Identification of a broad-spectrum flavivirus inhibitor targeting NS2A, a previously unidentified target

Identification of a broad-spectrum flavivirus inhibitor targeting NS2A, a previously unidentified target | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Identification of a broad-spectrum flavivirus inhibitor targeting NS2A, a previously unidentified target Olivia Goethals, Doortje Borrenberghs, Kitti Wing Ki Chan, Sven Van Brandt, and 15 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5979312/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Flaviviruses, such as dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV), Japanese encephalitis virus (JEV), and yellow fever virus (YFV), constitute a significant public health concern with billions of people at risk of infection. Climate change and the expanding geographical distribution of mosquito vectors transmitting flaviviruses have increased their potential to cause large-scale disease outbreaks. The frequency and severity of disease outbreaks highlights the urgent need for a broad-spectrum antiviral agent targeting flaviviruses. In this work, we conducted a comprehensive morphological profiling of approximately 200,000 small molecules through a fluorescence-based high-content imaging platform, which led to the identification of a singular small molecule exhibiting broad-spectrum activity against flaviviruses. Subsequent hit deconvolution against DENV serotype 2 (DENV-2) revealed NS2A as a novel therapeutic target and suggested a mechanism whereby the identified small molecule inhibits the interaction between NS2A and the prM protein, revealing a previously uncharacterized antiviral mechanism of action. Biological sciences/Drug discovery/Pharmaceutics Biological sciences/Microbiology/Virology/Dengue virus Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Flaviviruses, such as dengue virus (DENV), Zika virus (ZIKV), West Nile (WNV), yellow fever (YFV), and Japanese encephalitis virus (JEV) are mosquito-borne human pathogens known for causing a wide range of diseases in humans, including dengue fever, yellow fever, and viral encephalitis. Collectively, they threaten more than half of the global population and gained increasing attention in recent years due to their ability to cause significant outbreaks and global health concerns, especially in areas that are naïve to infection 1 . The development of vaccines and therapeutics against flaviviruses has proven challenging. While licensed vaccines are available for certain mosquito-borne flaviviruses, including DENV, YFV and JEV, the vaccine development process is complicated by several factors. These include suboptimal safety profiles, the need to establish a durable protective immune response, and the occurrence of cross-reactive antibodies, which can lead to antibody-dependent enhancement of disease 2 , 3 . The COVID-19 pandemic also revealed major points of concern regarding distribution and availability of vaccines to low-income countries. In addition, the pandemic highlighted the importance of developing drugs for antiviral treatment and/or prophylaxis. Currently, no antiviral is clinically available for prevention and treatment of any of these flavivirus infections although there is “no insurmountable scientific obstacle to develop safe and effective antiviral drugs” against them (Fauci, 2024) 4 . Flavivirus virions are 40 to 60 nm in diameter structures that contain a single-stranded plus-sense RNA genome (~ 11 kb), which is translated into a single polyprotein precursor, that is co- and post-translationally cleaved into three structural proteins (capsid [C], pre-membrane [prM] and envelope [E]) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) by host and viral (NS2B-NS3) proteases 5 , 6 . The non-structural proteins form the replication complex which is associated with the endoplasmic reticulum (ER) membrane 7 – 9 . Some of the NS proteins drive the viral replication process, such as NS1 10,11 , NS3 helicase 12 , NS2B-NS3 protease 12 – 14 , NS5 methyl-transferase 15 – 18 and NS5 RNA-dependent RNA polymerase 19 , 20 . Other NS proteins (NS2A, NS4A and NS4B) have no enzymatic activity ascribed but contain transmembrane domains associated with the ER membrane, serving as scaffolds for the replication complex 8 , 21 – 23 . Apart from serving as a membrane anchoring point and being a cofactor in the formation of the viral replication complex, the NS4A protein also interacts with host factors involved in immune responses, and plays a role in viral pathogenesis, inducing cellular stress responses and apoptosis 24 . Several important functions have been ascribed to NS4B 25 . Like NS4A, NS4B plays a crucial role in the formation and organization of the viral replication complexes by aiding in the remodeling of the host cell membrane to create vesicle packets. NS4B can modulate host immune responses and has been implicated in viral pathogenesis. More recently, in vitro studies revealed that NS4B dissociates NS3 from single-stranded RNA and enhances NS3 helicase activity 26 , 27 . NS2A plays a crucial role in various stages of the viral replication cycle. The protein appears to be a central hub in the virion packaging process, by coordination of viral and host factors, host antiviral response and recruitment of the viral RNA 5 , 7 , 21 , 28 – 41 . NS2A has a documented role in viral replication, by interacting with the 3’ untranslated region (3’UTR) of the viral RNA as well as with other viral components involved in virion assembly to coordinate genome encapsulation and assembly 7 , 39 , 41 . Furthermore, NS2A is also suggested to function as a viroporin, because of its oligomerization, potentially helping to direct nascent viral RNAs to the sites of virion assembly 42 . One single virion consists of multiple copies of capsid, prM and E, therefore each NS2A molecule may aid in the coordination of the complex virion assembly process 43 . Recent advances in high-content imaging (HCI), an image-based technology in high throughput format, allow both visualization of the virus and changes in cellular phenotypes induced by compound treatment 44 , 45 . This approach has been used in recent antiviral screening campaigns and has been successful in identifying inhibitors against various viruses, such as Hepatitis B virus 46 , coronavirus 47 , 48 , and ZIKV 49 . Here, a cellular-based HCI screening campaign resulted in the identification of a novel flavivirus broad-spectrum inhibitor. Through structure-activity relationship (SAR) optimization, a lead compound was identified with broad-spectrum anti-flavivirus potency and target engagement identified NS2A as a previously unidentified target. Results High-content imaging (HCI) antiviral assay against DENV-2/16681 In a quest for novel broad-spectrum flavivirus inhibitors, a fluorescence-based HCI platform was set up (Fig. 1 A, Supplementary Fig. 1A) using DENV-2 as a representative flavivirus. Using this optimized and validated platform (Supplementary Fig. 1BC), we screened approximately 200,000 compounds at a single concentration of 25 µM, originating from the Johnson & Johnson library and which were selected based on their biological and chemical diversity (Fig. 1 B). In addition to viral infection (eGFP expression) and cellular toxicity (cell count), image-based morphological profiling of cells was carried out during a first confirmation run. Morphological profiles were evaluated based on 30 cellular features determined during screen optimization, including intensity, texture, and shape properties (Fig. 1 C) 44 , 46 – 49 . The morphological profile induced by each compound in infected cells was compared to that of non-infected controls on the same plate and a “similarity to non-infected control” (SIM2NIC) score was calculated (Fig. 1 D, E). A SIM2NIC score close to 1 reflects a high correlation in morphology to non-infected control, and direct-acting antivirals are expected to have high SIM2NIC values close to 1. Compounds blocking virus infection by more than 50%, exhibiting a decrease in cell count of less than 70% and having a SIM2NIC > 0.6 compared to infected control cells, were selected for further confirmation screening. The hit rate from the primary screening after confirmation in 4 concentrations was 1.5%, resulting in 3,082 hits, which were next tested at 10 concentrations (Fig. 1 B). A total of 1,672 hits exhibited an antiviral activity of > 50%, a cell count > 30% and a SIM2NIC > 0.6. From this pool, 1,059 hits with a SIM2NIC threshold higher than 0.8 were further evaluated (Fig. 1 B). Next, compounds were clustered by chemical similarity. Frequent hitters (i.e., compounds frequently identified as “hits” across previous internal high-throughput screening (HTS) campaigns) were eliminated and the hits were ranked based on their activity, chemical attractiveness, and available SAR. The resulting hit classes were further explored for their potential broad-spectrum flavivirus activity against DENV-1/-3/-4, ZIKV, WNV, JEV and YFV (Fig. 1 B). Identified hit JNJ-3644 shows broad-spectrum flavivirus activity One hit, 7-[(6-chloro-2-methoxy-3-quinolyl)-(4-chlorophenyl)methyl]-5-phenyl-2,3,5,6,8,8a-hexahydro-1H-indolizin-7-ol (JNJ-3644; Fig. 2 A), exhibited nanomolar (nM) antiviral potency against DENV-2/16681-eGFP in various cell lines (Vero, Huh7 and THP1-DC sign), accompanied with high selectivity (SI, selective index) (Table 1 ). JNJ-3644 has 4 chiral centers, resulting in 8 different enantiomers. As shown in Fig. 2 B, the potency of the 8 different enantiomers to inhibit DENV-2/16681 replication in Vero-GFP cells varied. The hit compound had an EC 50 value of 0.18 µM with a SI of 72, while the other enantiomers had EC 50 values ranging from 0.85 to 4.6 µM, with selective indices between 4 and 7 (Fig. 2 B, Supplementary Table 1). Next, JNJ-3644 was tested in a tetravalent dengue antiviral assay using RT-qPCR as readout, and the EC 50 for the DENV serotypes ranged from 0.089 to 1.0 µM (Table 1 ). Subsequent testing of JNJ-3644 in different flavivirus assays, showed EC 50 values of around 0.77 µM (ZIKV, JEV), 1.96 µM (YFV) and 2.65 µM (WNV). No marked antiviral activity was detected against a selection of other RNA and DNA viruses (Supplementary Table 2). More than 300 compounds based on the same chemical series as JNJ-3644 were synthesized and tested in different flavivirus antiviral assays. The compound efficacy against some of the flaviviruses varied. For instance, compared to JNJ-3644, JNJ-4840 (Fig. 2 C) had a 4–5 fold better activity against DENV-2 and YFV but with a 3-fold lower efficacy against DENV-3. Another compound, JNJ-1953, demonstrated slightly improved in vitro broad-spectrum activity (Table 2 ) and improved microsomal clearance and mitochondrial toxicity compared to JNJ-3644 (Table 3 ). For this reason, most of the further experiments were executed using JNJ-1953 as the lead compound. Like the efficacy against DENV-2/16681, JNJ-1953 retained nM potency against DENV-2 clinical isolates DENV-2/EDEN3295 and DENV-2/38865Y10 (Table 2 , Fig. 2 C). Evaluation of JNJ-1953 antiviral activity against antibody-dependent enhancement (ADE) secondary infection revealed low nM potency (EC 50 = 50 nM; Table 2 ) of the compound, suggesting the potential of the compound in preventing severe disease. Table 1 Antiviral activity of JNJ-3644 against a set of different flaviviruses Cells Virus JNJ-3644 EC 50 [µM] CC 50 [µM] SI Vero DENV-2/16681 0.14 \(\:\:\pm\:\:\) 0.083 12 \(\:\:\pm\:\:\) 5.9 86 Huh7 DENV-2/16681 0.072 \(\:\pm\:\) 0.027 4.7 \(\:\:\pm\:\:\) 1.4 65 THP1-DC sign DENV-2/16681 0.12 \(\:\:\pm\:\:\) 0.027 7.8 \(\:\:\pm\:\:\) 4.5 65 Vero DENV-1/TC974 666 0.32 \(\:\:\pm\:\:\) 0.22 6.6 \(\:\:\pm\:\:\) 1.4 21 Vero DENV-2/16681 0.090 \(\:\pm\:\) 0.020 7.9 \(\:\:\pm\:\:\) 3.2 88 Vero DENV-3/H87 0.59 \(\:\:\pm\:\:\) 0.25 4.7 \(\:\:\pm\:\:\) 0.40 8 Vero DENV-4/H241 1.0 \(\:\:\pm\:\:\) 0.60 10 \(\:\:\pm\:\:\) 9.6 10 Vero WNV/NY-99 2.7 9.7 4 Vero-GFP WNV/B956 0.58 \(\:\:\pm\:\:\) 0.54 12 \(\:\:\pm\:\:\) 6.4 21 Vero ZIKV/MR766 0.77 8.2 11 Vero-GFP ZIKV/MP1751 3.4 \(\:\:\pm\:\:\) 2.1 15 \(\:\:\pm\:\:\) 7.1 4 Huh7 YFV/17D 1.9 20 10 Vero-GFP YFV/17D 2.7 \(\:\:\pm\:\:\) 2.6 12 \(\:\:\pm\:\:\) 5.2 4 Vero JEV/SA14-14-2 0.77 9.7 13 The initial antiviral data of JNJ-3644 for WNV/NY-99, ZIKV/MR766, YFV/17D and JEV/SA14-14-2 only represent one test experiment, subsequent testing of the compound against the flaviviruses was performed in the Vero-GFP antiviral assay where mean values from at least two independent experiments are shown. EC 50 : 50% effective concentration. CC 50 : 50% cytotoxic concentration. Selectivity index (SI): ratio CC 50 /EC 50 . Table 2 Antiviral activity of JNJ-4840 and JNJ-1953 against different flaviviruses Cells Virus JNJ-4840 JNJ-1953 EC 50 [µM] CC 50 [µM] SI EC 50 [µM] CC 50 [µM] SI Vero DENV-2/16681 0.035 \(\:\:\pm\:\) 0.010 5.1 \(\:\pm\:\) 0.91 146 0.14 \(\:\:\pm\:\:\) 0.070 12 \(\:\:\pm\:\:\) 11 86 Vero-GFP DENV-1/TC974 666 0.63 \(\:\pm\:\) 0.016 7.1 \(\:\:\pm\:\) 0.73 11 0.29 \(\:\:\pm\:\) 0.12 5.4 \(\:\:\pm\:\) 2.5 19 Vero-GFP DENV-2/16681 0.023 \(\:\pm\:\) 0.090 309 0.15 \(\:\:\pm\:\) 0.020 36 Vero-GFP DENV-3/H87 1.9 \(\:\:\pm\:\) 0.87 4 0.20 \(\:\:\pm\:\) 0.12 27 Vero-GFP DENV-4/H241 1.1 \(\:\pm\:\) 0.32 6 0.36 \(\:\:\pm\:\) 0.090 15 Vero-GFP WNV/B956 0.2 \(\:\pm\:\) 0.15 10 \(\:\:\pm\:\) 0.12 50 0.28 \(\:\:\pm\:\:\) 0.15 9.0 \(\:\pm\:\:\) 0.44 32 Vero-GFP ZIKV/MP1751 0.89 \(\:\pm\:\) 0.12 15 \(\:\pm\:\) 3.7 17 1.3 \(\:\:\pm\:\:\) 1.1 > 25 19 Vero-GFP YFV/17D 0.55 \(\:\:\pm\:\) 0.076 7.1 \(\:\pm\:\) 0.26 13 2.8 \(\:\:\pm\:\:\) 0.91 13 \(\:\:\pm\:\:\) 3.9 5 Huh7 DENV-2/EDEN3295​ NT 0.21 \(\:\pm\:\:\) 0.12 61​ 288 Huh7 DENV-2/38865Y10​ NT 0.34 \(\:\pm\:\:\) 0.09 178 THP1 ADE DENV-2/38865Y10​ NT 0.048 \(\:\pm\:\) 0.0059​ Antiviral data represent mean values from at least two independently performed experiments. The toxicity of Huh7 infected with DENV-2/EDEN3295 and DENV-2/38865Y10 in presence of JNJ-1953 represents one experiment. EC 50 : 50% effective concentration. CC 50 : 50% cytotoxic concentration. Selectivity index (SI): ratio CC 50 /EC 50 . NT: Not tested. Table 3 ADME-Tox profile of compounds JNJ-3644, JNJ-1953 and JNJ-4840 Parameter JNJ-3644 JNJ-1953 JNJ-4840 CHI logD cLogP pH 2.6 2.7 6.7 3.5 6.9 2.1 5.6 Equilibrium solubility HTeqSol pH 2 (µg/mL) pH 4 (µg/mL) pH 7.4 (µg/mL) ND ND ND 94 100 62 ND ND ND Intrinsic clearance cyprotex microsome Human – Clint (µL/min/mg Prot) Mouse – Clint (µL/min/mg Prot) 39 101 347 Plasma protein binding > 99% > 99% > 99% Cytotoxic screen in HepG2 cells IC 20 (µM) 11 11 15 Mitochondrial toxicity (Glu/Gal) ratio > 100/18 (> 5.7) 99/29 (3.4) 65/33 (2) CYP inhibition in human liver microsomes, Cytochrome P450 proteins family 1A2 – CEC (IC 50 µM) 2C8 – DBF (IC 50 µM) 2C9 – MFC (IC 50 µM) 2C19 – CEC (IC 50 µM) 2D6 – AMMC (IC 50 µM) 3A4 – DBF (IC 50 µM) > 20 > 20 > 20 > 20 15 > 20 > 20 > 20 > 20 > 20 12 > 20 NT NT NT NT NT NT NT: Not tested; CHI LogD: Chromatographic hydrophobicity index; Clint: Intrinsic clearance; CYP: Cytochrome P450 JNJ-1953 acts on a viral replication step before virus secretion. As a first step towards understanding the antiviral mechanism of JNJ-1953, a time-of-addition (TOA) experiment was performed to map the viral replication step(s) inhibited by the compound as depicted in the schematic (Fig. 3 ; Top). The Fig. 3 bottom panel shows the antiviral effect of JNJ-1953 added at different timings during infection. Pre-exposure of cells to 5 µM of JNJ-1953 2 hours before infection (pre-treatment, Pre-T, -2 hours) or co-treatment (Co-Treatment, Co-T, 0 hours) during virus infection resulted in > 90% virus inhibition which can be attributed to the high potency of the compound upon cell entry, similar to the well-characterized RNA virus polymerase inhibitor NITD-008 50 . Notably complete virus inhibition was observed when JNJ-1953 was added between 2 to 10 h post-infection (Post-Treatment, Post-T), a time window that corresponds to the release of viral RNA, protein translation and initiation of RNA synthesis 51 . Only a partial virus inhibition of ~ 40% was observed when the compound was added at 19 h post-infection, a timepoint when newly formed virions are thought to be secreted 51 . Collectively these results suggest that JNJ-1953 exerts its antiviral activity in replication step(s) before virus secretion. Resistance-associated mutations point NS2A as the target of JNJ-1953 To identify the molecular target of JNJ-1953, drug-resistant DENV-2 variants were selected during in vitro resistance selection (IVRS) experiments. CPE was observed starting at passage 18 and completely present at passage 28 in cells treated with 2.5 µM JNJ-1953, suggesting the emergence of JNJ-1953-resistant DENV-2 (Fig. 4 A). Following JNJ-1953 exposure, passage 18 and 28 viruses are subsequently characterized using next-generation sequencing (NGS). Sequence alignment between the parental and drug-resistant DENV-2 viruses revealed 12 different single-nucleotide amino acid substitutions (Supplementary Table 3), three single-nucleotide amino acid substitutions were found in NS2A: F18L, E21G, and A32V, which were not present in the in-parallel-passaged untreated cultures and compared to the other drug-resistant substitutions were present in all drug-resistant variants (Supplementary Table 3). NS2A has multiple transmembrane helixes and the three observed mutations (NS2A F 18 L , NS2A E 21 G and NS2A A 32 V ) are predicted to locate close to transmembrane domain 1 and 2 (Fig. 4 B). Alignment of 17,328 NS2A protein sequences of the different flaviviruses (DENV serotypes 1–4, JEV, WNV, YFV and ZIKV) collected from the Bacterial and Viral Bioinformatics Research center (BV-BRC) database (Fig. 4 C) showed 96.8% (99.8% among DENV-2 sequences) conservation for Glutamic acid (E) 21 (NS2A E 21 ). Interestingly, no Glycine (G) was observed in any of the flavivirus NS2A sequences at amino acid position 21 (Supplementary Table 4). For the amino acid at position 32, 99.8% of the DENV-2 NS2A sequences had an alanine (NS2A A 32 ) (Fig. 4 C), while 0.64% of the flavivirus NS2A proteins contained a valine (V) at this position (Supplementary Fig. 2). For the amino acid at position 18 of the NS2A protein, 99.35% of the DENV-2 NS2A sequences had a phenylalanine (NS2A F 18 ) at position 18 (Supplementary Table 4). However, the conservation of this F was remarkably lower (38%) among all flaviviruses (Fig. 4 C, Supplementary Fig. 2), and a leucine (L) is observed in approximately 16% of the different flavivirus NS2A protein sequences (Supplementary Fig. 2), and in 100% of the DENV-3 NS2A sequences (Supplementary Table 4). To confirm that the F18L, E21G, and A32V mutations within DENV-2 confer resistance to JNJ-1953, the three mutations (NS2A F 18 L , NS2A E 21 G , and NS2A A 32 V ) were inserted separately or in combination (NS2A E21G/A32V and NS2A F18L/E21G/A32V ) into a subgenomic DENV-2/16681 reporter replicon using site-directed mutagenesis. The antiviral activity of JNJ-1953 against these mutants was determined in a transient replicon assay. While JNJ-1953 efficiently inhibited wild-type (WT) DENV-2/16681 replication, all mutant viruses showed a 9–13 fold reduced susceptibility to JNJ-1953 (Table 4 ), suggesting that the mutations (NS2A F 18 L , NS2A E 21 G and NS2A A 32 V ) compromises viral replication. In addition, similar reduced susceptibility to compounds JNJ-3664 and JNJ-4840 are obtained (Supplementary Table 5). Given that L18 exists naturally in NS2A of some flaviviruses, only the single mutants (NS2A E 21 G and NS2A A 32 V ) and a double mutant virus containing (NS2A E21G/A32V ) were engineered into a cosmopolitan DENV-2/Eden3295 clinical isolate background. Analogous to the mutants of the subgenomic DENV-2/16681 reporter replicon, the DENV-2/Eden3295 NS2A single and double mutant virus were less susceptible to JNJ-1953 (NS2A E 21 G : 45-fold, NS2A A 32 V : 8-fold and NS2A E21G/A32V : 19-fold higher EC 50 ) compared to the WT virus (Table 4 ). Collectively these results clearly indicate that the IVRS-identified NS2A mutations are responsible for conferring JNJ-1953 resistance. Given the important functions of NS2A within the replication complex as well as acting as a chaperone in coordinating virion assembly, the impact of the NS2A mutations E21G and/or A32V on virus replication were examined. The single mutants NS2A E 21 G or NS2A A 32 V have no impact on the viral RNA synthesis or infectious virus production (Fig. 4 D) compared to WT virus. Interestingly the double mutant exhibited a ~ 2-log 10 lower RNA synthesis and infectious virus production as compared to WT virus (Fig. 4 E), suggesting that the mutations at the NS2A positions 21 and 32 may act synergistically to affect virus replication. Table 4 Antiviral activity of JNJ-1953 against wild-type and mutant DENV-2 subgenomic constructs and DENV-2/Eden3295 pFK-sgDVs-R2A DENV-2/Eden3295 Mutations EC 50 [µM] FC EC 50 [µM] FC Wild-type 0.38 0.12 E21G (0%) 4.5 12 5.4 45 A32V (< 0.2%) 4.0 10 0.9 8 F18L (16%) 3.5 9 ND E21G/A32V 4.4 12 2.3 19 E21G/A32V/F18L 5.0 13 ND ND: Not determined; FC: Fold change compared to wild-type. JNJ-1953 interferes with the recruitment of the prM viral structural protein As NS2A is reported to interact with viral prM and E proteins to orchestrate viral assembly 40 , a possible hypothesis is that JNJ-1953 plays a role in affecting the interaction between NS2A and prM. To test this hypothesis, a c-myc prM plasmid was co-transfected with either FLAG wild-type NS2A or the double mutant NS2A E21G/A32V plasmid (Fig. 5 A) into HEK293T cells for co-immunoprecipitation (CoIP) following treatment with 10 µM JNJ-1953 at 6 h post-transfection (Supplementary Fig. 2). CoIP showed that prM specifically pulled-down NS2A (Fig. 5 B; Lane 2), consistent with a previously reported finding 40 . A reduction in the amount of wild-type NS2A pulled down by prM was observed when JNJ-1953 was added during transfection (Fig. 5 B; Lane 3) and shown by densitometric analysis (Fig. 5 C), indicating that JNJ-1953 interferes with the interaction between prM and NS2A. Intriguingly, pull down of NS2A E21G/A32V by prM was less affected, albeit not significantly, upon JNJ-1953 treatment than that of wild-type NS2A (Fig. 5 B, lane 6 versus lane 3; Fig. 5 C), suggesting that the NS2A E21G/A32V interaction with prM is less sensitive to JNJ-1953 (Fig. 4 B). These experimental results corroborate with the finding that JNJ-1953 is less potent against the NS2A E21G/A32V double mutant virus (Table 4 ). We further confirmed the finding by performing a CoIP experiment of prM and wild-type NS2A interaction by comparing the effect of the active JNJ-1953 with one of its inactive enantiomers, JNJ-2005 (EC 50 = 5.0 µM; CC 50 = 8.6 µM). As shown in Fig. 5 B lane 4, the inactive enantiomer does not affect the interaction of prM with WT NS2A unlike JNJ-1953 (Fig. 5 C), confirming the specific inhibitory activity of JNJ-1953 on prM-NS2A interaction. Drug-like properties of JNJ-1953 Profiling of JNJ-1953, JNJ-4840 and JNJ-3644 in several first-line absorption, distribution, metabolism, and excretion (ADME)-Tox assays indicated suboptimal drug-like properties for the series (Table 3 ). The compounds are very lipophilic as shown by the high cLogP and CHI log D pH2.6 values. JNJ-1953 had good metabolic stability in human liver microsomes (< 7.7 µL/min/mg) and moderate stability in mouse liver microsomes (30 µL/min/mg) which was superior compared to the metabolic stability in human and mouse liver microsomes for JNJ-3644 and JNJ-4840 (Table 3 ). JNJ-1953 showed pH-dependent solubility, with good solubility at pH2 and lower solubility at physiological pH (62 µg/mL). Finally, the cytotoxicity in HepG2 cells was high and displayed potential for mitotoxicity for all compounds tested within the series. Discussion Flaviviruses have a large socio-economic burden of disease. Despite considerable efforts in the past two decades, no clinically approved antivirals are available for flavivirus treatment or prophylaxis, and currently, patients’ options for treatment are limited to measures that solely alleviate symptoms. Therefore, the search for new antivirals targeting endemic, emerging and potential future endemic flaviviruses remains a high priority (Fauci, 2024) 4 . Here, we present a novel series of broad-spectrum anti-flavivirus small molecule inhibitors targeting NS2A, with a novel mechanism of action. Resistance selection and reverse genetics studies pinpointed NS2A as the molecular target. No enzymatic activity has been shown to be associated with NS2A. We here demonstrated that the compound affects the NS2A-prM interaction, thereby hypothesizing its mechanism of action influences virion assembly. In this paper, a multi-parametric HCI-based HTS approach 44 – 49 , 52 , 53 based on DENV-2 is described to identify novel antiviral candidates active against a wide range of flaviviruses (Fig. 1 ). Compared to single-parametric, cell-based assays which are often used in phenotypic screening campaigns, the multi-parametric readout generated here is used to determine the individual compounds morphological profiles, thereby enabling an early deprioritization of hits with unfavorable mechanism of actions ( e.g. targeting the host cell instead of the virus) or undesirable cell phenotypes caused by the compound ( e.g. changes in nucleus, cytoplasm, toxicity). This approach maximized at the early stage of screening, the selection of direct-antivirals bocking the virus without any effect on the cell, thereby minimizing the risk of downstream failure, which led to the identification of JNJ-3644 (SIM2NIC: 0.98). This compound was identified with promising broad-spectrum flavivirus activity (Table 1 ; Fig. 2 ). Several compounds of the same series as JNJ-3644 were synthesized and JNJ-1953 was selected as lead compound because of its improved features over the other compounds, such as a good solubility at low pH and good metabolic stability in human (< 7.7 µL/min/mg) (Table 2 & 3 ; Fig. 2 ). Concerning the antiviral mechanism of action of the series to which JNJ-1953 belongs, an IVRS experiment identified three amino acid substitutions in NS2A (F18L, E21G, A32V) that confer DENV-2 resistance to JNJ-1953 (Fig. 4 A), pointing towards impairment of NS2A function as the mechanism of action of this compound. NS2A is a nonenzymatic integral membrane protein 43 , with three main functions reported (i) NS2A antagonizes the host immune response 30 , 31 , 33 , 36 , (ii) NS2A functions in viral RNA synthesis 7 and (iii) NS2A plays a role in virion assembly 21 , 29 , 39 . NS2A has a very low sequence identity among all flaviviruses (21–63%). The low conservation of the drug target can impose challenges for drug development such as a low specificity of the drug for the target, a low barrier to resistance of the drug and large differences in drug effectiveness against different strains or species. Sequence alignment of NS2A proteins from ZIKV, DENV-1-4, WNV, JEV, Saint Louis encephalitis virus (SLEV), tick-borne encephalitis virus (TBEV), and YFV revealed high diversity: only 31 out of 218–227 amino acids had a consensus of more than 80% among the different flaviviruses 43 , 54 . Interestingly most of the highly conserved residues are located between residues 12 to 100 of NS2A (i.e. N-terminal half of the protein), which is where we identified the resistance mutations for JNJ-1953. In this region two conserved basic residue clusters (residues 17–22 and 95–104) are known to be involved in virion assembly 28 . It is important to note that the three resistance mutations NS2A F 18 L , NS2A E 21 G and NS2A A 32 V are located near or within this conserved N-terminal basic cluster. Residue E21 is highly conserved among the different flaviviruses, except for YFV (V21; Fig. 4 C), and based on the IVRS experiment, we postulated that this residue plays a highly important role in the virus replication cycle. However, introduction of a Glycine on position 21 (NS2A E 21 G ), did not affect the viral RNA synthesis or plaque formation in reverse engineered viruses (Fig. 4 D and 4 E). This was confirmed in ZIKV, where Zhang et al . mutated E22 to A, where E22 corresponds to E21 in DENV. The E22A mutant replicated similarly as the wild-type virus, with comparable infectivity and infectious virus production to that of wild-type virus 43 . In contrast, Wu et al . concluded that the introduction of alanine substitutions at positions 21–23, showed a > 1,000-fold reduction in virus yield and an absence of plaque formation 55 . Intriguingly, the combination of the NS2A E 21 G and NS2A A 32 V mutations (NS2A E21G/A32V ) rather than the single mutant viruses (NS2A E 21 G and NS2A A 32 V ) exhibited an attenuated level of viral replication and plaque formation (Fig. 4 D, E), suggesting a synergistic effect of both mutations, underscoring the importance of the N-terminal basic cluster in virus replication. The structure of NS2A has yet to be determined and the latest AlphaFold2-predicted structures (Fig. 4 B) propose multiple membrane-spanning segments that contrasts with the predicted membrane topology models described in literature 28 , 37 , 43 , 54 . Based on the membrane topology models of NS2A from different flaviviruses, the location of the N-terminal basic cluster which encompasses the resistance mutations, may vary between viruses. For example, the NS2A residues ~ 20 to 25 from YFV are predicted to be cytoplasmic 37 , whereas for DENV and ZIKV this region is predicted to be in the ER lumen 43 , 54 . This could be a possible explanation why our medicinal chemistry exercise experienced difficulties in improving overall broad-spectrum flavivirus activity (Tables 1 and 2 ). Because of the differences in topology for the different flaviviruses, NS2A is suggested to mediate important protein-protein and protein-RNA interactions with factors present in the cytoplasm as well as in the ER 28 . NS2A is involved in various stages of the viral replication cycle, one of them being virion assembly. Here we showed that by time of drug additions studies that JNJ-1953 is acting on a step after replication is initiated (Fig. 1 3A, B). More specifically we showed that the addition of JNJ-1953 diminished the interaction between NS2A and prM (Fig. 5 B, C), suggesting that the mechanism of action of JNJ-1953 is related to virus assembly. Remarkably, when an inactive enantiomer of JNJ-1953 was used, the interaction between NS2A and prM was not affected (Fig. 5 B, C). In the same experiment, we also evaluated the interaction between the mutated NS2A (NS2A E21G/A32V ) and prM, which was not affected upon treatment with JNJ-1953 (Fig. 5 B, C). This aligns with the general model described by Xie et al . for flavivirus virion assembly, in which at late stage of flavivirus infection cycle, the NS2A molecules (bound to vRNA) at the assembly site recruit C-prM-E through binding to prM 40 . Whether disruption of the interaction between NS2A and prM is caused by the binding of the compound to NS2A and consequently blocking the association with prM, or by more indirect effects during the recruitment of host and viral proteins to the sites of virion assembly or other mechanisms remains to be determined. The antiviral flavivirus drug discovery has advanced significantly over the last years. Although there is currently no flavivirus antiviral treatment available, several compounds with different modes of action have been described 56 . The ER supports various steps throughout the whole flaviviral life cycle and provides different opportunities for anti-flaviviral drug development. The transmembrane proteins NS4A and NS4B are considered main drivers in the formation of the replication complex within the ER and were suggested to contribute to membrane rearrangements and stabilize the pore-like