https://orcid.org/0000-0003-2626-172X
This is an uncorrected proof.
Figures
Abstract
Zika virus (ZIKV), a member of the Flaviviridae family, causes significant public health concerns through congenital Zika syndrome and Guillain-Barré syndrome, yet no effective anti-ZIKV drugs or vaccines are available. To address this critical need, we conducted phenotypic, cytopathic effect-based high-throughput screening followed by medicinal chemistry optimization and discovered novel benzamide anti-ZIKV leads. Current best compounds demonstrated superior potency (EC50 values 40–400 nM, CC50 > 50 µM) compared to NITD-008, the most potent known anti-ZIKV agent. Time-of-addition assays, resistant virus selection studies, and biophysical binding experiments confirmed that NS4B interference constitutes the primary antiviral mechanism. Notably, resistance mutations mapped to the C-terminus of NS4B, distinct from other flavivirus NS4B inhibitors targeting dengue or yellow fever viruses, revealing novel insights into a critical function of the region. These findings establish NS4B as an Achilles’ heel for flaviviruses and support the development of pan-flavivirus therapeutics targeting this essential viral protein.
Author summary
Since its pandemic spread in 2015–2016, Zika virus infection remains a significant public health threat worldwide. The virus can cause severe brain damage in developing babies and serious neurological complications like Guillain-Barré syndrome in adults. Despite these devastating consequences, we currently lack effective medicines or vaccines to prevent the virus from spreading through communities or from mothers to their unborn children. To address this critical gap, we conducted a large-scale screening of chemical compounds and discovered a promising new class of molecules that can effectively stop Zika virus from replicating. Using medicinal chemistry techniques, we were able to make these compounds even more potent against the virus. In follow-up studies, we found that our compounds work by interfering with a specific viral protein called NS4B, which the virus needs to replicate its genome within the cell. Remarkably, other research teams studying related other flaviviruses (e.g., dengue and yellow fever virus) have independently discovered that this same protein is a vulnerable target. Our findings suggest that NS4B represents a universal weakness across the entire flavivirus family, making it an attractive target for developing broad-spectrum antiviral treatments.
Citation: Chung D, Otsuka Y, Kim E, Ullah S, Kennedy NM, Ngo KH, et al. (2026) Discovery of a potent anti-Zika virus benzamide series targeting the viral protein NS4B. PLoS Pathog 22(4): e1013609. https://doi.org/10.1371/journal.ppat.1013609
Editor: Volker Lohmann, Heidelberg University, GERMANY
Received: October 8, 2025; Accepted: March 21, 2026; Published: April 3, 2026
Copyright: © 2026 Chung et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All data are available in the main text or the supplementary materials.
Funding: This study was supported by funding from NIH grant U19AI171954 (to DC, LS, TPS, TDB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
1. Introduction
Zika virus (ZIKV) and other flaviviruses are important emerging pathogens of high pandemic potential. Most of these viruses use mosquitoes as a vector and are globally endemic in tropical and subtropical areas [1]. Due to climate change, the habitats of carrier mosquito species (i.e., Aedes albopictus and Aedes aegypti) are rapidly expanding along with regions of susceptibility to outbreaks and local epidemics of these diseases [2]. ZIKV was first isolated in Africa in 1947, spread to the Americas in 2015, and has been continuously circulating in many countries in South America. In year 2024, more than 44,000 cases have been confirmed in the Americas with >90% of cases in Brazil, by the Pan American Health Organization [3]. As of 2024, ZIKV infections had been reported in 92 countries; however, these infections pose a threat to ~60 additional countries based on the expanded range of the carrier mosquitoes [4], indicating a growing risk for multi-continent outbreaks.
ZIKV infection typically results in self-limiting febrile clinical outcomes [5]. However, 5–15% of infected pregnant women exposed to ZIKV have babies with Zika-related birth defects including infant microcephaly and other cognitive developmental defects, known collectively as congenital Zika syndrome. In adults, Guillain-Barré syndrome, stillbirth, and miscarriages have been reported [6–8]. ZIKV can spread via a human or urban cycle, in which infected humans drive its spread [9]. ZIKV urban epidemics occur mainly through the transmission of infectious virus in the blood to naïve individuals via peridomestic/domestic mosquitoes (Aedes spp.). Additionally, ZIKV can lead to a persistent infection, producing virus in bodily fluids that clears only after many months or even up to a year in certain cases [10–12]. ZIKV persistence in semen allows sexual transmission, passing to the mother and to a fetus, with resultant congenital Zika syndrome. Therefore, it is reasonably expected that early anti-ZIKV treatment which directly reduces the viral burden in the blood or bodily fluid of infected people will quickly minimize local viral spread, reducing pandemic potential. A treatment reducing ZIKV persistence could not only curb transmission but also reduce effects of congenital Zika syndrome, such as microcephaly. However, no antivirals nor vaccines for ZIKV infection have been approved, emphasizing a pressing need for such agents.
Phenotypic high-throughput based screening (HTS) approaches have often led to the discovery of novel antiviral compounds with new antiviral mechanisms of action, especially where no established targets are had been identified [13–15]. Prior phenotypic screens for anti-flaviviral agents identified small molecules targeting DENV (e.g., JNJ-A07, JNJ-1802 and NITD-688), and YFV (BDAA series) [16–19], several of which have demonstrated efficacy in preclinical studies and entered clinical development [20,21]. Interestingly, mechanistic studies established that the antiviral targets of each of these anti-DENV compounds is the viral NS4B protein, suggesting that targeting NS4B might also have utility for other flaviviruses. None of these molecules demonstrated anti-ZIKV activity, however, and no effective anti-ZIKV compounds are known thus far; though the nucleoside analog NITD-008, a viral polymerase inhibitor, has pan-flavivirus activity, including ZIKV [22].
Here, we present a novel benzamide lead series discovered from a phenotypic HTS for small molecules that inhibit ZIKV replication. Our mechanism of action studies showed that the series selectively targets ZIKV by binding to and disrupting the function of ZIKV NS4B. Initial screening hits with modest potency were optimized in a medicinal chemistry campaign, improving anti-ZIKV EC50 values to below 100 nM with greater than 1000-fold virus yield reduction following exposure to optimized compounds at 1 µM in cell-based assays. Our study establishes that the known importance of NS4B in DENV and YFV as a valid antiviral target extends to ZIKV. This suggests a similar key role for NS4B in other important members of the flavivirus family (e.g., WNV, JEV, and TBEV), further suggesting that opportunistically screening or optimizing currently available anti-NS4B compounds to target these other flaviviruses, for which limited or no treatment options are available, may be a viable strategy for anti-flaviviral drug development.
