concentrations of the indicated compounds (black bars) were applied simultaneously. At 72 h postransduction, luciferase activity was quantified and normalized to the DMSO control (white bars). Synagis: 0.1, 1.0, and 10.0 μg/mL; Compounds 3 and 12: 0.1, 1.0, and 10.0 μm. Means and SD from three independent replicates are shown.

FIG 3

Serial passaging of hRSV induces resistance against selected compounds. (a) Schematic representations of hRSV resistance selection assay. rHRSV-A-GFP, an hRSV A strain Long reporter virus (17), was passaged in the presence of selected hit compounds under increasing concentrations for 10 rounds. In parallel, rHRSV-A-GFP was passaged in the presence of DMSO. (b) Fluorescence microscopy analysis of compound-selected rHRSV-A-GFP infected HEp-2 cells. Cells were inoculated with passage 10 rHRSV-A-GFP virus populations and treated with the compound concentrations present during the final passage (Table S3). At 48 h after virus inoculation, cells were fixed, permeabilized, and stained for hRSV phosphoprotein. Blue, DAPI (4′,6-diamidino-2-phenylindole); orange, hRSV P protein; green, GFP reporter. (c) Phenotypic resistance analysis of virus populations selected with compounds. HEp-2 cells were infected with DMSO- or compound-selected virus populations and treated with the respective compound as given above in each panel. At 24 h postinoculation, cells were fixed, and the number of GFP-positive cells was quantified by fluorescence-activated cell sorter (FACS) and normalized to that observed with DMSO control infections of these virus populations. Gray, dose-response of DMSO-passaged virus population. Black: dose-response of virus population selected with the compound listed at the top of each panel. Mean values and SD of three independent experiments are shown. (d) Cross-resistance of compound 3- and 12-selected rHRSV-A-GFP virus populations. HEp-2 cells were infected with the DMSO-selected (left) compound 3-selected (middle), or compound 12-selected (right) virus populations in the presence of 100 μM compound 3 (black bars) or compound 12 (gray bars). At 24 h postinfection, cells were fixed, and the number of GFP-positive cells was quantified by FACS and normalized to the respective infection in the presence of DMSO. Mean and SD of three independent experiments are shown.

FIG 4

Sequence analysis of compound-selected virus populations and RSV F protein cell entry resistance. (a) Virus populations derived from the initial rHRSV-GFP reporter virus after 10 passages in the presence of DMSO or the given compounds were analyzed by Illumina sequencing. Coding (black circles) and noncoding (open circles) single nucleotide variants (SNVs) were detected based on comparing the sequence reads with the initial plasmid DNA sequence. The frequency of each variant is plotted, and each variant is shown at its respective position in the RSV genome. Lines depict the number of reads at the indicated genome position. Amino acid exchanges with a frequency of ≥5% (dotted line) are labeled. The coding region of the F protein is highlighted with gray shading. (b) Lentiviral hRSV F protein pseudoparticle resistance assay. Lentiviral particles with hRSV F proteins carrying the wild-type protein (WT) or F protein variants with the K68T, L142I, or F137Y mutation, respectively, were used to transduce Huh-7.5 cells in the presence of compound 3 (25 μM), compound 12 (64 μM) (white bars, respectively), or BMS-433771 (10 μM, gray bars). At 72 h postransduction, luciferase activity was quantified and normalized to the DMSO control. Mean values and SDs from three independent replicates are shown.

FIG 5

Anti-hRSV activity of selected hit compounds in air-liquid interface cultures of immortalized BCi-NS1.1 cells and RSV-strain dependence of antiviral activity. (a) Anti-hRSV activity of selected hit compounds was assessed in differentiated BCi-NS1.1 cells (23) cultured in an air-liquid interface configuration. Cells were treated from the basolateral side with compounds (concentrations used are summarized in Table S1; 2 and 0.2 μM for BMS-433771) and simultaneously infected with the recombinant rHRSV-A-GFP reporter virus. Basolateral treatment was repeated once daily. Apical washes were collected, and cells were lysed for determination of viral genome copies by reverse-transcription quantitative PCR (qRT-PCR). Genome copies in the mucus after 8 and 96 h and in cell lysate (96 hpi) are shown. Mean values and SD from a single experiment with duplicate measurements are depicted. (b) RSV strain-dependence of the antiviral activity of selected hit compounds. Clinical isolates of RSV-A and RSV-B were used to infect HEp-2 cells (multiplicity of infection [MOI] of 1 or 0.5; dots and triangles, respectively) in the presence of compounds (concentrations as in panel a). After 24 h, infection efficiency was determined by intracellular hRSV phosphoprotein staining and flow cytometry. Bars represent the mean values. Means and SDs are given. Four to seven independent replicates are shown.

FIG 6

Broad-spectrum antiviral activity of compound 5s. (a) Huh-7.5 cells were infected with a renilla luciferase expressing human coronavirus (hCoV-229E)-Rluc reporter virus (38) in the presence of compounds at the indicated concentrations for 48 h. The cytotoxicity of these compounds was assessed by MTT assays of uninfected Huh-7.5 cells exposed to the compounds for 48 h. Values were normalized to the DMSO control and averages of three independent replicates ± SD are shown. (b) Huh-7.5_ACE2 cells were transduced with recombinant vesicular stomatitis virus (VSV)-based pseudoparticles encoding firefly luciferase and resynthesized compounds 3s (25 μM) and 5s (17 μM) or DMSO. After 4 h postransduction, medium was changed, and luciferase activity was determined after 16 to 18 h as a measure of residual transduction. (c) Huh-7.5 cells were seeded in 96-well plates and inoculated with lentiviral pseudoparticles harboring the G proteins of VSV (black bar) or rabies virus (RABV; gray bar and compound 5s (17 μM), or DMSO and inoculum was removed after 16 h. Firefly luciferase activity was determined after another 72 h. Values of three independent biological replicates were normalized to their DMSO control and means and SD are shown. EBOV, ebola virus.