opening 57 , 58 . NS4B has been a well-known target 59 , with one molecule, Mosnodenvir, in phase 2 clinical trials targeting Dengue 26 . Mosnodenvir, a highly potent pan-dengue inhibitor blocks the NS3-NS4B interaction within the viral replication complex 60 . Mosnodenvir showed activity in vivo in mice and in non-human primates and was found to be safe and well tolerated in phase 1 clinical trials 26 . Furthermore, it was found to have a high barrier to resistance. Drugs targeting NS4A are also under evaluation, including compound B and SBI-0090799 which are active in vitro against DENV and ZIKV by preventing NS4A involvement in replication complex formation 61 , 62 . To our knowledge, no small molecules have been identified that target NS2A, thus highlighting the potential of this series of molecules targeting a novel mechanism of action. Furthermore, broad-spectrum activity is a desirable feature to prepare for a next flavivirus epidemic, which could emerge from as-yet unknown or neglected viruses. The design and development of new anti-flavivirus compounds must consider the broad-spectrum activity because the most promising candidates will be those capable of inhibiting a large panel of the most pathogenic flaviviruses, allowing their use in endemic areas in which multiple flaviviruses exist simultaneously. It is likely that an effective solution for combating flaviviruses will not arise from a single agent, but rather from a strategic combination of different measures. Considering the escape potential of RNA viruses, it is prudent to explore antiviral strategies that integrate targeting a viral component with modulation of host cell factors. Additionally, combination therapies that involve synergistic effects, vaccine development, or the use of monoclonal antibodies could further improve the overall management of flavivirus infections. Furthermore, employing measures to alleviate symptoms, such as supportive care and symptomatic treatments, can enhance patient outcomes. In conclusion, our study has successfully identified a series of small molecule inhibitors demonstrating broad-spectrum activity against flaviviruses by targeting the critical interaction between the NS2A and prM proteins. This finding positions NS2A as a promising novel target for future drug development efforts. However, to translate these discoveries into effective therapeutics, further research is essential. Deeper insights into the mechanism of action and the optimization of inhibitor potency will be pivotal for advancing these compounds into clinical development. By laying this groundwork, we open the door to innovative therapeutic strategies that could significantly improve outcomes for patients affected by flavivirus infections, ultimately contributing to global health efforts in combating these pervasive viral threats. Methods Cells and growth conditions Adenocarcinoma human alveolar basal epithelial cells (A549; CCL-185, ATCC) were cultured in RPMI-1640 (Gibco, Invitrogen Corp.) supplemented with 10% (V/V) fetal bovine serum (FBS, Biowest), 2 mM Ala-Glutamine (Sigma), 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes, Sigma) and 0.02 mg/mL gentamicin (Gibco). African green monkey kidney cells (Vero; CL 84113001, European Collection of Authenticated cell cultures (ECACC) and VeroE6; CRL-158, American Type Culture Collection), were cultured in Eagle’s minimal essential medium (MEM; Gibco) supplemented with 10% (V/V) FBS, 2 mM Ala-Glutamine (Sigma) and 0.02 mg/mL gentamicin (Gibco). Vero NS4B-NS5-Tat_LTR-eGFP/hRLuc stable cells, referred to as Vero-GFP, contain a stable expressed NS4B-NS5-Tat and an LTR-eGFP/hRLuc gene. Cells are maintained as described for Vero cells, supplemented additionally with 500 µg/mL geneticin (Gibco) and 200 µg/mL hygromycin (ant-hg-1, InvivoGen). In antiviral assays, the 10% (V/V) FBS is replaced by 2% (V/V) FBS and no hygromycin or geneticin is added. Huh7 hepatoma-derived cells (Sigma) were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco), supplemented with 10% FBS, 2 mM Ala-glutamine, 1 mM sodium pyruvate (Gibco) and 0.02 mg/mL gentamicin. In the antiviral assay, DMEM medium is used supplemented with 10% (V/V) FBS. BHK-21 cells (baby hamster kidney fibroblast cells, ATCC) was cultured in RPMI-1640 medium (Gibco) supplemented with 10% (V/V) FBS and 1% penicillin-streptomycin (P/S). HEK293T (human embryonic kidney, ATCC) cells were maintained in DMEM medium (Gibco) supplemented with 10% (V/V) FBS, 1% P/S and 4.5 g/L glucose. THP-1 dendritic cell-specific intracellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) cells (TIB-202n, ATCC) were propagated in RPMI-1640 supplemented with 10% (V/V) FBS, 2 mM Ala-Glutamine (Sigma), 25 mM Hepes and 0.02 mg/mL gentamicin. All above-described cells were cultured at 37°C with 5% CO 2 in a humified incubator. C6/36, an Aedes albopictus cell line (ATCC), was maintained in RPMI-1640 medium with 10% (V/V) FBS, 25 mM Hepes and 1% P/S at 28°C in the absence of CO 2 . All cell lines were regularly tested for mycoplasma contamination. Virus The following flavivirus strains and constructs were used in this study: DENV-1/TC974 #666 (National Collection of Pathogenic Viruses (NCPV) 0411281v; GenBank accession: AF180817), DENV-2/16681 (GenBank accession: NC_00174; licensed from Dr. R. Bartenschlager 63 , DENV-2/Eden3295 (GenBank accession: EU081177, 64 , DENV-2/38865Y10 (Obtained through an MTA with Environmental Health Institute, Singapore), DENV-3/H87 (NCPV 9911281v; GenBank accession: M93130), DENV-4/H241 (NCPV 9910102v; GenBank accession: AY947539), WNV/NY99 (UVE/WNV/1999/US/NY 385 − 99 (001v-EVA140); GenBank accession: AY842931; European Virus Archive(EVAg)), WNV Uganda B956 (UVE/WNV/1940/UG/UG 956 D117 (001v-EVA1461); GenBank accession: M12294; EvaG), ZIKV/MP1751 (NCPV 1308258v; GenBank accession: KY288905), ZIKV/MR766 (GenBank accession: DQ859059; EVAg), YFV/17D-204 Stamaril® vaccine, lot H5105 (GenBank accession: MN708488; Sanofi Pasteur). JEV/SA14-14-2 (GenBank accession: AF315119.1) was generated at KU Leuven using synthetic, overlapping DNA fragments, as described previously 65 . DENV-2/16681-eGFP, carrying an enhanced green fluorescent protein (eGFP) at the amino terminus of the capsid protein, was produced by transfection of in vitro -transcribed RNA of plasmid pFK-DV-G2A into Huh7 cells 63 . The DENV subgenomic reporter replicon (sgDVs-RLuc) consist out of a plasmid (denoted pFK, sgDVsR2A) which contains the non-structural genes NS1-NS5 of the DENV-2/16681 strain and the Renilla luciferase (RLuc) reporter gene. The sgDVs-RLuc was used to perform a transient DENV replicon assay 63 . Compounds A compound library at Johnson & Johnson consisting of 197,135 small-molecule antivirals was used for HCI-based screening. Compound JNJ-3644 and compounds of the same chemical class were synthesized in house. Reference compounds such as, Compound 24 66 , Ribavirin 67 , NITD-008 50 , 2-CMC 68 , JNJ-1A 69 , and Brequinar 70 were synthesized in-house. All compounds were > 95% pure, which was confirmed using liquid chromatography-mass spectrometry and proton nuclear magnetic resonance. High content imaging (HCI) For the primary HTS, 1,100 A549 cells/well were seeded in barcoded 384-well carrier plates (Cellcarrier-384, PerkinElmer), the cells were left to adhere at 37°C for 24 hours. Prior to infection, DENV-2/16681-eGFP virus was added to pre-spotted compound 384-well proxiplates (PerkinElmer). After 2 hours, the virus (MOI 0.15) preincubated with compound (25 µM) was transferred to the carrier plates. After 72 hours of incubation, cells were used for HCI assays. First, live staining was performed by incubating the cells with 50 nM MitoTracker Orange (Thermo Fischer Scientific) for 45 minutes at 37°C. Next, cells were fixed with formaldehyde (2% final concentration; Polyscience) at room temperature for 20 minutes and washed. Plates were then subjected to permeabilization (0.1% Triton-X100). For cell demarcation, nuclei were stained by Hoechst (3.5 µg/mL Hoechst 33258, Invitrogen) and entire cells by HCS CellMask ™ Deep Red (1 µg/mL CellMask ™ Deep red, Invitrogen) (Supplementary Fig. 1A). After staining, plates were imaged on the Cell Voyager 7000 (Yokogawa) confocal microscope, followed by data analysis. HCI data was analyzed with Phaedra HCI analysis software 71 . eGFP positive cells were determined based on the fluorescent signal compared with background signal from the non-infected control cells. The percentage of infected cells was calculated by taking the ratio of infected cells to the total number of cells determined by cell segmentation (Supplementary Fig. 1B). Assay quality was further assessed using the Z prime for the percentage of GFP fluorescent cells (Supplementary Fig. 1B). The assay was validated using reference compounds with established in vitro activity against DENV-2, including compound 24 66 , Ribavirin 67 , NITD-008 50 , 2-CMC 68 , and JNJ-1A 69 (Supplementary Fig. 1C). SIM2NIC analysis The similarity to non-infected control (SIM2NIC) analysis quantifies the extent to which the morphology of infected, compound-treated cells resembles the morphology of non-infected cells. A high similarity, with a SIM2NIC close to 1, indicates a clean antiviral. First, 600 features capturing intensity, shape and texture properties at the single-cell level were extracted using all 4 fluorescent channels (Hoechst, HCS CellMask™ Deep Red, MitoTracker Orange, eGFP reporter virus) and averaged across all cells per well using a custom-written Acapella (PerkinElmer) image analysis script. Then, features were normalized as z-scores relative to the infected controls/plate. Feature selection was done by data-driven minimum redundancy maximum relevance algorithm as described in Cox et al. (2020) 44 on all tested compounds with ≥ 2 replicates and ≥ 50% virus inhibition (at any concentration) and resulted in 30 reproducible and non-redundant features, which define the “morphological profile” of each treatment (compound at concentration) and control well. Finally, each morphological profile was compared to the median profile of the non-infected control wells on the same plate by Pearson correlation, resulting in SIM2NIC score. To summarize SIM2NIC at the compound level, the maximum SIM2NIC score overall concentrations at which the compound achieved ≥ 50% virus inhibition and retained ≥ 30% cell count (relative to infected control) was computed and plotted. Compounds with SIM2NIC score > 0.8 were considered for further screening. Antiviral assays After hit identification, the antiviral activity was determined against DENV-2/16681-eGFP on three different cell types (Vero, Huh7, and THP-1/DC-SIGN). In brief, 2,500 Vero or Huh7 cells or 7,500 THP-1/ DC-SIGN cells were seeded in 384-well black view plates (Corning, Sigma Aldrich) containing 200 nL of compounds in a 9-fold serially dilution. For Vero and Huh7 cells, the seeded plates were first incubated for 24 h at 37°C, before being infected with DENV-2/16681-eGFP at a multiplicity of infection (MOI) of 0.5 (Vero) or 5 (Huh7). THP-1/DC-SIGN cells were infected immediately after seeding of the cells with DENV-2/16681-eGFP (MOI 0.5). After three days of incubation at 37°C, viral replication was quantified by measuring eGFP fluorescence using the acumen Cellista (TtpLabtech). The 50% effective concentration (EC 50 ) was calculated using dose-dependent inhibition curves. The cytotoxic effect was determined in the same plates after the eGFP-based readout, except for THP1 cells where toxicity is measured in a non-infected plate. Cytotoxicity was measured using an ATPlite ™ cell viability luminescence assay (PerkinElmer), and the luminescence signal was detected with the ViewLux ™ imaging system (PerkinElmer). The 50% cytotoxic concentration (CC 50 ) was calculated using dose-dependent inhibition curves. The selective index (SI) was calculated as the ratio of CC 50 /EC 50 . The antiviral assay with JEV, WNV and ZIKV were conducted in a similar way. VeroE6 cells were seeded in a 96-well plate at a density of 1 × 10 5 cells/well. The next day, a 3-fold (for ZIKV) or 5-fold serial dilution of the compounds was added to the plates. Lastly, the virus was added to the plates (JEV, MOI:0.1; WNV, MOI:0.1; ZIKV, MOI:0.2). After 1 week of incubation in a humified incubator at 37°C with 5% CO 2 , virus-induced cytopathic effect (CPE) was determined by means of the MTS readout method (Promega), as described previously 72 . In the JEV antiviral assay, however, virus-induced CPE was determined using ATPlite ™ , according to the manufacturer’s protocol. The protocol of the YFV antiviral assay was essentially the same as for ZIKV with some differences: cells (Huh7) were seeded at a density of 5,500 cells/well and virus-induced CPE was determined (using MTS) on day 4 post-infection. Vero-GFP cells (2,500 cells/well) were seeded in 384-well black view plates (Corning®) containing 200 nL of compounds in a 9-fold serially dilution and then placed at 37°C for 24 hours. Next, the cells were infected with the different flaviviruses and corresponding amount of virus (DENV-1/TC974 #666, MOI:1; DENV-2/16681, MOI: 0.5; DENV-3/H87, MOI:0.5; DENV-4/H241, MOI:0.5; WNV/Uganda B956, MOI:0.5; ZIKV/MP1751, MOI:0.16; YFV/17D, MOI:0.06). Three or five days in case of YFV post-infection, the eGFP signal was measured using the acumen Cellista (TtpLabtech). After the eGFP-based readout, the cytotoxic effect was determined in the same plates using ATPlite ™ as described above for Vero cells, except for ZIKV for which the cytotoxic effect was measured on a non-infected plate. For the testing of the clinical isolate (DENV-2/Eden3295), Huh7 cells were seeded in a 24-well plate at 1 × 10 5 cells per well. Cells were first infected with DENV-2/Eden3295 at a MOI of 0.3 for 1 hour. Virus inoculums were then removed and fresh medium containing the compounds at concentrations ranging from 0.0 1 µM to 50 µM were added. Cells were incubated for additional 48 hours at 37°C and the supernatants were collected. Virus titers in the supernatants were determined by standard plaque assay on BHK-21 cells. Standard plaque assay on BHK-21 was performed as previously described 73 . EC 50 values were determined using a sigmoidal dose response (variable slope) non-linear regression model in GraphPad Prism software. For antibody-dependent enhanced (ADE) infection, DENV-2/38865Y10 infection at MOI 10 and humanized 4G2 (0.05 µg) were mixed and incubated on ice for 1 hour to allow the formation of immune complexes. THP-1 cells (1×10 5 ) were infected with the immune complexes for 2.5 hours at 37°C with shaking. Cells were then washed once with PBS before resuspending in RPMI-1640 medium containing the compound at concentrations ranging from 0.1 nM to 25 µM followed by a further incubation of 48 hours. After 48 hours, supernatants were harvested and subjected to virus titer determination by standard BHK-21 plaque assay. Tetravalent duplex real-time quantitative polymerase chain reaction (RT-qPCR) The tetravalent antiviral RT-qPCR was based on as previously described protocol 74 . In brief, Vero cells (10,000 cells/well) were seeded in 96-well plates containing a serial dilution of the test compound. 