2. Results
1. Identification of anti-ZIKV hits from a CPE-based HTS campaign
We previously developed a phenotypic, CPE-based HTS assay for antiviral discovery for ZIKV [23]. To avoid finding host-targeting pyrimidine synthesis inhibitors, which have been often identified as poorly tractable hits in other cell-based phenotypic antiviral HTS campaigns [14,24–27], the assay media was supplemented with 50 µM of uridine. Uridine supplementation effectively subverted the antiviral effects of brequinar, a cellular dihydroorotate dehydrogenase inhibitor, in our assay (See S1 Fig). For executing an automated HTS campaign to discover new compounds with anti-ZIKV activity, the assay was successfully miniaturized to a 1536-well plate format. The assay performance was robust with the overall Z′ > 0.8, and the antiviral activity of NITD-008 (EC50 = 0.8 ± 0.037 µM, n = 10) agreed with previously published data [22].
The UF Scripps diversity small molecule library of 650,000 compounds was screened at a single concentration in the assay (S2 Fig). Active “cherry pick” compounds were retested for reproducible activity at a higher cutoff while druglikeness filters (e.g., removing high M.W. compounds, PAINS, and frequent assay hitters) removed some hits from follow-up consideration. The remaining 91 compounds of interest were repurchased from vendors and were retested (along with selected purchased structural analogs) using several lines of independent assays, including a triage of two orthogonal secondary concentration-dependent assays (a CPE-readout and a reporter-based ZIKV assay) and finally a CPE-based anti-CHIKV counter assay. These efforts gave a small set of ~25 compounds of interest with activity in one or both ZIKV assay formats, no activity against CHIKV (an unrelated alphavirus), and low cellular cytotoxicity suggestive of valid antiviral activity. We found several hits sharing a common benzamide core having significant antiviral activity in the two orthogonal assays (EC50 < 2 µM, Table 1), activity that was not compound batch dependent.
Table 1 compares data for top hits, the most potent being the low molecular weight (245) benzamide MWAC-3475, which showed EC50 values comparable to NITD-008. All compounds had no observed activity against CHIKV, where the same cell line (Vero 76) was used, suggesting that the anti-ZIKV activity of the series is not host-dependent broad-spectrum, antiviral activity, rather virus-specific.
2. Assessment of ZIKV-specific antiviral activity
To validate the observed anti-ZIKV activity and to understand the mechanism of action (MOA) of the benzamide series (termed the MWAC-3475 series hereafter), we first sought to evaluate its antiviral activity against various strains of ZIKV and against other flaviviruses, including DENV, Yellow fever virus (YFV), Japanese encephalitis virus (JEV), and West Nile virus (WNV). MWAC-3475 series compounds showed antiviral activities for all the ZIKV strains tested with an EC50 range of 0.9-9.95 µM but showed no measurable antiviral activity for other flaviviruses up to 50 µM (Table 2). This differs from NITD-008, which demonstrated a broad-spectrum antiviral activity for all tested flaviviruses with a low micromolar EC50 values. This result showed the MWAC-3475 series is a ZIKV-specific antiviral, potentially targeting a viral motif unique to ZIKV.
3. The MWAC-3475 series possesses excellent anti-ZIKV efficacy
Next, we used MWAC-3475 as a model compound for the characterization of the series in detail. Treatment of ZIKV-infected Vero 76 cells with MWAC-3475 resulted in significant, concentration-dependent, reduction in progeny virus titers, > 1000-fold reduction at 5 µM and reaching a 4.2-log reduction (or 1.78 x 104-fold) at 20 µM (Fig 1A). To validate its antiviral activity in other cells, MWAC-3475 was tested in ZIKV-infected Huh 7.5.1 cells and demonstrated strong antiviral activity, leading to a similar degree of virus titer reduction at 5 µM as NITD-008 (Fig 1B), implying that the antiviral activity of this series is independent of cell type. The antiviral activity was further confirmed at the viral protein expression level by detecting viral E protein using the microscopy approach. The intensity and abundance of viral E protein detected with the 4G2 antibody decreased in a concentration-dependent manner. (Fig 1B).
(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.
4. Compounds in the lead series inhibit viral replication in the middle stage and require de novo protein synthesis
We conducted a time-of-addition study to determine the viral replication stage at which compounds in MWAC-3475 series intervene. We first measured viral RNA levels in cells at various times after a synchronous infection with a high MOI (MOI = 3) of ZIKV (Fig 2A). A significant accumulation of viral RNA synthesis initiated at 12 h.p.i. and peaked at 36 h.p.i., indicating the most productive phase of viral replication (i.e., the middle phase) occurs during this period. Detection of viral protein by western blot also confirmed the active viral protein synthesis during this period (S3 Fig). When a test compound was added at different timepoints in relation to the infection, we found that its addition to infected cultures could be delayed without loss of antiviral efficacy as long as active intracellular viral RNA synthesis had not initiated (i.e., at 12 h.p.i., Fig 2B) without loss of antiviral efficacy. When the inhibitor was added after onset of viral RNA synthesis (i.e., 16 h.p.i.), a gradual loss of its antiviral activity was noted, suggesting an interference with the viral RNA replication step in the middle phase of replication, which was similar to the pattern of NITD-008 (Fig 2B). To understand if MWAC-3475 can inhibit viral RNA synthesis by the preexisting replication complexes or if newly synthesized viral proteins are required for its antiviral action, we tested the effect of cycloheximide co-administration, which allows to monitor RNA synthesis by the pre-existing replication complexes only. Pre-treatment of cycloheximide during the middle of viral RNA synthesis abolished the antiviral RNA synthesis-inhibitory effect of MWAC-3475, indicating the antiviral mechanism of MWAC-3475 requires new protein synthesis (Fig 2C).
(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.
