Enhanced broad spectrum in vitro antiviral efficacy of 3-F-4-MeO-Bn, 3-CN, and 4-CN derivatives of lipid remdesivir nucleoside monophosphate prodrugs

Figures

• 

  Figure 1:. Structures of V2043 and RVn lipid prodrugs containing sn-1 alkyl ether or sn-2 glycerol modifications. (A) V2043 structure and R1 and R2 modifications of RVn lipid prodrug analogues. (B) Structures of R2 modifications.

• 

  Figure 2:. 3-F-4-MeO-Bn, 3-CN, and 4-CN R2 substituted compounds have increased antiviral activity against DENV and ZIKV in Huh7.5, a human hepatocyte cell line. (A-F) Antiviral dose-response curves comparing compounds that differ solely by a 3-F-4-MeO-Bn modification (A and B V2043 versus V2051), (C and D V2052 versus V2053), and (E and F V2054 versus V2055) during (A, C, E) DENV and (B, D, F) ZIKV infection of Huh7.5 cells. Antiviral dose-response curves comparing compounds that differ solely by a 3-CN-Bn or 4-CN-Bn compared to V2043 in (G) DENV or (H) ZIKV infection of Huh7.5 cells. Immunofluorescence was used to quantify changes in intracellular viral antigen, detected by staining for pan-flavivirus envelope epitope, 4G2. Data shown are generated from at least 3 independent experiments performed in duplicate. Error bars represent standard deviation. Log10EC50 values from each experiment were compared by unpaired t test (A-F) or compared to V2043 by one-way ANOVA (G and H) with Dunnett’s correction for multiple comparisons * P < 0.05, ** P < 0.01, ***P < 0.001.

• 

  Figure 3:. 3-F-4-MeO-Bn, 3-CN, and 4-CN R2 substituted compounds have increased antiviral activity against DENV and ZIKV in primary human monocyte-derived dendritic cells (moDC). (A-H) Antiviral dose-response curves for indicated compounds during (A, C, E, G) DENV and (B, D, F, H) ZIKV infection of moDCs. Flow cytometry was used to quantify changes in intracellular viral antigen, detected by staining for pan-flavivirus envelope epitope, 4G2. (I-J) Data shown are generated from at least 3 independent experiments performed in duplicate. Error bars represent standard deviation. Log10EC50 values from each experiment were compared by unpaired t test (A-F) or compared to V2043 by one-way ANOVA (G and H) with Dunnett’s correction for multiple comparisons * P < 0.05, ** P < 0.01, ***P < 0.001.

• 

  Figure 4:. 3-F-4-MeO-Bn, 3-CN, and 4-CN R2 substituted compounds have increased antiviral activity over V2043 against EBOV, NiV, and HeV. Antiviral dose-response curves for indicated compounds in in either HSAEC1-KT or TIME cells infected with recombinant reporter rEBOV-ZsG (A, E,) and rNiV-ZsG (B, F) viruses or with wild-type NiV-B (C) and HeV (D). Cells infected with recombinant viruses expressing ZsGreen1 protein (ZsG) were assayed for levels of fluorescence normalized to levels observed from DMSO-treated virus infected controls. HSAEC1-KT cells infected with wild-type NiV-B and HeV were assayed for cytopathic effect (CPE) based on cellular ATP levels using CellTiterGlo 2.0. CPE was normalized to cellular ATP levels observed in DMSO-treated uninfected controls. Data shown are representative at least 3 independent experiments performed in triplicate. Error bars represent standard deviation. LogEC50 values from each experiment were compared to V2043 by one-way ANOVA with Dunnett’s correction for multiple comparisons * P < 0.05, ** P < 0.01, ***P < 0.001.

• 

  Figure 5:. Antiviral activity of RDV, V2043, and 3-F-4-MeO-Bn and CN R2 substituted compounds against RSV and human coronavirus 229E. Antiviral dose-response curves for indicated compounds during (A-B) RSV and (C) HCoV 299E infection of indicated cell types. Data shown are generated from at least 3 independent experiments performed in duplicate. Error bars represent standard deviation. Log10EC50 values from each experiment were compared to V2043 by one-way ANOVA with Dunnett’s correction for multiple comparisons * P < 0.05, ** P < 0.01, ***P < 0.001.

• 

  Figure 6:. Relative antiviral activity of 3-F-4-MeO-Bn and CN R2 substituted compounds compared to V2043 and RVn against RNA viruses of clinical concern in several cell types. The relative average EC50 of lipid monophosphate prodrugs for the indicated virus and cell type compared to (A) V2043 or (B) RVn and median are shown. Average EC50 data are derived from at least 3 independent experiments performed in duplicate.