24 Hours after seeding, the cells were infected with DENV (DENV-1/TC974 #666, MOI:0.1; DENV-3/H87, MOI:0.025; DENV-4/H241, MOI:0.6), and incubated at 37°C for three days. Intracellular RNA was measured by washing the adherent cells of the plates without supernatant with cold PBS and plates were incubated at -80°C for at least 24 hours. After 24 hours the cells were lysed with Cells-to-CT Bulk Lysis Reagents kit (Thermo Fisher Scientific) and the cell lysates were used to prepare cDNA (using Expand Reverse Transcriptase) of the target sequences, the 3’-untranslated region (3’UTR) of DENV (Forward primer: 5′-GGCCAGGTCATCACCATT-3′, Reverse primer: 5′-GAGACAGCAGGATC TCTGGTC-3′, Probe: FAM-5′-AAGGACTAGAGGTTAGAGGAGACCCCCC-3′-BHQ1), and the cellular housekeeping reference gene β-actin (Forward primer: 5′-GGCCAGGTCATCACCATT-3′, Reverse primer: 5′-ATGTCCACGTCACACTTCATG-3′, Probe: HEX-5'-TTCCGCTGC(ZEN)CCTGA GGCTCTC-3IABkFQ). Subsequently, a duplex RT-qPCR was performed on a Lightcycler480 II instrument (Roche) at the following conditions: 10 minutes at 95°C, followed by 40 cycles of 10 seconds at 95°C, 1 minute at 60°C. Time-of-addition assay The time-of-addition (TOA) assay was performed as shown in the schematics of Fig. 3 75 . Briefly, 1 × 10 5 Huh7 cells were infected with DENV-2/Eden3295 at MOI 1 for 1 hour followed by treatment with 5 µM JNJ-1953 at 1, 2, 4, 6, 10 and 19 h post-infection (post-treatment). For pre-treatment, the cells were exposed to 5 µM of the respective compounds for 2 hours prior to infection. For co-treatment, the cells were infected with the virus that was mixed with 5 µM of the compounds for 1 hour and subsequently replaced with media. The endpoint assessment is by quantifying the infectious virus production after 24 hours post-infection. DENV-2/16681 in vitro resistance selection Vero cells were seeded at a density of 1 × 10 5 cells/well in a 96 well plate. The next day, cells were infected (MOI of 0.1) with DENV-2/16681 and incubated for 96 hours at 37°C in the presence of a 2-fold serial dilution of JNJ-3644 (5-0.039 µM) for 4 days at 37°C. After 4 days, cells were microscopically checked for CPE, and the supernatant from two adjacent wells showing 30–70% CPE was collected and pooled. The collected supernatant was subsequently used to infect freshly seeded cells. The remaining supernatant was stored at − 80°C until further analysis. Virus was passaged twice a week. During passaging of the virus, the start concentration of the compound was gradually increased. This procedure was repeated until the observed EC 50 value approached the cytostatic concentration of the compound. To check for spontaneous and/or tissue-culture-adapted mutations, part of the wells served as wild-type virus controls to which no compound was added. Wild-type DENV-2/16681 was passaged using Vero cells in a similar way to compound-treated virus. Further analysis includes next-generation sequencing of the viral RNA isolated from cell culture supernatant (140 µL) using a QIAamp Viral RNA Mini kit (Qiagen) per the manufacturer’s protocol. Viral RNA was amplified into double-stranded DNA using a NuGEN Trio RNA-Sequence kit per manufacturer’s protocol. Full DENV-2/16681 genome was sequenced using next-generation sequencing technology (Illumina). Sequences were filtered for viral content by aligning the reads to DENV-2/16681 viral genome (GenBank accession: NC_00174). A coverage cut-off value of 100 and a 15% read frequency cut-off were used for the reliable detection of amino acid variants. Transient DENV replication assay Mutant subgenomic DENV reporter replicons (sgDVs-RLuc) each containing a NS2A single, double or triple resistance mutation (Epoch Life Science) were used to determine the compound resistance imposed by each of the mutations. First, each resistance mutation was inserted separately into the sgDVs-RLuc replicon. The plasmid (denoted pFK-sgDVs-R2A) contains the non-structural genes NS1-NS5 of the DENV-2/16681 strain with cell-adaptive mutations in NS3 (A546V and H451P), NS4A (I116M) and NS5 (E892K), and the Renilla luciferase (RLuc) reporter gene 63 . The plasmids of both wild-type and mutant sgDVs-Rluc were used to produce in vitro transcribed (IVT) DENV RNA as described previously and electroporated into Huh7 cells 63 . The RLuc activity was measured using the Renilla-Glo® Luciferase assay system (Promega) following the manufacturer’s instructions and detected using the ViewLux ™ . Full-length DENV-2/Eden3295 cDNA clone (GenBank accession: EU081177) used in this study has been previously described 76 . Site-directed mutagenesis was used to generate the different NS2A mutations (single mutants E21G or A32V and the double mutant E21G A32V) on the full-length DENV-2/Eden3295 clone. IVT RNA was obtained from the full-length cDNA clone using T7 mMESSAGE mMACHINE kit (Ambion) and transfected into C6/36 using the previously described electroporation conditions 76 . Supernatants from the transfected C6/36 cells were collected on day 7 after transfection (P0 virus) and passaged once in C6/36 to obtain the P1 virus stock for compound efficacy evaluation assays. The IVT RNAs of DENV-2 wild-type or the various NS2A mutants were transfected into BHK-21 cells as previously described 76 to profile its replication kinetics over a course of 3 days. Supernatants were collected for infectious virus quantification by standard plaque assay while the transfected cells were washed once prior to lysing with RLT buffer (Qiagen) for intracellular viral RNA quantification by RT-qPCR. Construction of DENV-2 prM and NS2A mammalian expression plasmids The mammalian expression plasmids of DENV-2/Eden3295 (EU081177) prM and NS2A were designed and constructed as described in 40 (see schematic in Fig. 5 A). Briefly, the signal peptide from Gaussia luciferase (Gluc) together with the last 16-amino acids (aa) of NS1 is fused to the N-terminus of NS2A to ensure correct processing and membrane topology of NS2A. This NS2A construct (wild-type, WT) is then cloned into a C-terminal Flag-tagged pcDNA3.1 + vector (Genscript) using Nhe I and Xho I restriction sites. The WT NS2A plasmid was subjected to reverse-engineering of the E21G/A32V double mutations (NS2A E21G/A32V ) using Quikchange II XL site-directed mutagenesis kit (Stratagene) according to manufacturer’s instructions. For the prM construct, the anchor C signal peptide (AnC-14aa) is retained at the N-terminus of prM to ensure correct targeting to the ER membrane. This prM construct is then cloned into a N-terminal Myc pcDNA3.1 + vector (Genscript) with Hind III and Xho I restriction sites. Co-immunoprecipitation, SDS-Page and Western blot The co-immunoprecipitation was performed as described in 40 . Briefly, single, or various combinations of expression plasmids carrying wild-type NS2A or NS2A E21G/A32V and prM (each at a concentration of 5 µg) were transfected into HEK293T cells (2.5 × 10⁵ cells per 10-cm dish) using the Fugene 6 transfection reagent (Promega). At 6 hours post-transfection, the cells were treated with 10 µM of JNJ-1953 or its inactive enantiomer (JNJ-2005). Following a 44-hour incubation period, the cells were lysed in 0.5 mL immunoprecipitation (IP) buffer (20 mM Tris, pH 7.5, 100 mM NaCl, 0.5% DDM, and EDTA-free protease inhibitor cocktail [Roche]) with rotation at 4°C for one hour. The lysates were then clarified by centrifugation at 21,000 × g and 4°C for 30 minutes. The supernatants (200 µL) were combined with IP buffer (20 mM Tris [pH 7.5], 0.5% DDM), and NaCl was added to achieve a final concentration of 400 mM. 2 µg of rabbit anti-c-myc antibody (Sigma) were then added to the mixture, followed by agitation overnight at 4°C (end-to-end shaker) to form immune complexes. The immune complexes were captured by the addition of 30 µL of Pierce™ Protein A/G Plus Agarose (Thermo Scientific), and the mixtures were tumbled further for 1–2 hours. Thereafter, the beads-bound immune complexes were collected by centrifuging at 900 × g at 4°C for 3 min and washed five times with PBS containing 0.1% Tween 20 (PBS-T). The beads-bound immune complexes were eluted by boiling in 5× sodium dodecyl sulfate (SDS) (Bio Basic Asia Pacific) sample buffer supplemented with 50 mM dithiothreitol (DTT) at 100°C for 10 min. The tubes were briefly vortexed and then subjected to centrifugation at 10,000 × g for one minute. A total of 40 µL of the sample was loaded onto a 4–20% SDS-PAGE gel (Bio-Rad, Cat # 4561094). Subsequently, the proteins were resolved and transferred onto a nitrocellulose membrane (Bio-Rad) using the Bio-Rad Blotting System. To prevent the light chain (size 26 kDa) from obscuring the prM or NS2A protein bands (size approx. 24 kDa), the protein blot was stained with Ponceau as previously described 77 and trimmed precisely at 25 kDa. Subsequently, the blot was incubated for 1 h in a blocking buffer containing 5% skim milk in PBS-T. The blot was washed twice with PBS-T and incubated with a primary antibody, either rabbit anti-Myc (Sigma) or mouse anti-Flag (Sigma), and an anti-GAPDH antibody (Thermo Fisher), for approximately 16 hours at 4°C while shaking. Following three washes with PBS-T buffer, the protein blot was incubated with horseradish peroxidase (HRP)-conjugated rabbit or mouse antibody for one hour at room temperature on a shaker. To demonstrate equal expression and loading, the protein blot with inputs (10%) was probed with an anti-Myc, anti-Flag, or anti-GAPDH antibody. Subsequently, the blots were subjected to three comprehensive washes with PBS-T buffer. Thereafter, the ECL substrates (Advansta Inc.) were applied in accordance with the instructions provided. Subsequently, the chemiluminescence signals were detected using the ChemiDoc system (Bio-Rad). Statistical analysis Densitometric analysis of the band intensities of the CoIP were determined for 4 individual experiments, combined and compared using unpaired Student’s t-test (p-value = 0.096; degrees of freedom = 6). Declarations Data availability All data generated or analyzed during this study are included in this published article and its supplementary information. The uncropped images of the western blot shown in Fig. 5 are presented in Supplementary Fig. 2. Code availability The natural occurrence of the mutations was retrieved from the Bacterial and Viral Bioinformatics Research center (BV-BRC) database. Graphs and figures were generated using Microsoft PowerPoint or GraphPad Prism (v.9.0.0 and v.7.04); the software was made available by Johnson & Johnson. Acknowledgements We thank Natalene Hui Shan Yuen, Sze Woei Ng, Nanthini Ramanathan at the center of global health discovery for technical assistance; Caroline Collard and Elke Maas for technical assistance at the KU Leuven; Edgar Jacoby for the Alphafold model of NS2A. We also thank Drs Lee Ching Ng and Judith Wong (National Environmental Agency, Singapore) for providing the DENV2/38865Y10 strain used in this study. We received funding from the Flanders Agency Innovation & Entrepreneurship (VLAIO O&O grants HBC.2021.1131 and HBC. 2017.0947. This work was further supported by the Center of Global Health Discovery (CGHD) fund 2022-1723 administrated by Johnson & Johnson as well as the IAF-ICP I2301E0019 by Agency for Science, Technology and Research. Author contributions D.B, O.G and D.P. planned, coordinated and performed the experimental virology work at Johnson & Johnson. P.G, A.E.B, D.L, S.M, J.V.D.V, P.V performed the experimental virology work at Johnson & Johnson. K.W.K.C, M.M.C and S.G.V planned, coordinated and performed the experimental virology work at the center for global health discovery in Singapore. P.B. performed experimental virology work at the center for global health discovery in Singapore. S.J.F.K. and J.N. planned, coordinated and performed the experimental virology work at KU Leuven. S.V.B planned, coordinated and performed the medicinal chemistry work at Johnson & Johnson. B.S. planned and coordinated the pharmacokinetics and pharmacodynamics work at Janssen Johnson & Johnson. S.J. and D.P performed data analysis on the High-throughput screen at Johnson & Johnson. O.G. designed and initiated the project at Johnson & Johnson. A.K., O.G., D.B. and S.G.V secured funding from external organizations. D.B wrote the manuscripts with contributions from S.G.V, K.W.K.C, M.M.C, S.J, S.J.F.K, B.S and O.G and with comments from all the authors. Competing interests D.B., S.V.B., S.J, J.V.D.V, P.V., D.P., B.S., A.K. and G.O. are full-time employes of Johnson & Johnson and potential stockholders of Johnson and Johnson. The other authors declare no competing interests. References Tabachnick, W. J. Climate Change and the Arboviruses: Lessons from the Evolution of the Dengue and Yellow Fever Viruses. Annu Rev Virol 3 , 125-145, doi:10.1146/annurev-virology-110615-035630 (2016). Dutta, S. K. & Langenburg, T. A Perspective on Current Flavivirus Vaccine Development: A Brief Review. Viruses 15 , doi:10.3390/v15040860 (2023). Halstead, S. B. 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08:40:36","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5979312/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5979312/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":76877754,"identity":"587906fa-3d71-44f3-9fa9-8c2f0632e4ca","added_by":"auto","created_at":"2025-02-21 16:23:54","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":668915,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHigh-content imaging antiviral assay based on DENV-2/166681-GFP in A549 cells. a\u003c/strong\u003e Experimental set up of the used HCI fluorescence platform. Representative images of DENV-2/16681-GFP infective A549 cells. Images from CV7000 Yokagawa showing eGFP signal coming from the DENV‑2/16681-GFP infection (eGFP, green), Hoechst staining (nuclei, blue), MitoTracker orange staining (mitochondria, Orange) and Cell-Mask\u003csup\u003eTM\u003c/sup\u003e Deep red staining (cytoplasm and nucleus, Red). Scale bar: 50 µM. \u003cstrong\u003eb\u003c/strong\u003e High level flow of the screening funnel from the primary screen to the identification of hits with orthoflavivirus broad-spectrum potential. \u003cstrong\u003ec\u003c/strong\u003e A morphological profile based on 30 features of each compound, here shown for Brequinar\u003csup\u003e70\u003c/sup\u003e and compound 24\u003csup\u003e66\u003c/sup\u003e. \u003cstrong\u003ed\u003c/strong\u003e Results of the hit deprioritization using SIM2NIC. The maximum SIM2NIC score over all concentration at which a compound achieved ≥ 50% virus inhibition and retained cell count ≥ 30% (relative to infected control (0-line) was computed and plotted. Non-infected control cells had a SIM2NIC value of 0.99 (median of individual control wells. The SIM2NIC values obtained for the reference compounds Brequinar and compound 24 were 0.20 and 0.98, respectively. \u003cstrong\u003ee\u003c/strong\u003e T-SNE clustering of full feature analysis of hits from the confirmation screen identifies clusters of compounds with similar phenotypes. The phenotypic cluster of hits with a SIM2NIC \u0026gt; 0.8 closely correlates with the non-infected control (NIC; star symbol).\u003c/p\u003e","description":"","filename":"Picture1.png","url":"https://assets-eu.researchsquare.com/files/rs-5979312/v1/c64728696cdfd4f514f98e13.png"},{"id":76877753,"identity":"01475786-046c-401f-b391-71e73b46ffea","added_by":"auto","created_at":"2025-02-21 16:23:54","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":193822,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBroad-spectrum orthoflavivirus hits, JNJ-3644 and 1953. a\u003c/strong\u003e Structure of compound JNJ-3644. \u003cstrong\u003eb\u003c/strong\u003eAntiviral activity of JNJ-3644 and the seven other isomers against DENV-2/16681 in Vero cells based on GFP expression. Data shown for one representative experiment. \u003cstrong\u003ec\u003c/strong\u003e Structure of compounds JNJ-4840 and JNJ-1953.\u003c/p\u003e","description":"","filename":"Picture2.png","url":"https://assets-eu.researchsquare.com/files/rs-5979312/v1/d811a8897612498c4a94d284.png"},{"id":76877744,"identity":"248ad052-5609-4d65-883d-604710118c15","added_by":"auto","created_at":"2025-02-21 16:23:54","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":101800,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTime of drug addition studies of JNJ-1953. \u003c/strong\u003eHuh7 cells were either treated with JNJ-1953 (orange symbol/line) 2 h prior to DENV-2/Eden3295 infection (pre-treatment; Pre-T), infected with DENV-2/Eden3295 in the presence of the compound (co-treatment; Co-T), or post-treated with the compound after 1 h DENV-2/Eden3295 infection at the indicated hours post-infection (post-treatment; Post-T) as shown in the schematic. At 24 hours post-infection, plaque quantification of the culture supernatants was assessed. NITD-008 was included as reference compound \u003csup\u003e50\u003c/sup\u003e (grey symbol/line). Data is presented as line graph showing the average percentage of infection with standard deviation (percentage of virus infection)\u003cem\u003e \u003c/em\u003eof the treated samples compared to the untreated infected control (grey dotted line) obtained from two independent experiments with duplicates.