5. Resistance studies implicate the viral protein NS4B as the molecular target of the MWAC-3475 series
Identifying mutants arising in selection of resistance studies is a useful method for target identification. Accordingly, we sought to select MWAC-3475-resistant isolates and to identify mutations conferring resistance. ZIKV was passed 10 times in the presence of MWAC-3475, gradually increasing concentration from 1 µM to 20 µM. During the serial passages the population maintained its infectivity, as high as > 107 pfu/mL (Fig 3A). After the final passage, the viral population showed resistance to MWAC-3475, as evidenced by the observation of plaque generation despite the presence of the compound in the overlay media (Fig 3B). Sequencing of the passage 9 virus populations from three independent passages identified MWAC-3475-specific mutations mapped within the C-terminal region of NS4B, aa 241–248 (V241L, A245T, and V248G. Fig 3C). These mutations map to the transmembrane domain 5 (TM-5). Interestingly, this site is distal from the site conferring resistance in DENV to previously developed DENV NS4B inhibitors (Fig 3D) [16–19]. Sequence homology analysis of the identified residues (Fig 3E) showed a low consensus sequence homology across members of the flavivirus family, in accord with the ZIKV-specific activity we have observed (Table 2).
(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.
For further validation, we used a reverse-genetics approach, introducing these mutations into a molecular clone derived strain (ZIKV-RLuc) and measuring susceptibility to MWAC-3475. We found that introducing these mutations within NS4B resulted in a 6- to 55-fold decrease in potency of MWAC-3475 (Fig 3F). This phenotypic resistance resulting from the mutations clearly indicates direct involvement of these ZIKV NS4B residues in the antiviral activity of the MWAC-3475 series.
6. 19F NMR studies provide support for ZIKV NS4B as the target
The flavivirus protein NS4B is membrane bound [28], a property that has hindered structure elucidation for complexes of DENV NS4B [29] with clinical anti-DENV compounds found to target NS4B. 19F-NMR spectroscopy can be used with such targets, however and was the method we chose for studying interactions of fluorine-containing small molecules with ZIKV NS4B. We first validated the method using DENV NS4B and JNJ-1802, a fluorine-containing pan-DENV NS4B inhibitor [30]. When the 19F-NMR spectra of JNJ-1802 (at 50 µM) combined with NS4B (5 µM) was compared to that of free JNJ-1802, a chemical shift perturbation and significant line broadening was seen, which are both strong evidence for direct interaction (Fig 4A). A similar study using our series of anti-ZIKV compounds required a fluorinated analog, MWAC-3533, which has modest anti-ZIKV activity (RLuc EC50 ~ 16 µM). 19F-NMR signals for this compound were shifted by the presence of ZIKV NS4B in a concentration-dependent manner (Fig 4B). Ionic strength/pH was not significantly altered in dilutions. Kd determination using 19F shifts in general aligned with the activity seen in the RLuc assay (Fig 4C). Together, these observations further support the hypothesis that this series directly binds to and inhibits the function of ZIKV NS4B.
(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.
7. FAM fluorescence labeling provides further support for ZIKV NS4B as the target
While the phenotypic resistance study strongly implicates NS4B as the molecular target of the series, we sought to further support this plausible mechanism using an in situ microscopy approach. We utilized a miniRFPnano3-tagged ZIKV NS4B and prepared a fluorescence-labelled analog of MWAC-3489, the racemic a-methyl p-methoxy benzyl derivative (Table 1). The anti-ZIKV activity and lack of cytotoxicity seen with MWAC-3986 justified a strategy of using the alkyne to link a FAM fluorophore to give a derivative suitable for direct imaging (see probe MWAC-4163, Fig 5A). First, we prepared individual isomers and found that activity resides in the S-isomer of MWAC-4168 instead of the R-isomer of MWAC-4169, that the p-methoxy group is preferred over meta (MWAC-4170) or ortho (MWAC-4138) substitution, and that extension of the OMe group to a longer alkyl chain bearing a clickable alkyne handle (MWAC-3986) slightly augments rather than erodes activity (S3 Table).
(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.
Following a period of 24–36 hours after delivery of the 2K-miniRFPnano3-NS4B polypeptide-expressing plasmid (miniRFP-NS4B, hereafter), cells were fixed, permeabilized, and incubated with MWAC-4163. The FAM fluorescence signal from the compound was shown in a punctuated pattern in cells without ZIKV NS4B expression (Fig 5C). In cells expressing miniRFP-ZIKV NS4B, however, a strong colocalization between ZIKV NS4B (shown in red) and MWAC-4163 (shown in green) was detected (Fig 5C). In a FACS-based approach with a similar set up, we found that cells expressing miniRFP-NS4B showed a significantly higher degree of binding to MWAC-4169 compared to the controls (miniRFP alone or miniRFP-NS5) or to the ZIKV NS4B-negative cell populations in the same samples (Fig 5D). A sample construct with V241L mutation showed a decrease in the MWAC-4169 positive populations, although it was not statistically significant, implying the amino acid residues in the C-terminal region of the NS4B could be responsible for the interaction with the lead small molecule series.
8. Investigation of drug synergy potential: NS4B inhibition by MWAC-3475 may synergize with viral polymerase inhibition
Combinations of antiviral drugs acting on different but critically important pathways are attractive with respect to robust and sustained efficacy with reduced resistance potential. Accordingly, we surmised that targeting ZIKV NS4B in combination with another anti-ZIKV agent with an unrelated mechanism of action would be an effective strategy. To quantify multi-drug effects, we used the concentration-responsive, CPE-based anti-ZIKV assay and evaluated synergy scores for a combination of MWAC-3475 with NITD-008 (Fig 6). Overall, the mean synergy scores were relatively moderate (e.g., ZIP score 5.47); however, we found a significant synergistic effect between the two compounds within a concentration range near their EC50 for both compounds, with ZIP and HSA synergy scores of 42.08 and 48.08, respectively (S1 Table). Combination significance scores were high (>79), indicating a potential synergistic or at least a robust additive effect from the combination [31,32]. When MWAC-3475 was instead tested together with an analog in the same series, synergy (as expected) was not observed.
(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.
