Fig 1.
High throughput screening of nucleoside analogs identifies antivirals against DENV in different cell models: A.
Representative fluorescence microscopy images of DENV2 infection in the indicated cell models (A549, IMR90, HepG2, and Huh7.5).Cells were treated with vehicle or the indicated compounds and either left uninfected or infected with DENV2 at MOI 2 (Huh7.5 and A549), MOI 1 (HepG2), and MOI 5 (IMR90). At 24 hours post-infection (hpi), cells were fixed and stained for viral antigen (4G2, green) and nuclei (Hoechst 33342, blue). Images were acquired at 10 × magnification. B. Schematic of the screening strategy. 384-well format high-throughput screen was conducted using A549, IMR90, HepG2, and Huh7.5 cells to evaluate a library of 1,101 nucleoside analogs for their ability to inhibit DENV2 infection at 50 μM. Cells were treated with compounds for 2 h then infected with DENV2 at the MOI in A, followed by fixation at 24 hours post-infection. Automated microscopy was used to quantify percent of infection and cell numbers normalized to vehicle control. Percent of control (POC) for DENV2 infection in C. Huh7.5 D. HepG2 cells E. A549 cells and F. IMR90 cells. G. Venn diagram representing 68 non-redundant candidates that showed >80 inhibition of infection; > 60% viability in at least one of the screened cell models (C-F). H. Summary of hit selection and downstream validation showing the progression from 68 initial hits identified in four cell models to 23 candidates with SI > 5 based on dose–response analysis, followed by qPCR validation in selected cell models.
Fig 2.
Dose response and orthogonal validation of nucleoside analogs: Table presents the comprehensive validation panel of IC50, CC50 (ATPlite), and SI values for indicated nucleoside analogs including the 23 primary hits from the DENV2 primary screen (n = 68) that exhibited a selectivity index (SI) > 5 in at least one cell model. The panel includes 2 positive controls (NITD-008 and AT-527). The serial numbers (S. no.) correspond to compounds listed in S1 Table. Blue SI > 5 and orange SI > 10. ATPlite data shown for each drug in each cell type at 72 h. SI shown for CC50 (ATPlite)/IC50. The log fold change from qPCR assessment in cells pretreated with the indicated compounds (10 μM) vs DMSO vehicle control infected DENV (MOI = 0.05) and subject to RT-qPCR 24 hours post-infection. Data are presented as mean ± SD, showing viral RNA levels relative to the vehicle control (n ≥ 1–3 independent biological replicates). Statistical significance was determined on n ≥ 3 by one-way ANOVA with Dunnett’s correction for multiple comparisons on log10-transformed values (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).
Fig 3.
Nucleoside analog activity across DENV serotypes: A. Representative microscopy images for DENV1, DENV3 and DENV4 infection in the Huh7.5 cells were treated with either DMSO or NITD-008. Cells were fixed 24 hpi and stained for viral infection (4G2, green) and cell number (Hoechst 33342, blue).
10 × magnification. B. Table of IC50, CC50 (ATPlite), and SI values for selected drug candidates active against DENV2, evaluated in Huh7.5 cells infected with DENV1, DENV2, DENV3, or DENV4. Huh7.5 cells were infected with DENV1 (MOI = 1), DENV2 (MOI = 0.05), DENV3 (MOI = 1), or DENV4 (MOI = 0.5). The serial numbers (S. no.) correspond to in S1 Table. ATPlite data shown for each drug in each cell type at 72 h. SI shown for CC50 (ATPlite)/IC50 with blue SI > 5 and orange SI > 10. The log fold change from qRT-PCR analysis of dengue serotypes DENV1, DENV2, DENV3 and DENV4 infection for 24 h in Huh7.5 cells pretreated with the indicated compounds (10 μM) vs DMSO vehicle control. Data are presented as mean ± SD, showing viral RNA levels relative to the vehicle control (n ≥ 3 independent biological replicates). Statistical significance was determined for n ≥ 3 by one-way ANOVA with Dunnett’s correction for multiple comparisons on log10-transformed values (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).
Fig 4.
Identification of nucleoside analogs active across diverse flaviviruses: A. Representative microscopy images for Huh7.5 cells infected with WNV (NY2000) or ZIKA (MR766) and Caco-2 cells infected with YFV (17D) pretreated with either DMSO or NITD-008 (10uM). Cells were fixed 24 hpi and stained for viral infection (4G2, green) and cell number (Hoechst 33342, blue).
10 × magnification. Percent of control (POC) for (B.) WNV infection and (C.) ZIKA infection in Huh7.5 cells screened against the library of nucleoside analogs. D. Venn diagram showing 41 non-redundant candidate compounds with >80% inhibition of infection and >60% cell viability against DENV2, WNV or ZIKV, in Huh7.5 cells. E. Table of IC50, CC50 (ATPlite), and SI values for selected drug candidates against WNV, ZIKA, DENV2, infection in Huh7.5 cells and YFV in Caco2 cell with SI > 5 at least against one virus. The serial numbers (S. no.) correspond to S1 Table. ATPlite data shown for each drug in each cell type at 72 h. SI shown for CC50 (ATPlite)/IC50 with blue SI > 5 and orange SI > 10. The log fold change from qRT-PCR analysis of WNV infection; ZIKA infection YFV infection at MOI = 0.5 and DENV2 infection at MOI = 0.05 each in Huh7.5 cells pretreated with the indicated compounds (10 μM) or DMSO vehicle control at 24 hpi. qPCR data are presented as mean ± SD, showing viral RNA levels relative to the vehicle control (n ≥ 1-3 independent biological replicates). Statistical significance was determined for n ≥ 3 by one-way ANOVA with Dunnett’s correction for multiple comparisons on log10-transformed values (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).
