Table 1.
Antiviral activity against ZIKV and CHIKV, and cytotoxicity of the benzamide series hits.
Table 2.
Anti-flavivirus activity of the MWAC-3475 series.
Fig 1.
Anti-ZIKV activity of MWAC-3475.
(A) Titers of progeny viruses in the supernatants (n = 4) from ZIKV-infected Vero76 cells (0.05 m.o.i. for two days, left) and Huh 7.5.1 cells (3 m.o.i. for one day, right) in the presence of various concentrations of MWAC-3475 or NITD-008. L.O.D., limit of detection. *, P < 0.05, **** P < 0.0001, One-way ANOVA Dunnett’s multiple comparisons test. (B) Decrease of the viral protein expression by MWAC-3475 treatment in ZIKV-infected Vero76 cells at 2 days post infection. Nucleus stain (blue) and anti-4G2 signal (green) were imaged using a fluorescence microscope.
Fig 2.
Inhibition of ZIKV replication by MWAC-3475.
(A) Kinetics of viral RNA synthesis in ZIKV-infected cells. Vero cells infected with ZIKV (m.o.i. = 3) were harvested at times indicated (x-axis) and the viral RNA amount was quantitated with real-time PCR. (B). Time-of-addition. Compound was added at different timepoints with respect to virus infection and harvested at 36 h.p.i. Viral RNA was measured using the real-time PCR and normalized using the 2-ddCq method. (C). ZIKV-infected cells were treated with cycloheximide (CHX) 30 minutes prior to the treatment of MWAC-3475 and incubated for 6 hours. Viral RNA was quantitated as described before.
Fig 3.
Identification of mutations conferring resistance to MWAC-3475.
(A) Virus passage scheme with increasing concentrations of MWAC-3475 for 10 passages (top). Three independent series were passaged, and the average viral titers with S.D. at each passage were depicted. (B) Plaques of the 10th passage virus populations in DMSO or MWAC-3475 were developed in the presence (bottom) or the absence (top) of MWAC-3475 in the agarose-overlay media. After three days, cells were fixed and stained with neutral red dye. (C) Locations and frequencies of mutations of virus populations. Bars represent mutations identified from population passed in the presence of mock (DMSO, blue) and MWAC-3475 (red). Mutations of interest clustered within NS4B were noted with amino acid sequences and their frequency at passage 9 with their passage series information (color coded). (D) Locations of mutations conferring resistance to various anti-flavivirus inhibitors. 2K and transmembrane (TM) domains depicted as cylinders. Anti-DENV inhibitors (JNJ-A07 and NITD-668, green and orange circle), anti-YFV inhibitor (BDAA, cyan circle), and MWAC-3475 (yellow star) resistant mutations are depicted in with respect to their relative locations to each NS4B topology. (E) Sequence alignment around the C-terminal region of flavivirus NS4B. Red boxes represent the locations of amino acids related to the activity of MWAC-3475. (F) Phenotypic resistance by the NS4B mutations.
Fig 4.
(A). Overlaid 19F-NMR spectra of JNJ-1802 in absence (blue) and presence of DENV NS4B (red). Both chemical shift perturbation and line broadening are evidence for target engagement. (B). Interaction between ZIKV NS4B and MWAC-3533 shown in 19F-NMR. (C). Measurement of Kd through titration and chemical shift perturbation analysis.
Fig 5.
In situ interaction between the benzamide series and ZIKV NS4B.
(A). Structure of MWAC-4163. (B) Diagrams of plasmids used for the experiment. (C) Plasmid encoding miniRFP-NS4B was introduced into Vero cells for expression for 30 hrs. After fixation and permeabilization, cells were incubated with MWAC-4163 (10 µM), then imaged with a confocal microscope. Cell boundaries were manually traced with blue or orange dashed lines for NS4B-negative and NS4B-positive cells, respectively. (D) 293T cells transfected with plasmids expressing proteins noted in the Fig were processed as described in above and analyzed with FACS. Cell populations were gated first based on the target protein expression (miniRFP-pos and -neg) and frequency of cells stained with MWAC-4163 (FAM signal) were calculated. ** Two-way ANOVA test for mini-RFP positive groups, Tukey multiple comparisons, p < 0.01.
Fig 6.
Synergistic effects for a combination of MWAC-3475 with NITD-008.
(A) Performance of MWAC-3475 and NITD-008 as a single agent in the dose-responses anti-ZIKV assay. (B) Dose response of antiviral effect at various concentration matrix (n = 3, per point). (C) Contour diagram of the predicted ZIP synergy scores. The analysis and graphs were generated by using SynergyFinder Plus without a baseline correction.
Fig 7.
VYR: Virus Yield reduction (Log 10) at 1 µM in Huh 7.5.1 cells. > 10-fold reduction highlighted in red.
Table 3.
Phenotypic resistance by the NS4B mutations.
Table 4.
Modification of the phenyl substituent.