9. Improvement of antiviral potency by medicinal chemistry
The potency, efficacy, ease of synthesis (from the addition of commercial p-(t-butyl) benzoyl chloride to commercial amino cyclopentane in the presence of a base), and small size of MWAC-3475 (MW 275) together justified further study and optimization efforts. We thus studied both commercial and internally prepared analogs to gauge structural features important for anti-ZIKV activity with low cellular cytotoxicity. Increasing size of the amide group from cyclopentyl to cyclohexyl to cycloheptyl gave increased anti-ZIKV activity (Fig 7), though MWAC-4001 showed appreciable cellular cytotoxicity. A commercial bicycloheptyl analog (MWAC-3508, tested as a mixture of four isomers) showed promise. Preparation of the separate endo- and exo- racemic compounds showed specificity for the exo-isomer (MWAC-3989). Several attempts to increase water solubility in the series by introducing heteroatoms into the amide region eroded antiviral activity (bottom row, Fig 7). Some of the compounds showed a significant virus yield reduction efficacy with greater than a 10-fold reduction at 1 µM in a different cell line (i.e., Huh 7.5.1), validating their antiviral activities.
VYR: Virus Yield reduction (Log 10) at 1 µM in Huh 7.5.1 cells. > 10-fold reduction highlighted in red.
To assess whether the single mutations identified with the hit compound, MWAC-3475, also confer resistance to other compounds from the series or not, we evaluated the phenotypic resistance of these advanced compounds (Table 3). All tested compounds showed a similar profile as that of MWAC-3475 with the maximum and minimum resistance by V241L and V248G, respectively, indicating the series interacts with the same binding site.
Encouraged by the antiviral activity and low cytotoxicity seen for MWAC-3989, we retained this exo-bicyclic amide group and probed the preference for the para substituent on the phenyl ring. In a focused set of seven analogs (Table 4), we found no suitable replacement for the para-t-butyl group. Further studies will broaden exploration in each region.
10. Preliminary in vitro DMPK assessment of the series
All analogs in the series were compliant with common druglikeness rules (e.g., Lipiniski’s and Veber’s rules, and their extensions) [33–35], but further in vitro assessments were warranted to validate the potential of the series for advancement. Properties and in vitro DMPK of the early bicycloheptane lead MWAC-3508 were analyzed (S2 Table). Areas for improvement may include decreasing plasma protein binding, improving stability to liver metabolism in mice, and augmenting water solubility. Nevertheless, this lead and the series appear to have potential for further development.
3. Discussion
Since the “re-emergence” of ZIKV around 2014 in Asia and the Americas, ZIKV has become endemic on a global scale. In the Americas between 2022 and 2025, an average of approximately 40,000 cases of ZIKV have been reported per year, with highest case numbers in Brazil and neighboring countries. ZIKV infection is particularly concerning due to its devastating outcomes to infants born to mothers infected by ZIKV during the pregnancy. Approximately 5% of infants born to mothers infected with ZIKV during pregnancy develop Zika-associated birth defects, including microcephaly and other various developmental deficits [36]. The horizontal transfer of ZIKV from mother’s blood to fetal brain has been shown to drive these complications [37,38]. Hence, an effective treatment to substantially lower ZIKV levels in the mother would be expected to avert development of congenital Zika syndrome in the fetus, a premise supported by results from a monoclonal antibody treatment in mouse models [39]. In addition, infected humans are a critical contributor to ZIKV urban transmission. Therapeutic or preemptive prophylactic treatments of people in epidemic regions could lower the risk of viral transmission to pregnant mothers and further contribute to curtailing of viral spread. However, no effective therapeutics or potential candidates are currently available for ZIKV. Further studies are warranted to ascertain the most effective treatment strategies, such as acute vs. prophylactic treatments. With further study, compounds related to the agents here disclosed may show promise, alone or in combination with other antivirals, in thwarting the spread of ZIKV outbreaks.
Our phenotypic HTS assay successfully discovered the direct-acting, benzamide-based antiviral hit compounds. Cellular pyrimidine synthesis inhibitors have been reported often from antiviral HTS campaigns by our group and others [14,24–26,40–42]. Our strategy of supplementing with uridine appeared successful as evidenced by the fact that only one compound from the 91 compounds selected for the concentration-response assay (Fig 1) showed a non-ZIKV specific antiviral activity (EC50-CHIKV = 6 µM).
Collectively, we demonstrated that the antiviral mechanism of action of MWAC-3475 series is interfering with NS4B through resistant mutation biochemical, and biophysical assays. Previously, NS4B has been reported as the antiviral target of hits discovered by other phenotypic assay-based HTS campaigns for flaviviruses, such as JNJ-A07 [16,30], NITD-618 [43], Compound 14a [20], NITD-668 [19] for DENV, and BDAA series [18] for yellow fever virus. It is quite intriguing to see NS4B as a convergent target from these successful various flavivirus antiviral discovery campaigns, despite large differences in chemical structures and the phenotypic resistant profiles of each. This could be due to the multifaceted biological roles of NS4B. Flavivirus NS4B is a membrane protein without known catalytic activity. It forms homomultimers [44–46] and interacts with various viral non-structural proteins, including NS1 [47], NS2B [48], NS3 [21,49,50], NS4A [51], and host proteins [52] (see a review article for the comprehensive list of proteins [53]. NS4B is expected to play critical roles in the formation of viral replication organelles (ROs) and to serve as a foundation for building ROs for RNA synthesis. These diverse interactions with critical viral proteins centered around NS4B could provide multiple druggable sites, leading to the identification of multiple hits targeting various regions of NS4B. These NS4B inhibitors including our series shares common aspects such as 1) high specificity to the target virus, and 2) inhibition of de novo formation of the viral replicase complex [17] or membrane remodeling [17], resulting in effective viral RNA synthesis inhibition. However, the resistant mutations are specific to the corresponding viruses, indicating the mode of action at the molecular level might be unique for each antiviral and its target NS4B.
The benzamide series-resistant mutations (a.a. positions of 241, 245, and 248) were found between the terminus of the potential transmembrane domain 5 (pTMD5) and the C-terminus of NS4B (a.a. 251) [46,54]. Interestingly, this region in DENV NS4B has been reported important for stable expression of NS4B on the ER via interaction with the ER membrane complex (EMC) [55]. DENV2 with NS4B N246Y (i.e., A/T mutation at 7558 n.t.) mutation was able to restore the replication ability in EMC4-deficient cells, implying the region might be critical to interact with the EMC for proper topology of potential transmembrane domains (pTMDs). The motif seems to be important for virus replication as we found that the resistant mutations affect the fitness by decreasing the peak titers between 10 and 1000-fold (S4 Table). In addition, the sequences near the C-terminal region of the NS4B are highly conserved among various ZIKV clinical isolates (S4 Fig).