\u003c/p\u003e","description":"","filename":"Picture3.png","url":"https://assets-eu.researchsquare.com/files/rs-5979312/v1/8c9e335cd6b382abb7a3b557.png"},{"id":76877752,"identity":"11f1ef38-8bda-4414-8e21-14e659d02f07","added_by":"auto","created_at":"2025-02-21 16:23:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":451658,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIn vitro resistance selection experiments identify NS2A as target. a\u003c/strong\u003e Allele frequency distributions of the polyprotein of two passaged (p18 and p28) DENV-2/16681 viruses which were exposed to increasing drug concentrations of JNJ-1953, highlighting mutations within NS2A (pink region). \u003cstrong\u003eb\u003c/strong\u003e AlphaFold 2 model (Polg_DEN26 p29990 DENV-2) was used to predict the structure for NS2A. The three amino acids that confer DENV-2 resistance (NS2A\u003csup\u003eF18\u003c/sup\u003e, NS2A\u003csup\u003eE21\u003c/sup\u003e and NS2A\u003csup\u003eA32\u003c/sup\u003e) are highlighted. \u003cstrong\u003ec\u003c/strong\u003e Sequence alignment of a part of the NS2A proteins among DENV 4 serotypes, ZIKV, WNV, JEV, and YFV.\u0026nbsp; The consensus and conservation percentage are indicated below each amino acid. \u003cstrong\u003ed-e\u003c/strong\u003e BHK-21 cells were electroporated with DENV2 wild-type (WT) or NS2A mutants (NS2A\u003csup\u003eE21G\u003c/sup\u003e, NS2A\u003csup\u003eA32V\u003c/sup\u003e and NS2A\u003csup\u003eE21G/A32V\u003c/sup\u003e) IVT RNA and the replication profile were assessed over a time-course of 72 hours. \u003cstrong\u003ed\u003c/strong\u003e Intracellular viral RNA replication of WT and the NS2A mutants (NS2A\u003csup\u003eE21G\u003c/sup\u003e (green), NS2A\u003csup\u003eA32V\u003c/sup\u003e (purple) and NS2A\u003csup\u003eE21G/A32V \u003c/sup\u003e(red)) determined by RT-qPCR. \u003cstrong\u003ee\u003c/strong\u003e Infectious-virus titers of WT and mutant viruses (NS2A\u003csup\u003eE21G\u003c/sup\u003e (green), NS2A\u003csup\u003eA32V\u003c/sup\u003e (purple) and NS2A\u003csup\u003eE21G/A32V\u003c/sup\u003e (red)) in the culture supernatants of electroporated cells as determined by standard BHK-21 plaque assay.\u003c/p\u003e","description":"","filename":"Picture4.png","url":"https://assets-eu.researchsquare.com/files/rs-5979312/v1/ad3aa1e2ee6015ef2a906bca.png"},{"id":76877749,"identity":"32407b81-a28d-4b2e-af46-579ac262be8c","added_by":"auto","created_at":"2025-02-21 16:23:54","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":178723,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eJNJ-1953 destabilizes prM binding to NS2A. a\u003c/strong\u003e Schematic showing the construct design of DENV-2 NS2A and prM. The constructs design followed Xie \u003cem\u003eet al.\u003c/em\u003e\u003csup\u003e40\u003c/sup\u003e \u003cstrong\u003eb\u003c/strong\u003e HEK293T cells were transfected with 5 µg each of Myc-prM and Flag-WT or NS2A\u003csup\u003eE21G/A32V\u003c/sup\u003e, followed by treatment with the active JNJ‑1953 compound or with its inactive enantiomer, JNJ-2005, at 6 hours post-transfection. Cell lysates were harvested at 44 hours post-transfection and subjected to co-immunoprecipitation using Myc or IgG control antibody. Western blots showing the detection of Flag-NS2A WT and Flag-NS2A\u003csup\u003eE21G/A32V\u003c/sup\u003e immunoprecipitated with Myc-prM in the absence and presence of JNJ-1953 or JNJ-2005. GAPDH was used as a loading control. \u003cstrong\u003ec\u003c/strong\u003e Densitometric analysis of the band intensities of WT or NS2A\u003csup\u003eE21G/A32V\u003c/sup\u003e normalized to prM for the co-immunoprecipitated samples upon treatment with JNJ-1953 or JNJ-2005 compared to untreated. Data is presented as bar graphs showing mean with standard deviation from 4 independent experiments (n=4) and differences between WT and NS2A\u003csup\u003eE21G/A32V\u003c/sup\u003e is compared by unpaired Student’s t-test with df = 6.\u003c/p\u003e","description":"","filename":"Picture5.png","url":"https://assets-eu.researchsquare.com/files/rs-5979312/v1/a43c9d297cc305337e2a74ca.png"},{"id":98622535,"identity":"262f60a2-c691-41c4-a848-2df347528430","added_by":"auto","created_at":"2025-12-19 16:57:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3320365,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5979312/v1/21e6acb4-e08c-481f-a46b-420dd8959c8e.pdf"},{"id":76877750,"identity":"90b71ea5-89ef-49eb-988a-ecfd4a6b4161","added_by":"auto","created_at":"2025-02-21 16:23:54","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":343726,"visible":true,"origin":"","legend":"Supplementary information","description":"","filename":"SupplementaryinformationDoortjeBorrenberghs.docx","url":"https://assets-eu.researchsquare.com/files/rs-5979312/v1/2ec10393751fb428e9f2b06a.docx"},{"id":76877745,"identity":"ff75f355-8fc1-4343-9e39-e82fdd16a398","added_by":"auto","created_at":"2025-02-21 16:23:54","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1666417,"visible":true,"origin":"","legend":"Reporting Summary","description":"","filename":"20250127nrreportingsummary.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5979312/v1/26b7777cecac633fe8e4fe3c.pdf"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nD.B., S.V.B., S.J, J.V.D.V, P.V., D.P., B.S., A.K. and G.O. are full-time employees of Johnson \u0026 Johnson and potential stockholders of Johnson and Johnson. The other authors declare no competing interests.","formattedTitle":"Identification of a broad-spectrum flavivirus inhibitor targeting NS2A, a previously unidentified target","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFlaviviruses, such as dengue virus (DENV), Zika virus (ZIKV), West Nile (WNV), yellow fever (YFV), and Japanese encephalitis virus (JEV) are mosquito-borne human pathogens known for causing a wide range of diseases in humans, including dengue fever, yellow fever, and viral encephalitis. Collectively, they threaten more than half of the global population and gained increasing attention in recent years due to their ability to cause significant outbreaks and global health concerns, especially in areas that are na\u0026iuml;ve to infection \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe development of vaccines and therapeutics against flaviviruses has proven challenging. While licensed vaccines are available for certain mosquito-borne flaviviruses, including DENV, YFV and JEV, the vaccine development process is complicated by several factors. These include suboptimal safety profiles, the need to establish a durable protective immune response, and the occurrence of cross-reactive antibodies, which can lead to antibody-dependent enhancement of disease \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The COVID-19 pandemic also revealed major points of concern regarding distribution and availability of vaccines to low-income countries. In addition, the pandemic highlighted the importance of developing drugs for antiviral treatment and/or prophylaxis. Currently, no antiviral is clinically available for prevention and treatment of any of these flavivirus infections although there is \u0026ldquo;no insurmountable scientific obstacle to develop safe and effective antiviral drugs\u0026rdquo; against them (Fauci, 2024)\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFlavivirus virions are 40 to 60 nm in diameter structures that contain a single-stranded plus-sense RNA genome (~\u0026thinsp;11 kb), which is translated into a single polyprotein precursor, that is co- and post-translationally cleaved into three structural proteins (capsid [C], pre-membrane [prM] and envelope [E]) and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) by host and viral (NS2B-NS3) proteases \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The non-structural proteins form the replication complex which is associated with the endoplasmic reticulum (ER) membrane \u003csup\u003e\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Some of the NS proteins drive the viral replication process, such as NS1 \u003csup\u003e10,11\u003c/sup\u003e, NS3 helicase \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, NS2B-NS3 protease \u003csup\u003e\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e, NS5 methyl-transferase \u003csup\u003e\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e and NS5 RNA-dependent RNA polymerase \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Other NS proteins (NS2A, NS4A and NS4B) have no enzymatic activity ascribed but contain transmembrane domains associated with the ER membrane, serving as scaffolds for the replication complex \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Apart from serving as a membrane anchoring point and being a cofactor in the formation of the viral replication complex, the NS4A protein also interacts with host factors involved in immune responses, and plays a role in viral pathogenesis, inducing cellular stress responses and apoptosis \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Several important functions have been ascribed to NS4B \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Like NS4A, NS4B plays a crucial role in the formation and organization of the viral replication complexes by aiding in the remodeling of the host cell membrane to create vesicle packets. NS4B can modulate host immune responses and has been implicated in viral pathogenesis. More recently, \u003cem\u003ein vitro\u003c/em\u003e studies revealed that NS4B dissociates NS3 from single-stranded RNA and enhances NS3 helicase activity \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. NS2A plays a crucial role in various stages of the viral replication cycle. The protein appears to be a central hub in the virion packaging process, by coordination of viral and host factors, host antiviral response and recruitment of the viral RNA \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan additionalcitationids=\"CR29 CR30 CR31 CR32 CR33 CR34 CR35 CR36 CR37 CR38 CR39 CR40\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. NS2A has a documented role in viral replication, by interacting with the 3\u0026rsquo; untranslated region (3\u0026rsquo;UTR) of the viral RNA as well as with other viral components involved in virion assembly to coordinate genome encapsulation and assembly \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Furthermore, NS2A is also suggested to function as a viroporin, because of its oligomerization, potentially helping to direct nascent viral RNAs to the sites of virion assembly \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. One single virion consists of multiple copies of capsid, prM and E, therefore each NS2A molecule may aid in the coordination of the complex virion assembly process \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRecent advances in high-content imaging (HCI), an image-based technology in high throughput format, allow both visualization of the virus and changes in cellular phenotypes induced by compound treatment \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. This approach has been used in recent antiviral screening campaigns and has been successful in identifying inhibitors against various viruses, such as Hepatitis B virus \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e, coronavirus \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, and ZIKV \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eHere, a cellular-based HCI screening campaign resulted in the identification of a novel flavivirus broad-spectrum inhibitor. Through structure-activity relationship (SAR) optimization, a lead compound was identified with broad-spectrum anti-flavivirus potency and target engagement identified NS2A as a previously unidentified target.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eHigh-content imaging (HCI) antiviral assay against DENV-2/16681\u003c/h2\u003e \u003cp\u003eIn a quest for novel broad-spectrum flavivirus inhibitors, a fluorescence-based HCI platform was set up (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, Supplementary Fig.\u0026nbsp;1A) using DENV-2 as a representative flavivirus. Using this optimized and validated platform (Supplementary Fig.\u0026nbsp;1BC), we screened approximately 200,000 compounds at a single concentration of 25 \u0026micro;M, originating from the Johnson \u0026amp; Johnson library and which were selected based on their biological and chemical diversity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In addition to viral infection (eGFP expression) and cellular toxicity (cell count), image-based morphological profiling of cells was carried out during a first confirmation run. Morphological profiles were evaluated based on 30 cellular features determined during screen optimization, including intensity, texture, and shape properties (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan additionalcitationids=\"CR47 CR48\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. The morphological profile induced by each compound in infected cells was compared to that of non-infected controls on the same plate and a \u0026ldquo;similarity to non-infected control\u0026rdquo; (SIM2NIC) score was calculated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, E). A SIM2NIC score close to 1 reflects a high correlation in morphology to non-infected control, and direct-acting antivirals are expected to have high SIM2NIC values close to 1. Compounds blocking virus infection by more than 50%, exhibiting a decrease in cell count of less than 70% and having a SIM2NIC\u0026thinsp;\u0026gt;\u0026thinsp;0.6 compared to infected control cells, were selected for further confirmation screening. The hit rate from the primary screening after confirmation in 4 concentrations was 1.5%, resulting in 3,082 hits, which were next tested at 10 concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). A total of 1,672 hits exhibited an antiviral activity of \u0026gt;\u0026thinsp;50%, a cell count\u0026thinsp;\u0026gt;\u0026thinsp;30% and a SIM2NIC\u0026thinsp;\u0026gt;\u0026thinsp;0.6. From this pool, 1,059 hits with a SIM2NIC threshold higher than 0.8 were further evaluated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Next, compounds were clustered by chemical similarity. Frequent hitters (i.e., compounds frequently identified as \u0026ldquo;hits\u0026rdquo; across previous internal high-throughput screening (HTS) campaigns) were eliminated and the hits were ranked based on their activity, chemical attractiveness, and available SAR. The resulting hit classes were further explored for their potential broad-spectrum flavivirus activity against DENV-1/-3/-4, ZIKV, WNV, JEV and YFV (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIdentified hit JNJ-3644 shows broad-spectrum flavivirus activity\u003c/h3\u003e\n\u003cp\u003eOne hit, 7-[(6-chloro-2-methoxy-3-quinolyl)-(4-chlorophenyl)methyl]-5-phenyl-2,3,5,6,8,8a-hexahydro-1H-indolizin-7-ol (JNJ-3644; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), exhibited nanomolar (nM) antiviral potency against DENV-2/16681-eGFP in various cell lines (Vero, Huh7 and THP1-DC sign), accompanied with high selectivity (SI, selective index) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). JNJ-3644 has 4 chiral centers, resulting in 8 different enantiomers. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, the potency of the 8 different enantiomers to inhibit DENV-2/16681 replication in Vero-GFP cells varied. The hit compound had an EC\u003csub\u003e50\u003c/sub\u003e value of 0.18 \u0026micro;M with a SI of 72, while the other enantiomers had EC\u003csub\u003e50\u003c/sub\u003e values ranging from 0.85 to 4.6 \u0026micro;M, with selective indices between 4 and 7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, Supplementary Table\u0026nbsp;1). Next, JNJ-3644 was tested in a tetravalent dengue antiviral assay using RT-qPCR as readout, and the EC\u003csub\u003e50\u003c/sub\u003e for the DENV serotypes ranged from 0.089 to 1.0 \u0026micro;M (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Subsequent testing of JNJ-3644 in different flavivirus assays, showed EC\u003csub\u003e50\u003c/sub\u003e values of around 0.77 \u0026micro;M (ZIKV, JEV), 1.96 \u0026micro;M (YFV) and 2.65 \u0026micro;M (WNV). No marked antiviral activity was detected against a selection of other RNA and DNA viruses (Supplementary Table\u0026nbsp;2). More than 300 compounds based on the same chemical series as JNJ-3644 were synthesized and tested in different flavivirus antiviral assays. The compound efficacy against some of the flaviviruses varied. For instance, compared to JNJ-3644, JNJ-4840 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC) had a 4\u0026ndash;5 fold better activity against DENV-2 and YFV but with a 3-fold lower efficacy against DENV-3. Another compound, JNJ-1953, demonstrated slightly improved \u003cem\u003ein vitro\u003c/em\u003e broad-spectrum activity (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and improved microsomal clearance and mitochondrial toxicity compared to JNJ-3644 (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). For this reason, most of the further experiments were executed using JNJ-1953 as the lead compound. Like the efficacy against DENV-2/16681, JNJ-1953 retained nM potency against DENV-2 clinical isolates DENV-2/EDEN3295 and DENV-2/38865Y10 (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Evaluation of JNJ-1953 antiviral activity against antibody-dependent enhancement (ADE) secondary infection revealed low nM potency (EC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;50 nM; Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) of the compound, suggesting the potential of the compound in preventing severe disease.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntiviral activity of JNJ-3644 against a set of different flaviviruses\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCells\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eVirus\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eJNJ-3644\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEC\u003csub\u003e50\u003c/sub\u003e [\u0026micro;M]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCC\u003csub\u003e50\u003c/sub\u003e [\u0026micro;M]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSI\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVero\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDENV-2/16681\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.14\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e0.083\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e5.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e86\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHuh7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDENV-2/16681\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.072 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.027\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.7\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTHP1-DC sign\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDENV-2/16681\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.12\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e0.027\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.8\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e4.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVero\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDENV-1/TC974 666\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.32\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.6\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e1.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVero\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDENV-2/16681\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.090 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.9\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e3.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e88\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVero\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDENV-3/H87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.59\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.7\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVero\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDENV-4/H241\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e0.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e9.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVero\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWNV/NY-99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVero-GFP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWNV/B956\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.58\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e0.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e6.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVero\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZIKV/MR766\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVero-GFP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZIKV/MP1751\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.4\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e2.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e7.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHuh7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYFV/17D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVero-GFP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYFV/17D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.7\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e2.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e5.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVero\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eJEV/SA14-14-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eThe initial antiviral data of JNJ-3644 for WNV/NY-99, ZIKV/MR766, YFV/17D and JEV/SA14-14-2 only represent one test experiment, subsequent testing of the compound against the flaviviruses was performed in the Vero-GFP antiviral assay where mean values from at least two independent experiments are shown. EC\u003csub\u003e50\u003c/sub\u003e: 50% effective concentration. CC\u003csub\u003e50\u003c/sub\u003e: 50% cytotoxic concentration. Selectivity index (SI): ratio CC\u003csub\u003e50\u003c/sub\u003e/EC\u003csub\u003e50\u003c/sub\u003e.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntiviral activity of JNJ-4840 and JNJ-1953 against different flaviviruses\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCells\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eVirus\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eJNJ-4840\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e \u003cp\u003eJNJ-1953\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEC\u003csub\u003e50\u003c/sub\u003e [\u0026micro;M]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCC\u003csub\u003e50\u003c/sub\u003e [\u0026micro;M]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eEC\u003csub\u003e50\u003c/sub\u003e [\u0026micro;M]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCC\u003csub\u003e50\u003c/sub\u003e [\u0026micro;M]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSI\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVero\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDENV-2/16681\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.035\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.010\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.1 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e146\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.14\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e0.070\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e86\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVero-GFP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDENV-1/TC974 666\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.63 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.016\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.1\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.29\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.4\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 2.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVero-GFP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDENV-2/16681\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.023 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.090\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e309\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.15\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVero-GFP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDENV-3/H87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.9\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.20\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVero-GFP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDENV-4/H241\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.1 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.36\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.090\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVero-GFP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWNV/B956\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.2 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e10\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.28\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e0.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9.0 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVero-GFP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZIKV/MP1751\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.89 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 3.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.3\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e1.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVero-GFP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYFV/17D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.55\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.076\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.1 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2.8\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e0.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e13\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e3.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHuh7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDENV-2/EDEN3295​\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.21 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e61​\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e288\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHuh7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDENV-2/38865Y10​\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.34 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e178\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTHP1 ADE\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDENV-2/38865Y10​\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.048 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.0059​\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003eAntiviral data represent mean values from at least two independently performed experiments. The toxicity of Huh7 infected with DENV-2/EDEN3295 and DENV-2/38865Y10 in presence of JNJ-1953 represents one experiment. EC\u003csub\u003e50\u003c/sub\u003e: 50% effective concentration. CC\u003csub\u003e50\u003c/sub\u003e: 50% cytotoxic concentration. Selectivity index (SI): ratio CC\u003csub\u003e50\u003c/sub\u003e/EC\u003csub\u003e50\u003c/sub\u003e. NT: Not tested.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eADME-Tox profile of compounds JNJ-3644, JNJ-1953 and JNJ-4840\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eJNJ-3644\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eJNJ-1953\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eJNJ-4840\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCHI logD\u003c/p\u003e \u003cp\u003ecLogP\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epH 2.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.7\u003c/p\u003e \u003cp\u003e6.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003cp\u003e6.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.1\u003c/p\u003e \u003cp\u003e5.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEquilibrium solubility\u003c/p\u003e \u003cp\u003eHTeqSol\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003epH 2 (\u0026micro;g/mL)\u003c/p\u003e \u003cp\u003epH 4 (\u0026micro;g/mL)\u003c/p\u003e \u003cp\u003epH 7.4 (\u0026micro;g/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eND\u003c/p\u003e \u003cp\u003eND\u003c/p\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e94\u003c/p\u003e \u003cp\u003e100\u003c/p\u003e \u003cp\u003e62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eND\u003c/p\u003e \u003cp\u003eND\u003c/p\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIntrinsic clearance cyprotex microsome\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHuman \u0026ndash; Clint (\u0026micro;L/min/mg Prot)\u003c/p\u003e \u003cp\u003eMouse \u0026ndash; Clint (\u0026micro;L/min/mg Prot)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e39\u003c/p\u003e \u003cp\u003e101\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;7.7\u003c/p\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e115\u003c/p\u003e \u003cp\u003e\u0026gt;\u0026thinsp;347\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePlasma protein binding\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;99%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;99%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;99%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCytotoxic screen in HepG2 cells\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIC\u003csub\u003e20\u003c/sub\u003e (\u0026micro;M)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMitochondrial toxicity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e(Glu/Gal) ratio\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;100/18 (\u0026gt;\u0026thinsp;5.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e99/29 (3.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e65/33 (2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCYP inhibition in human liver microsomes, Cytochrome P450 proteins family\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1A2 \u0026ndash; CEC (IC\u003csub\u003e50\u003c/sub\u003e \u0026micro;M)\u003c/p\u003e \u003cp\u003e2C8 \u0026ndash; DBF (IC\u003csub\u003e50\u003c/sub\u003e \u0026micro;M)\u003c/p\u003e \u003cp\u003e2C9 \u0026ndash; MFC (IC\u003csub\u003e50\u003c/sub\u003e \u0026micro;M)\u003c/p\u003e \u003cp\u003e2C19 \u0026ndash; CEC (IC\u003csub\u003e50\u003c/sub\u003e \u0026micro;M)\u003c/p\u003e \u003cp\u003e2D6 \u0026ndash; AMMC (IC\u003csub\u003e50\u003c/sub\u003e \u0026micro;M)\u003c/p\u003e \u003cp\u003e3A4 \u0026ndash; DBF (IC\u003csub\u003e50\u003c/sub\u003e \u0026micro;M)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;20\u003c/p\u003e \u003cp\u003e\u0026gt;\u0026thinsp;20\u003c/p\u003e \u003cp\u003e\u0026gt;\u0026thinsp;20\u003c/p\u003e \u003cp\u003e\u0026gt;\u0026thinsp;20\u003c/p\u003e \u003cp\u003e15\u003c/p\u003e \u003cp\u003e\u0026gt;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;20\u003c/p\u003e \u003cp\u003e\u0026gt;\u0026thinsp;20\u003c/p\u003e \u003cp\u003e\u0026gt;\u0026thinsp;20\u003c/p\u003e \u003cp\u003e\u0026gt;\u0026thinsp;20\u003c/p\u003e \u003cp\u003e12\u003c/p\u003e \u003cp\u003e\u0026gt;\u0026thinsp;20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNT\u003c/p\u003e \u003cp\u003eNT\u003c/p\u003e \u003cp\u003eNT\u003c/p\u003e \u003cp\u003eNT\u003c/p\u003e \u003cp\u003eNT\u003c/p\u003e \u003cp\u003eNT\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eNT: Not tested; CHI LogD: Chromatographic hydrophobicity index; Clint: Intrinsic clearance; CYP: Cytochrome P450\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eJNJ-1953 acts on a viral replication step before virus secretion.