Fig 5.
Nucleoside analog activity in primary cells: A. Representative fluorescence microscopy images of primary human keratinocytes and fibroblasts pretreated with either DMSO or 10 μM NITD-008 for 1 hour prior to infection with DENV2 (MOI = 1 for keratinocytes; MOI = 10 for fibroblasts).
48 hpi, cells were fixed and stained for viral antigen (4G2, green) and nuclei (Hoechst 33342, blue). Images captured at 10 × magnification. B. Representative fluorescence microscopy images of primary human keratinocytes pretreated with either DMSO or 10 μM NITD-008 for 1 hour prior to infection with WNV (MOI = 1). 48 hpi, cells were fixed and stained for viral antigen (4G2, green) and nuclei (Hoechst 33342, blue). Images captured at 10 × magnification. C. Table of IC50, CC50 (ATPlite), and SI values for selected drug candidates active against DENV2, and WNV evaluated in Huh7.5 cells. The log fold change from RT-qPCR assay in primary keratinocytes and primary fibroblasts pretreated with the indicated compounds (10 μM) vs DMSO vehicle control were infected with DENV2 (MOI = 0.5) or WNV (MOI = 0.5) for 48hpi. For all qPCR, data are presented as mean ± SD, showing viral RNA levels relative to the vehicle control (n ≥ 1-3 independent biological replicates). Statistical significance was determined for n ≥ 3 by one-way ANOVA with Dunnett’s correction for multiple comparisons on log10 transformed values (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).
Fig 6.
Nucleoside analog activity in the presence of exogenous nucleosides: Dose–response analysis of DENV2 infection of Huh7.5 cells with the reference (A.) and test (B.) nucleoside analogs in the presence of increasing concentrations of pyrimidine (C + U) or purine (A + G) nucleosides.
At each concentration of the indicated nucleoside analogs (0.103, 0.309, 0.926, 2.778, 8.333, and 25 µM), corresponding concentrations of purine (A + G) were co-administered at 0.21, 0.62, 1.85, 5.56, 16.67, and 50 µM, respectively, and pyrimidine nucleosides (C + U) at 0.412, 1.23, 3.70, 11.11, 33.33, and 100 µM, respectively.
Fig 7.
Resistance profiles to nucleoside analogs: A. Growth kinetics of KUNV strains amplified in Huh7.5 cells in the presence of increasing concentrations of the indicated nucleoside analogs. Red numbers = IC50s B. RT-qPCR analysis of KUNV resistant mutants (MOI = 0.5) in Huh7.5 cells pretreated with the indicated compounds (10 μM NITD-008, MK-0608, UPGNUC558; 2 uM UPGNUC255) or DMSO vehicle control 24hpi.
Data are presented as mean ± SD, showing viral RNA levels relative to the vehicle control (n = 3 independent biological replicates). Statistical significance was determined by one-way ANOVA with Dunnett’s correction for multiple comparisons on log10-transformed values (*P < 0.05, **P < 0.01, ***P < 0.001). C. Chemical structures of nucleoside analogs related to UPGNUC558 and UPGNUC255. D. Dose response curves for Huh7.5 cells pretreated with the indicated drugs infected with the KUNV strains (MOI = 1) 24hpi with black showing POC infection and green showing POC cell viability. E. Table of the sensitivity profile of each selected KUNV strain compared to wild type for the indicated nucleoside analogs (IC50 Resistant strain/ Wild type) indicating the extent of cross-resistance to structurally diverse nucleoside inhibitors.
Fig 8.
UPGNUC558 and UPGNUC255 interact with RdRp to confer antiviral activity: A. Sequencing of selected KUNV strains showing percent amino acid for each strain, gene and domain. “+” indicates that the mutation was identified by Sanger sequencing B. Modeling shows that the S604T substitution (green, with blue arrow) is localized near the active site (yellow) of the RNA-dependent RNA polymerase (RdRP) domain within the NS5 protein.
The RNA template and incoming nucleoside in the primers site are also shown (grey circle). The A534 residue (green) is located far from the active site. C. Modeling shows that the R355Q mutation (green, blue arrow) is situated in the vicinity of the RdRP active site (yellow) of RdRp region of NS5. The RNA template is also shown with the incoming nucleoside (grey circle). D. Cells transfected with wild-type or specified alleles were treated with the nucleoside analogs as indicated. Mean±SD shown (n ≥ 3) and are normalized either to wild-type controls (indicated in green stars) or to the untreated condition of the respective samples (indicated in red stars). Statistical significance was determined using one-way ANOVA with Dunnett’s correction for multiple comparisons (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).