In our attempt to decipher the molecular mechanisms of our benzamide series, we tested if MWAC-3475 for antiviral mechanisms demonstrated by other NS4B inhibitors such as the induction of interferon in the infected cells or disruption of NS3-NS4B interaction as shown in YFV inhibition by the BDAA series and NITD-688 in DENV inhibition. However, MWAC-3475 did not inhibit either of these steps [56]. We also tested if MWAC-3475 can affect the cleavage between NS4B and NS5, considering the fact that the resistance mutations are close to the C-terminus of NS4B. However, no such effect was detected in our assay either (S5 Fig). A follow up mechanism of action study will be warranted to understand how the series interacts with ZIKV NS4B and how the interactions result in the inhibition of viral replication in detail.
NS4B, a critical protein for replication of flaviviruses (an emerging family of RNA viruses of high pandemic potential), had been validated as an antiviral target with clinical anti-DENV compounds. Thus, it is not surprising that perturbation of ZIKV NS4B function inhibits ZIKV replication, though no prior reports of ZIKV NS4B inhibitors have emerged. Given that NS4B is involved in various functions, including protein-protein interactions, we also explored combination strategies to enhance antiviral efficacy. MWAC-3475 showed synergistic effects when used with a viral polymerase inhibitor but not with structurally similar compounds. This suggests the potential of combining NS4B inhibitors with agents targeting different pathways, focusing on enzyme inhibition of proteins such as NS3 and NS5, which often exhibit low druggability. Our discovery once again demonstrated NS4B as a vulnerable point (i.e., Achilles heels) for flaviviruses, establishing it as the valid antiviral target. Despite being an integral membrane protein lacking enzymatic activity, which complicates structure-based drug discovery, our findings provide a framework for overcoming these challenges. A well-designed screening strategy is critical for identifying active compounds and confirming the activity of the developed compounds. Iterative medicinal chemistry optimization is indispensable for improving potency and pharmacological properties. Determining the molecular target is crucial for understanding the mechanism of action and for designing potential combination treatment strategies. Both biochemical and biophysical validation assays are necessary to confirm direct compound-target interactions. Despite the progress made in current studies, further structural characterization of NS4B–inhibitor complexes will provide binding mechanisms of this series of inhibitors and support rational design of next-generation antivirals.
Our discovery showcases that a robust workflow, including phenotypic screening, compound optimization, target identification through generating resistant viruses, and NMR structural biology, can quickly lead to novel potent antiviral compounds for development. Also, our discovery of anti-ZIKV compound targeting NS4B validates NS4B as an antiviral target for flaviviruses. However, so far none of the reported NS4B inhibitors have shown a pan-flavivirus activity, indicating a challenge in developing a broad-spectrum antiviral for flavivirus targeting the NS4B. While a pan-flaviviruses drug discovery effort is warranted, we present our benzamide series as a therapeutic development candidate to address the current unmet gap for a ZIKV therapeutic. A future study to develop the benzamide series into a therapeutic candidate for ZIKV, as well as for pan-flaviviruses, would be warranted.
4. Materials and methods
Virus and cells
Vero 76 Cells (ATCC CRL-158) were maintained in Modified Eagle’s Medium with Earle’s Balanced Salt Solution and L-glutamine (MEM-E) supplemented with 10% fetal bovine serum (FBS) (Corning CellGro). Huh-7.5.1 cells (a gift from Dr. Andrew Tai, University of Michigan), HEK 293T cells (BEI, NR-9313), and HEK-Blue IFN-α/β cells (InvivoGen) were maintained in Dulbecco’s Modified Eagle Medium with 4.5g/L glucose, L-glutamine, and sodium pyruvate supplemented with 10% FBS. Cells were maintained at 37 °C in humidified incubators with 5% CO2.
Virus stocks were obtained from BEI resources and amplified once before being used for experiments. Amplified stock virus was aliquoted and stored at -80 °C until used. The ZIKV strains MR766 (GenBank: AY632535) and PRVABC59 (GenBank: KX087101) were provided by Dr. Barbara Johnson at the CDC. The following reagents were obtained through BEI Resources, NIAID, NIH: ZIKV strains of PLCal_ZV (Human/2013/Thailand; GenBank: KF993678; BEI NR-50234), Ib H30656 (GenBank:HQ234500: BEI NR-50066), FLR (GenBank: KX087102; BEI NR-50183), DENV Type4 (H241/TC; GenBank: AY947539; BEI NR-86), and YFV strain 17D (GenBank: X03700; BEI NR-116). CHIKV 181/25 was rescued from the pCHIKV181/25 plasmid (a gift from Dr. Sokoloski) following a procedure described previously [57].
A plasmid pZIKV-RLuc was a gift from the World Reference Center for Emerging Viruses and Arboviruses, The University of Texas Medical Branch. It has the cDNA of Zika virus isolate FSS13025 genome (GenBank: MN755621.1) and the Renilla luciferase gene as a reporter. The whole genome and reporter cassette was subcloned into the CopyControl pCCBAC vector by using a standard PCR-based cloning approach. Plasmid (pCC-ZIKV-RLuc) linearized with FesI was used as a template for in vitro RNA synthesis using (mMessage mRNA synthesis) following the manufacturer’s protocol with addition of 2 µL of GTP mix in a 20 µL reaction. Produced RNA was purified and approximately 5 µg of RNA was transfected to Vero 76 cells grown in a 6-well plate with Lipofectamine MessengerMax (Thermo Fisher Scientific Inc.). Rescued virus (ZIKV-RLuc hereafter) was harvested at 5 days post transfection and was amplified once for use. Virus titration was done by the TCID-50 method based on the RLuc expression with the Renilla-Glo assay system (Promega, USA).
Specific mutant viruses were generated using the Quick-change mutagenesis with the pCC-ZIKV-RLuc template (See Supportive information for the primer sequences, S5 Table). The full sequence of the resultant clone was validated prior to rescuing the virus. Virus rescue was performed as described above.