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs a first step towards understanding the antiviral mechanism of JNJ-1953, a time-of-addition (TOA) experiment was performed to map the viral replication step(s) inhibited by the compound as depicted in the schematic (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; Top). The Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e bottom panel shows the antiviral effect of JNJ-1953 added at different timings during infection. Pre-exposure of cells to 5 \u0026micro;M of JNJ-1953 2 hours before infection (pre-treatment, Pre-T, -2 hours) or co-treatment (Co-Treatment, Co-T, 0 hours) during virus infection resulted in \u0026gt;\u0026thinsp;90% virus inhibition which can be attributed to the high potency of the compound upon cell entry, similar to the well-characterized RNA virus polymerase inhibitor NITD-008 \u003csup\u003e50\u003c/sup\u003e. Notably complete virus inhibition was observed when JNJ-1953 was added between 2 to 10 h post-infection (Post-Treatment, Post-T), a time window that corresponds to the release of viral RNA, protein translation and initiation of RNA synthesis \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Only a partial virus inhibition of ~\u0026thinsp;40% was observed when the compound was added at 19 h post-infection, a timepoint when newly formed virions are thought to be secreted \u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. Collectively these results suggest that JNJ-1953 exerts its antiviral activity in replication step(s) before virus secretion.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eResistance-associated mutations point NS2A as the target of JNJ-1953\u003c/h3\u003e\n\u003cp\u003eTo identify the molecular target of JNJ-1953, drug-resistant DENV-2 variants were selected during \u003cem\u003ein vitro\u003c/em\u003e resistance selection (IVRS) experiments. CPE was observed starting at passage 18 and completely present at passage 28 in cells treated with 2.5 \u0026micro;M JNJ-1953, suggesting the emergence of JNJ-1953-resistant DENV-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Following JNJ-1953 exposure, passage 18 and 28 viruses are subsequently characterized using next-generation sequencing (NGS). Sequence alignment between the parental and drug-resistant DENV-2 viruses revealed 12 different single-nucleotide amino acid substitutions (Supplementary Table\u0026nbsp;3), three single-nucleotide amino acid substitutions were found in NS2A: F18L, E21G, and A32V, which were not present in the in-parallel-passaged untreated cultures and compared to the other drug-resistant substitutions were present in all drug-resistant variants (Supplementary Table\u0026nbsp;3). NS2A has multiple transmembrane helixes and the three observed mutations (NS2A\u003csup\u003eF\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003eL\u003c/sup\u003e, NS2A\u003csup\u003eE\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003eG\u003c/sup\u003e and NS2A\u003csup\u003eA\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003eV\u003c/sup\u003e) are predicted to locate close to transmembrane domain 1 and 2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Alignment of 17,328 NS2A protein sequences of the different flaviviruses (DENV serotypes 1\u0026ndash;4, JEV, WNV, YFV and ZIKV) collected from the Bacterial and Viral Bioinformatics Research center (BV-BRC) database (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) showed 96.8% (99.8% among DENV-2 sequences) conservation for Glutamic acid (E) 21 (NS2A\u003csup\u003eE\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e). Interestingly, no Glycine (G) was observed in any of the flavivirus NS2A sequences at amino acid position 21 (Supplementary Table\u0026nbsp;4). For the amino acid at position 32, 99.8% of the DENV-2 NS2A sequences had an alanine (NS2A\u003csup\u003eA\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), while 0.64% of the flavivirus NS2A proteins contained a valine (V) at this position (Supplementary Fig.\u0026nbsp;2). For the amino acid at position 18 of the NS2A protein, 99.35% of the DENV-2 NS2A sequences had a phenylalanine (NS2A\u003csup\u003eF\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e) at position 18 (Supplementary Table\u0026nbsp;4). However, the conservation of this F was remarkably lower (38%) among all flaviviruses (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, Supplementary Fig.\u0026nbsp;2), and a leucine (L) is observed in approximately 16% of the different flavivirus NS2A protein sequences (Supplementary Fig.\u0026nbsp;2), and in 100% of the DENV-3 NS2A sequences (Supplementary Table\u0026nbsp;4). To confirm that the F18L, E21G, and A32V mutations within DENV-2 confer resistance to JNJ-1953, the three mutations (NS2A\u003csup\u003eF\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003eL\u003c/sup\u003e, NS2A\u003csup\u003eE\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003eG\u003c/sup\u003e, and NS2A\u003csup\u003eA\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003eV\u003c/sup\u003e) were inserted separately or in combination (NS2A\u003csup\u003eE21G/A32V\u003c/sup\u003e and NS2A\u003csup\u003eF18L/E21G/A32V\u003c/sup\u003e) into a subgenomic DENV-2/16681 reporter replicon using site-directed mutagenesis. The antiviral activity of JNJ-1953 against these mutants was determined in a transient replicon assay. While JNJ-1953 efficiently inhibited wild-type (WT) DENV-2/16681 replication, all mutant viruses showed a 9\u0026ndash;13 fold reduced susceptibility to JNJ-1953 (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), suggesting that the mutations (NS2A\u003csup\u003eF\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003eL\u003c/sup\u003e, NS2A\u003csup\u003eE\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003eG\u003c/sup\u003e and NS2A\u003csup\u003eA\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003eV\u003c/sup\u003e) compromises viral replication. In addition, similar reduced susceptibility to compounds JNJ-3664 and JNJ-4840 are obtained (Supplementary Table\u0026nbsp;5). Given that L18 exists naturally in NS2A of some flaviviruses, only the single mutants (NS2A\u003csup\u003eE\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003eG\u003c/sup\u003e and NS2A\u003csup\u003eA\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003eV\u003c/sup\u003e) and a double mutant virus containing (NS2A\u003csup\u003eE21G/A32V\u003c/sup\u003e) were engineered into a cosmopolitan DENV-2/Eden3295 clinical isolate background. Analogous to the mutants of the subgenomic DENV-2/16681 reporter replicon, the DENV-2/Eden3295 NS2A single and double mutant virus were less susceptible to JNJ-1953 (NS2A\u003csup\u003eE\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003eG\u003c/sup\u003e: 45-fold, NS2A\u003csup\u003eA\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003eV\u003c/sup\u003e: 8-fold and NS2A\u003csup\u003eE21G/A32V\u003c/sup\u003e: 19-fold higher EC\u003csub\u003e50\u003c/sub\u003e) compared to the WT virus (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Collectively these results clearly indicate that the IVRS-identified NS2A mutations are responsible for conferring JNJ-1953 resistance. Given the important functions of NS2A within the replication complex as well as acting as a chaperone in coordinating virion assembly, the impact of the NS2A mutations E21G and/or A32V on virus replication were examined. The single mutants NS2A\u003csup\u003eE\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003eG\u003c/sup\u003e or NS2A\u003csup\u003eA\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003eV\u003c/sup\u003e have no impact on the viral RNA synthesis or infectious virus production (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) compared to WT virus. Interestingly the double mutant exhibited a\u0026thinsp;~\u0026thinsp;2-log\u003csub\u003e10\u003c/sub\u003e lower RNA synthesis and infectious virus production as compared to WT virus (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), suggesting that the mutations at the NS2A positions 21 and 32 may act synergistically to affect virus replication.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntiviral activity of JNJ-1953 against wild-type and mutant DENV-2 subgenomic constructs and DENV-2/Eden3295\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003epFK-sgDVs-R2A\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eDENV-2/Eden3295\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMutations\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEC\u003csub\u003e50\u003c/sub\u003e [\u0026micro;M]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFC\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eEC\u003csub\u003e50\u003c/sub\u003e [\u0026micro;M]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFC\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWild-type\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e0.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE21G (0%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eA32V (\u0026lt;\u0026thinsp;0.2%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eF18L (16%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE21G/A32V\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e4.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e19\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eE21G/A32V/F18L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eND\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eND: Not determined; FC: Fold change compared to wild-type.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e\n\u003ch3\u003eJNJ-1953 interferes with the recruitment of the prM viral structural protein\u003c/h3\u003e\n\u003cp\u003eAs NS2A is reported to interact with viral prM and E proteins to orchestrate viral assembly \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, a possible hypothesis is that JNJ-1953 plays a role in affecting the interaction between NS2A and prM. To test this hypothesis, a c-myc prM plasmid was co-transfected with either FLAG wild-type NS2A or the double mutant NS2A\u003csup\u003eE21G/A32V\u003c/sup\u003e plasmid (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) into HEK293T cells for co-immunoprecipitation (CoIP) following treatment with 10 \u0026micro;M JNJ-1953 at 6 h post-transfection (Supplementary Fig.\u0026nbsp;2). CoIP showed that prM specifically pulled-down NS2A (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB; Lane 2), consistent with a previously reported finding \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. A reduction in the amount of wild-type NS2A pulled down by prM was observed when JNJ-1953 was added during transfection (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB; Lane 3) and shown by densitometric analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), indicating that JNJ-1953 interferes with the interaction between prM and NS2A. Intriguingly, pull down of NS2A\u003csup\u003eE21G/A32V\u003c/sup\u003e by prM was less affected, albeit not significantly, upon JNJ-1953 treatment than that of wild-type NS2A (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, lane 6 versus lane 3; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), suggesting that the NS2A\u003csup\u003eE21G/A32V\u003c/sup\u003e interaction with prM is less sensitive to JNJ-1953 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). These experimental results corroborate with the finding that JNJ-1953 is less potent against the NS2A\u003csup\u003eE21G/A32V\u003c/sup\u003e double mutant virus (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). We further confirmed the finding by performing a CoIP experiment of prM and wild-type NS2A interaction by comparing the effect of the active JNJ-1953 with one of its inactive enantiomers, JNJ-2005 (EC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;5.0 \u0026micro;M; CC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;8.6 \u0026micro;M). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB lane 4, the inactive enantiomer does not affect the interaction of prM with WT NS2A unlike JNJ-1953 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), confirming the specific inhibitory activity of JNJ-1953 on prM-NS2A interaction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eDrug-like properties of JNJ-1953\u003c/h3\u003e\n\u003cp\u003eProfiling of JNJ-1953, JNJ-4840 and JNJ-3644 in several first-line absorption, distribution, metabolism, and excretion (ADME)-Tox assays indicated suboptimal drug-like properties for the series (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The compounds are very lipophilic as shown by the high cLogP and CHI log D pH2.6 values. JNJ-1953 had good metabolic stability in human liver microsomes (\u0026lt;\u0026thinsp;7.7 \u0026micro;L/min/mg) and moderate stability in mouse liver microsomes (30 \u0026micro;L/min/mg) which was superior compared to the metabolic stability in human and mouse liver microsomes for JNJ-3644 and JNJ-4840 (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). JNJ-1953 showed pH-dependent solubility, with good solubility at pH2 and lower solubility at physiological pH (62 \u0026micro;g/mL). Finally, the cytotoxicity in HepG2 cells was high and displayed potential for mitotoxicity for all compounds tested within the series.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFlaviviruses have a large socio-economic burden of disease. Despite considerable efforts in the past two decades, no clinically approved antivirals are available for flavivirus treatment or prophylaxis, and currently, patients\u0026rsquo; options for treatment are limited to measures that solely alleviate symptoms. Therefore, the search for new antivirals targeting endemic, emerging and potential future endemic flaviviruses remains a high priority (Fauci, 2024) \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Here, we present a novel series of broad-spectrum anti-flavivirus small molecule inhibitors targeting NS2A, with a novel mechanism of action. Resistance selection and reverse genetics studies pinpointed NS2A as the molecular target. No enzymatic activity has been shown to be associated with NS2A. We here demonstrated that the compound affects the NS2A-prM interaction, thereby hypothesizing its mechanism of action influences virion assembly.\u003c/p\u003e \u003cp\u003eIn this paper, a multi-parametric HCI-based HTS approach \u003csup\u003e\u003cspan additionalcitationids=\"CR45 CR46 CR47 CR48\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e based on DENV-2 is described to identify novel antiviral candidates active against a wide range of flaviviruses (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Compared to single-parametric, cell-based assays which are often used in phenotypic screening campaigns, the multi-parametric readout generated here is used to determine the individual compounds morphological profiles, thereby enabling an early deprioritization of hits with unfavorable mechanism of actions (\u003cem\u003ee.g.