Antiviral assays
Compounds were dissolved in DMSO at 20 mM and stored at -20 °C. Antiviral potency assays (i.e., EC50 assays) were done in the 96-well format. Briefly, Vero 76 cells seeded in a 96-well plate one day prior were infected with virus at a virus-specific optimal MOI in the presence of test compounds, which were serially diluted to 8 different concentrations. The final concentration of DMSO was maintained at 0.25%. For CPE-based assays, infected cells were incubated for virus-specific optimal incubation times prior to measuring cell viability with CellTiter-Glo (Promega) to assess protection from virus-induced CPE. A different combination of an MOI and an incubation time that was optimized for consistent and robust performance was used for each virus assay. For ZIKV, an MOI of 0.05 and a 3-day incubation was used while for YFV 17D and DENV, an MOI of 0.05 and 0.1 was used with an incubation time of 4 and 6 days, respectively. For ZIKV-RLuc-based assays, cells infected with ZIKV-RLuc (MOI 0.1) were incubated for three days to measure the RLuc expression with the Renilla-Glo assay system (Promega, USA). For the virus yield reduction assay, (also known as titer reduction assay) 12-well plates seeded with 105 cells/well grown overnight were infected with ZIKV PLCal at MOI of 0.05 for Vero 76 or MOI of 3 for Huh.7.5.1 cells, then incubated for 1 hour at 37°C. The cells were washed with PBS and replenished with cell growth media supplemented 25 mM HEPES (Corning) containing test compound or equivalent volume of DMSO (final concentration of 0.25%) in triplicate. After 24 hours, supernatant was harvested, and the viral titers were determined by a standard plaque assay using methylcellulose overlay media described elsewhere [23].
Compound library
The UF Scripps Drug Discovery Library (SDDL) contains over 650,000 unique, drug-like compounds curated from 20+ commercial and academic sources, including 20,000 unique to Scripps. Compounds are selected for scaffold novelty, physical properties, and spatial connectivity, and organized into libraries focused on either specific drug-target interactions or broad compound diversity to accelerate hit-to-lead efforts. The SDDL’s diversity mirrors that of large pharmaceutical collections while incorporating insights from successful drug discovery and emerging HTS trends. It includes focused sub-libraries for major target classes (kinases, GPCRs, ion channels, nuclear receptors, hydrolases, transporters) and the Anti-viral compound library with anti-virus bioactivity, an appropriate tool for drug repurposing for new anti-virus drug discovery based on common host cellular mechanisms to promote discrete stages in life cycles. The SDDL also includes diverse chemistries (click-chemistry, PAINS-free, Fsp³-enriched, covalent inhibitors, natural products) with desirable physical properties (Rule-of-Five/Three, polar surface area). Additional sub-libraries support target de-orphaning and assay validation, including pharmacologically characterized collections (Sigma LOPAC, Tocriscreen, Prestwick) and clinically relevant/FDA-approved drugs as well as the MMV Pathogen box for neglected tropical diseases. All compounds undergo LC-MS and/or NMR confirmation to ensure quality and structural integrity for HTS campaigns.
CPE-based phenotypic high-throughput screen
To begin, 600 cells at 2.5 μL/ well of Vero 76 cells in MEM-E supplemented with 10% FBS and 1X Antibiotic-Antimycotic (Gibco 15240-062) were dispensed to 1536 well plate (Aurora EBB0-40000A) and incubated at 37 °C in humidified incubators with 5% CO2 for 24 hours. After that, 30 nL of each compound or vehicle (75% DMSO) were transferred using pintool and further incubated for 2 hours. Then, 2.5 μL/well of ZIKV PLCal at MOI 0.5 in virus dilution medium (MEM-E supplemented with 10% FBS, 12.5 mM HEPES, 100 μM uridine and 1X Antibiotic-Antimycotic) was dispensed. For the negative control wells (N = 24/plate), only virus dilution media was dispensed. Positive control wells contained virus and cells (N = 24/plate). After incubation at 37 °C in humidified incubators with 5% CO2 for 72 hours, plates were incubated for 10 minutes at room temperature, followed by dispensing 5 μL/well CellTiter Glo (Promega). Luminescent signal was detected using PHERAstar FSX (BMG Labtech).
In the primary screen, compounds were tested at a single concentration in singlicate at a final nominal concentration of 6.95 μM. Raw assay data was imported into UF Scripps’ corporate database and subsequently analyzed using Symyx software. Activity of each compound was calculated on a per-plate basis using the following equation: Percent Response of compound = 100*((Test Well-Median Data Wells)/ (Median High Control - Median Data Wells)).
A summary of the results of the primary screening assay is shown in S2 Fig. A mathematical algorithm was used to determine active compounds on a per plate basis. Three values were calculated: (1) the average activity value for all sample wells (2) 3 times the standard deviation value for the same set of wells (3) The sum of these two values was used as a cutoff parameter, i.e., any compound that exhibited greater percent inhibition than the cutoff parameter was declared active; again, this was applied to each individual plate. For the confirmation assay, we applied the same formula to determine the hit cut-off. For the titration assays, a four-parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (Symyx Technologies Inc.). The reported EC50 values were generated from fitted curves by solving for the X-intercept value at the 50% inhibition level of the Y-intercept value. Compounds with an EC50 greater than 10 μM were considered inactive, while compounds with an EC50 less than 10 μM were considered active.
Test compounds
The following compounds, listed in order of appearance in the manuscript, were purchased from the indicated vendor and tested as provided: MWAC-3400 (ChemDiv, K781-6490), MWAC-3415 (Chembridge, 7435251), MWAC-3417 (Chembridge, 7268948), MWAC-3481 (Life Chemicals, F1175-0153), MWAC-3489 (Vitas-M Labs, STL197485), MWAC-3475 (Enamine, Z32413960), MWAC-3508 (Chembridge, 6486655), MWAC-3509 (Chembridge, 7945795), MWAC-3506 (Chembridge, 5551926), MWAC-3512 (Chembridge, 9297289), MWAC-3510 (Chembridge, 9054690), and MWAC-3533 (Chembridge, 69155285).
Non-commercial compounds were prepared by direct amide coupling of a para-substituted benzoic acid, such as 4-t-butyl benzoic acid, with a commercially available amine, or alternatively by acylating the amine with a para-substituted benzoyl chloride (see Supplementary material).
Property assessments of MWAC-3508
Calculated properties (e.g., MW, LogP, tPSA) were assigned based upon input structure by the CDD (Collaborative Drug Discovery) Vault database using accepted algorithms. In vitro property assessments (CYP450 inhibition, kinetic solubility, liver microsomal stability, plasma protein binding) were conducted by WuXi AppTec, Inc. following established protocols.
Western blot
ZIKV-infected Vero 76 cells were harvested at various points post-infection and suspended in 200 µL of RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton-X100, 0.1% SDS) for lysis. Proteins in the lysates were separated by SDS-PAGE and transferred on to PVDF membranes (BioRad). After a blocking with 5% non-fat dry milk and 1% of normal goat serum in TBS-T for one hour, blots were probed with following primary antibodies in TBS-T with 5% non-fat dry milk overnight: anti-ZIKV NS4B (Invitrogen MA5-47129 1:1000), anti-ZIKV NS5 (Invitrogen PA5-143441, 1: 1000), anti-ZIKV NS3 (GeneTex, GTX13309, 1: 2000), anti-actin (Cell Signaling, 12262S, 1:1000). After washing in TBS-T, blots were incubated with HRPO-conjugated goat anti-rabbit secondary antibody (Santacruz, Sc-2054, 1: 10,000) then imaged with chemiluminescent substrate using an imager (Azure).
Realtime PCR
Total RNA from the infected cells were isolated by using RNAzol-RT (MRC, Inc.) following the manufacturer’s instruction. RNA was suspended in THE RNA Storage Solution (Invitrogen) in 50 µL/sample, and RNA was reverse transcribed to cDNA with Maxima reverse transcriptase primed using a mixture of random hexamers (0.5 µM) and oligo-dT (0.31 µM) following the manufacturer’s protocol. cDNA was subjected to real-time PCR in a multiplex format with 2X TaqMan Gene Expression Master Mix (ABI), 500 nM of ZIKV-specific primers and 250 nM of probes (FAM-tagged) [58], and a Vero GAPDH-specific primer/probe set (SUN-tagged, See Supplementary information for sequences). Realtime PCR was performed with Quanta7Pro (ABI) and analyzed by using the 2-ddCq method with three biological replicates and three technical replicates.
Time-of-addition study
Vero 76 cells cultured in 6-well plates overnight were infected with virus at an MOI of 3 for one hour. After removal of virus, cells were rinsed with PBS and replenished with fresh media (T = 0). Compound (10 µM of MWAC-3475) or DMSO was added at the times indicated in the study and the total cells were harvested at 36 h.p.i. for total RNA isolation. Quantitation of viral RNA was performed as described above with three biological replicates.
For cycloheximide treatment, cells were treated with cycloheximide (Sigma) at 12.5 µg/mL or DMSO (n = 3) at 13.5 h.p.i. At 14 h.p.i., test compound was added, and the infected cells were incubated at 37 °C for 6 hours (i.e., 20 h.p.i.). Total RNA was harvested from the cells and subjected to real-time PCR for RNA quantitation as described above.
Serial passage and mutant virus sequencing
For the initial infection, Vero 76 cells grown in 6-well plates were infected by incubating the cells with ZIKV PLCal at MOI of 1 for one hour at 37 °C. The cells were washed with PBS and replenished with virus growth media containing either MWAC-3475 or DMSO. 0.125mL of the cell supernatant was harvested to blindly inoculate for the next round of infection. The remainder of the passages followed the procedure described above. The progeny viral titers were enumerated as described above, and infected cells were resuspended in 1mL RNAzol RT (Molecular Research Center, Inc.) for total RNA purification following the manufacturer’s protocol. The cDNA was synthesized by using approximately 2 µg RNA whose secondary structures were removed by heating to 65°C for 5 minutes and cooling on ice for 5 more minutes, 2 µM random hexamers (IDT), RT Buffer, RNaseOUT Ribonuclease Inhibitor, 0.5mM dNTPs, and Maxima H-minus Reverse Transcriptase (Thermo Scientific). The ZIKV genome was PCR amplified by employing the ZIKV tiling strategy; with 0.5 µM of different primer pools [59], 0.2 mM dNTPs (IDT), and Q5 High-Fidelity DNA Polymerase and Buffer (NEBNext) on a S1000 Thermal Cycler (Bio-Rad) utilizing a protocol of: 98 °C for 30 seconds, 35 cycles of 98 °C for 15 seconds and 65 °C for 5 minutes. The amplicons from each sample were pooled and bead purified using KAPA Pure magnetic beads with a 0.8x bead-to-sample ratio (Roche) following the manufacturer’s protocol. The samples from each passage were combined to 1 mg for sequencing library preparation using the Nanopore Ligation Sequencing kit and Native Barcoding Expansion kit. Sequencing was performed using MinION Mk1b and Mk1c sequencers with R9.4.1 Flonges for each passage for 18 hours with high accuracy basecalling (Oxford Nanopore Technologies). The demultiplexed fastq files that that met the Phred quality score threshold were aligned to the reference genome sequence (ZIKV PLCal GenBank: KF993678.1) using Minimap2 [60] {REF: PMID 29750242}, and mutations and their frequencies were identified by using mpileup from samtools (now bcftools) [61]. For the sequenced samples, the average sequencing depth was 2632x, and the average coverage was 99.90%.
Purification of ZIKV NS4B
Purification of ZIKV NS4B was performed using the method described previously with slight modifications [29]. The cDNA encoding full-length ZIKV NS4B was cloned into pET15b and transformed into E. coli BL21(DE3) Rosetta T1R cells. The cells were grown in 2xYT medium, and protein was induced by adding 0.5 mM β-D-1-thiogalactopyranoside (IPTG). The cells were then cultured at 18 °C, 180 rpm for 24 hours. Recombinant protein was then purified in the presence of lyso-myristoyl phosphatidylglycerol (LMPG) micelles as described previously [29]. Protein was flash-frozen and stored in the SEC buffer containing 20 mM HEPES pH 8.0, 150 mM NaCl, 0.05% LMPG, 2 mM DTT at -80 °C.
19F-NMR experiments
To investigate the molecular interactions between NS4B and the test compounds, 19F-NMR experiments were performed on a Bruker spectrometer operating at a proton frequency of 400 or 700 MHz and equipped with a BBO probe and a cryoprobe. All measurements were conducted at 298K using standard pulse sequences from the Bruker pulse program library. 19F spectra were recorded for the compounds at a concentration of 50 µM. The changes of the spectra in the absence and presence of NS4B were recorded to probe the molecular interactions. To estimate the binding affinity, a titration experiment was conducted at a fixed concentration of 50 µM MWAC-3533 in NMR buffer (20 mM HEPES pH 8.0, 150 mM NaCl, 0.1% LMPG, 2 mM DTT, 5% DMSO, 10% D2O) in vary ZIKV NS4B concentration of 0, 2.5, 5, 7.5, 10, 15, and 20 µM. The resulting concentration-dependent chemical shift perturbations were analyzed using global nonlinear regression [62].
Microscopy analysis
For microscopy analysis plasmids were delivered to Vero 76 cells grown in cover slips by using Lipofectamine 3000 according to the manufacturer’s protocol. At 30-hours post-transfection, cells were fixed with 2% paraformaldehyde (PFA) and permeabilized with 0.1% Tween-20 in PBS for 20 minutes. Then, cells were incubated with MWAC-4163 at 10 µM in PBS for 30 minutes, followed by washing and nucleus counter staining with Hoest 33342. After mounting with a mounting media (ProLong Gold Antifade Mountant with DNA Stain DAPI, ThermoFisher), slides were imaged using a confocal microscope (Zeiss, LSM710 AxioObserver with a 63x objective).
Flow cytometric assay
HEK-293T cells plated in 12-well plates at 70–80% confluency were transfected with plasmid DNA (1 µg/well) using PEI (Polyethylenimine, 3 µg/well) following a protocol described elsewhere [63]. At 24 hours post transfection, cells were harvested by trypsinization, fixed with 2% PFA, and permeabilized with 0.1 Tween 20. After blocking for one hour with Intercept Protein-Free blocking buffer (Li-Cor), cells were incubated with MWAC-4163 in PBS with 10% blocking buffer for 30 minutes. After extensive washing, cells were analyzed on an Accuri C6 flow cytometer (BD Sciences) using channel 1 for FAM (ex488 nm/em533) and channel 4 (ex640 nm/em675LP) for miniRFP.
Combination treatment and synergy scoring
Antiviral effect was tested in antiviral assays with combinations of an 8 × 10 dose matrix format, with one column and row dedicated for a single compound treatment. Each were serially diluted 2-fold by starting from a top concentration of 4 µM. CPE-protection activity was calculated as above and synergy effect was calculated by using SynergyFinder R package (version 3.16) without baseline correction [31,32].
Construction of NS protein expressing plasmids
ZIKV NS protein expressing plasmids were constructed by using standard molecular techniques. First, a plasmid harboring ZIKV cDNA covering the region from NS2B to NS5, pCDNA-Z-NS2B/NS5, was constructed by using the Gibson cloning method with PCR amplicons of NS2B/NS5 (insert) derived from pCC-ZIKV-RLuc (see above) and pCDNA 3.1(vector). Each part was PCR-amplified by using Phusion thermostable polymerase (NEB) with primers with overlapping sequences. Other plasmids expressing various NS proteins were constructed by using the QuickChange method with pCDNA-Z-NS2B/NS5 as the template. The sequences of the final constructs were verified by the whole plasmid sequencing.
Co-immunoprecipitation
HEK-293T cells plated in 12-well plates at 70–80% confluency were transfected with plasmid DNA (1 µg/well) using PEI (Polyethylenimine, 3 µg/well) as described above. At 6 hours post transfection, cells were treated with compound by adding media containing the compound or DMSO at a final concentration of 10 µM for compound test or 0.125% of DMSO for vehicle control. The next day, the media was replaced with fresh media containing compound or DMSO and incubated further until at 30 hours post transfection. Cells were harvested and lysed in a lysis buffer (20 mM Tris pH 8.0, 150 mM NaCl, 1% DDM) with a protease inhibitor cocktail (Protease Inhibitor Cocktail III- Mammalian, RPI Corp), followed by an immunoprecipitation (IP) process using anti-DYKDDDDK Magnetic Agarose kit (Pierce) following the manufacturer’s protocol. Samples were analyzed using the Western Blot method as described above.
Interferon response measurement
Huh 7.5.1. cells were infected with ZIKV with/without test compound treatment, and the supernatants were harvested at 24 h.p.i. and tested for interferon- α/β (IFN- α/β) following the manufacturer’s protocol. HEK-Blue IFN-α/β cells (5000 cells/well) were incubated in 96-well plates with 20 µL of test samples for overnight. The following day, 20 µL of the cell supernatant from the HEK-Blue IFN-α/β cells were further incubated with 180 µL of pre-warmed Quanti-blue SEAP reagent (Invivogen) for 2 hours and the O.D. A620 was measured using a Cytation 1 (Agilent). As a positive and negative control, recombinant human IFN-α2b (InvivoGen, 30 pg/ml) and conditioned media from mock-infected Huh 7.5.1. were used.
Supporting information
S1 Fig. Effect of supplementary uridine on antiviral effect.
https://doi.org/10.1371/journal.ppat.1013609.s001
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S2 Fig. A flow diagram of various assays used in the HTS of 650K compounds and follow-up assays.
https://doi.org/10.1371/journal.ppat.1013609.s002
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S3 Fig. Time course of viral protein expression.
https://doi.org/10.1371/journal.ppat.1013609.s003
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S4 Fig. Sequence alignment of the C-terminal region of ZIKV NS4B.
https://doi.org/10.1371/journal.ppat.1013609.s004
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S5 Fig. Effect of MWAC-3475 on potential antiviral mechanisms.
https://doi.org/10.1371/journal.ppat.1013609.s005
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S1 Text. Chemistry protocols for the synthesis and characterization of non-commercial compounds that were evaluated.
https://doi.org/10.1371/journal.ppat.1013609.s006
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S1 Table. Synergy scores for MWAC-3475 and NITD-008.
https://doi.org/10.1371/journal.ppat.1013609.s007
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S2 Table. Physical properties and in vitro DMPK characteristics of MWAC-3508.
https://doi.org/10.1371/journal.ppat.1013609.s008
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S3 Table. Modification in the MWAC-3489 subseries for target ID.
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S4 Table. Peak titers of ZIKV-RLuc virus with resistant mutations.
https://doi.org/10.1371/journal.ppat.1013609.s010
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