\u003c/em\u003e targeting the host cell instead of the virus) or undesirable cell phenotypes caused by the compound (\u003cem\u003ee.g.\u003c/em\u003e changes in nucleus, cytoplasm, toxicity). This approach maximized at the early stage of screening, the selection of direct-antivirals bocking the virus without any effect on the cell, thereby minimizing the risk of downstream failure, which led to the identification of JNJ-3644 (SIM2NIC: 0.98). This compound was identified with promising broad-spectrum flavivirus activity (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Several compounds of the same series as JNJ-3644 were synthesized and JNJ-1953 was selected as lead compound because of its improved features over the other compounds, such as a good solubility at low pH and good metabolic stability in human (\u0026lt;\u0026thinsp;7.7 \u0026micro;L/min/mg) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026amp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eConcerning the antiviral mechanism of action of the series to which JNJ-1953 belongs, an IVRS experiment identified three amino acid substitutions in NS2A (F18L, E21G, A32V) that confer DENV-2 resistance to JNJ-1953 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), pointing towards impairment of NS2A function as the mechanism of action of this compound. NS2A is a nonenzymatic integral membrane protein \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, with three main functions reported (i) NS2A antagonizes the host immune response \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e, (ii) NS2A functions in viral RNA synthesis \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e and (iii) NS2A plays a role in virion assembly \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. NS2A has a very low sequence identity among all flaviviruses (21\u0026ndash;63%). The low conservation of the drug target can impose challenges for drug development such as a low specificity of the drug for the target, a low barrier to resistance of the drug and large differences in drug effectiveness against different strains or species. Sequence alignment of NS2A proteins from ZIKV, DENV-1-4, WNV, JEV, Saint Louis encephalitis virus (SLEV), tick-borne encephalitis virus (TBEV), and YFV revealed high diversity: only 31 out of 218\u0026ndash;227 amino acids had a consensus of more than 80% among the different flaviviruses \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Interestingly most of the highly conserved residues are located between residues 12 to 100 of NS2A (i.e. N-terminal half of the protein), which is where we identified the resistance mutations for JNJ-1953. In this region two conserved basic residue clusters (residues 17\u0026ndash;22 and 95\u0026ndash;104) are known to be involved in virion assembly \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. It is important to note that the three resistance mutations NS2A\u003csup\u003eF\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003eL\u003c/sup\u003e, NS2A\u003csup\u003eE\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003eG\u003c/sup\u003e and NS2A\u003csup\u003eA\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003eV\u003c/sup\u003e are located near or within this conserved N-terminal basic cluster. Residue E21 is highly conserved among the different flaviviruses, except for YFV (V21; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), and based on the IVRS experiment, we postulated that this residue plays a highly important role in the virus replication cycle. However, introduction of a Glycine on position 21 (NS2A\u003csup\u003eE\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003eG\u003c/sup\u003e), did not affect the viral RNA synthesis or plaque formation in reverse engineered viruses (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). This was confirmed in ZIKV, where Zhang \u003cem\u003eet al\u003c/em\u003e. mutated E22 to A, where E22 corresponds to E21 in DENV. The E22A mutant replicated similarly as the wild-type virus, with comparable infectivity and infectious virus production to that of wild-type virus \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. In contrast, Wu \u003cem\u003eet al\u003c/em\u003e. concluded that the introduction of alanine substitutions at positions 21\u0026ndash;23, showed a\u0026thinsp;\u0026gt;\u0026thinsp;1,000-fold reduction in virus yield and an absence of plaque formation \u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. Intriguingly, the combination of the NS2A\u003csup\u003eE\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003eG\u003c/sup\u003e and NS2A\u003csup\u003eA\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003eV\u003c/sup\u003e mutations (NS2A\u003csup\u003eE21G/A32V\u003c/sup\u003e) rather than the single mutant viruses (NS2A\u003csup\u003eE\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003eG\u003c/sup\u003e and NS2A\u003csup\u003eA\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003eV\u003c/sup\u003e) exhibited an attenuated level of viral replication and plaque formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, E), suggesting a synergistic effect of both mutations, underscoring the importance of the N-terminal basic cluster in virus replication.\u003c/p\u003e \u003cp\u003eThe structure of NS2A has yet to be determined and the latest AlphaFold2-predicted structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB) propose multiple membrane-spanning segments that contrasts with the predicted membrane topology models described in literature \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. Based on the membrane topology models of NS2A from different flaviviruses, the location of the N-terminal basic cluster which encompasses the resistance mutations, may vary between viruses. For example, the NS2A residues\u0026thinsp;~\u0026thinsp;20 to 25 from YFV are predicted to be cytoplasmic \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e, whereas for DENV and ZIKV this region is predicted to be in the ER lumen \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. This could be a possible explanation why our medicinal chemistry exercise experienced difficulties in improving overall broad-spectrum flavivirus activity (Tables\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Because of the differences in topology for the different flaviviruses, NS2A is suggested to mediate important protein-protein and protein-RNA interactions with factors present in the cytoplasm as well as in the ER \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. NS2A is involved in various stages of the viral replication cycle, one of them being virion assembly. Here we showed that by time of drug additions studies that JNJ-1953 is acting on a step after replication is initiated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e 3A, B). More specifically we showed that the addition of JNJ-1953 diminished the interaction between NS2A and prM (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C), suggesting that the mechanism of action of JNJ-1953 is related to virus assembly. Remarkably, when an inactive enantiomer of JNJ-1953 was used, the interaction between NS2A and prM was not affected (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C). In the same experiment, we also evaluated the interaction between the mutated NS2A (NS2A\u003csup\u003eE21G/A32V\u003c/sup\u003e) and prM, which was not affected upon treatment with JNJ-1953 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C). This aligns with the general model described by Xie \u003cem\u003eet al\u003c/em\u003e. for flavivirus virion assembly, in which at late stage of flavivirus infection cycle, the NS2A molecules (bound to vRNA) at the assembly site recruit C-prM-E through binding to prM \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Whether disruption of the interaction between NS2A and prM is caused by the binding of the compound to NS2A and consequently blocking the association with prM, or by more indirect effects during the recruitment of host and viral proteins to the sites of virion assembly or other mechanisms remains to be determined.\u003c/p\u003e \u003cp\u003eThe antiviral flavivirus drug discovery has advanced significantly over the last years. Although there is currently no flavivirus antiviral treatment available, several compounds with different modes of action have been described \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. The ER supports various steps throughout the whole flaviviral life cycle and provides different opportunities for anti-flaviviral drug development. The transmembrane proteins NS4A and NS4B are considered main drivers in the formation of the replication complex within the ER and were suggested to contribute to membrane rearrangements and stabilize the pore-like opening \u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. NS4B has been a well-known target \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e, with one molecule, Mosnodenvir, in phase 2 clinical trials targeting Dengue \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Mosnodenvir, a highly potent pan-dengue inhibitor blocks the NS3-NS4B interaction within the viral replication complex \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. Mosnodenvir showed activity \u003cem\u003ein vivo\u003c/em\u003e in mice and in non-human primates and was found to be safe and well tolerated in phase 1 clinical trials\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Furthermore, it was found to have a high barrier to resistance. Drugs targeting NS4A are also under evaluation, including compound B and SBI-0090799 which are active \u003cem\u003ein vitro\u003c/em\u003e against DENV and ZIKV by preventing NS4A involvement in replication complex formation \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e,\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. To our knowledge, no small molecules have been identified that target NS2A, thus highlighting the potential of this series of molecules targeting a novel mechanism of action. Furthermore, broad-spectrum activity is a desirable feature to prepare for a next flavivirus epidemic, which could emerge from as-yet unknown or neglected viruses. The design and development of new anti-flavivirus compounds must consider the broad-spectrum activity because the most promising candidates will be those capable of inhibiting a large panel of the most pathogenic flaviviruses, allowing their use in endemic areas in which multiple flaviviruses exist simultaneously. It is likely that an effective solution for combating flaviviruses will not arise from a single agent, but rather from a strategic combination of different measures. Considering the escape potential of RNA viruses, it is prudent to explore antiviral strategies that integrate targeting a viral component with modulation of host cell factors. Additionally, combination therapies that involve synergistic effects, vaccine development, or the use of monoclonal antibodies could further improve the overall management of flavivirus infections. Furthermore, employing measures to alleviate symptoms, such as supportive care and symptomatic treatments, can enhance patient outcomes.\u003c/p\u003e \u003cp\u003eIn conclusion, our study has successfully identified a series of small molecule inhibitors demonstrating broad-spectrum activity against flaviviruses by targeting the critical interaction between the NS2A and prM proteins. This finding positions NS2A as a promising novel target for future drug development efforts. However, to translate these discoveries into effective therapeutics, further research is essential. Deeper insights into the mechanism of action and the optimization of inhibitor potency will be pivotal for advancing these compounds into clinical development. By laying this groundwork, we open the door to innovative therapeutic strategies that could significantly improve outcomes for patients affected by flavivirus infections, ultimately contributing to global health efforts in combating these pervasive viral threats.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCells and growth conditions\u003c/h2\u003e \u003cp\u003eAdenocarcinoma human alveolar basal epithelial cells (A549; CCL-185, ATCC) were cultured in RPMI-1640 (Gibco, Invitrogen Corp.) supplemented with 10% (V/V) fetal bovine serum (FBS, Biowest), 2 mM Ala-Glutamine (Sigma), 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes, Sigma) and 0.02 mg/mL gentamicin (Gibco). African green monkey kidney cells (Vero; CL 84113001, European Collection of Authenticated cell cultures (ECACC) and VeroE6; CRL-158, American Type Culture Collection), were cultured in Eagle\u0026rsquo;s minimal essential medium (MEM; Gibco) supplemented with 10% (V/V) FBS, 2 mM Ala-Glutamine (Sigma) and 0.02 mg/mL gentamicin (Gibco). Vero NS4B-NS5-Tat_LTR-eGFP/hRLuc stable cells, referred to as Vero-GFP, contain a stable expressed NS4B-NS5-Tat and an LTR-eGFP/hRLuc gene. Cells are maintained as described for Vero cells, supplemented additionally with 500 \u0026micro;g/mL geneticin (Gibco) and 200 \u0026micro;g/mL hygromycin (ant-hg-1, InvivoGen). In antiviral assays, the 10% (V/V) FBS is replaced by 2% (V/V) FBS and no hygromycin or geneticin is added. Huh7 hepatoma-derived cells (Sigma) were maintained in Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM, Gibco), supplemented with 10% FBS, 2 mM Ala-glutamine, 1 mM sodium pyruvate (Gibco) and 0.02 mg/mL gentamicin. In the antiviral assay, DMEM medium is used supplemented with 10% (V/V) FBS. BHK-21 cells (baby hamster kidney fibroblast cells, ATCC) was cultured in RPMI-1640 medium (Gibco) supplemented with 10% (V/V) FBS and 1% penicillin-streptomycin (P/S). HEK293T (human embryonic kidney, ATCC) cells were maintained in DMEM medium (Gibco) supplemented with 10% (V/V) FBS, 1% P/S and 4.5 g/L glucose. THP-1 dendritic cell-specific intracellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) cells (TIB-202n, ATCC) were propagated in RPMI-1640 supplemented with 10% (V/V) FBS, 2 mM Ala-Glutamine (Sigma), 25 mM Hepes and 0.02 mg/mL gentamicin. All above-described cells were cultured at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e in a humified incubator. C6/36, an \u003cem\u003eAedes albopictus\u003c/em\u003e cell line (ATCC), was maintained in RPMI-1640 medium with 10% (V/V) FBS, 25 mM Hepes and 1% P/S at 28\u0026deg;C in the absence of CO\u003csub\u003e2\u003c/sub\u003e. All cell lines were regularly tested for mycoplasma contamination.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eVirus\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe following flavivirus strains and constructs were used in this study: DENV-1/TC974 #666 (National Collection of Pathogenic Viruses (NCPV) 0411281v; GenBank accession: AF180817), DENV-2/16681 (GenBank accession: NC_00174; licensed from Dr. R. Bartenschlager \u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e, DENV-2/Eden3295 (GenBank accession: EU081177, \u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e, DENV-2/38865Y10 (Obtained through an MTA with Environmental Health Institute, Singapore), DENV-3/H87 (NCPV 9911281v; GenBank accession: M93130), DENV-4/H241 (NCPV 9910102v; GenBank accession: AY947539), WNV/NY99 (UVE/WNV/1999/US/NY 385\u0026thinsp;\u0026minus;\u0026thinsp;99 (001v-EVA140); GenBank accession: AY842931; European Virus Archive(EVAg)), WNV Uganda B956 (UVE/WNV/1940/UG/UG 956 D117 (001v-EVA1461); GenBank accession: M12294; EvaG), ZIKV/MP1751 (NCPV 1308258v; GenBank accession: KY288905), ZIKV/MR766 (GenBank accession: DQ859059; EVAg), YFV/17D-204 Stamaril\u0026reg; vaccine, lot H5105 (GenBank accession: MN708488; Sanofi Pasteur). JEV/SA14-14-2 (GenBank accession: AF315119.1) was generated at KU Leuven using synthetic, overlapping DNA fragments, as described previously \u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. DENV-2/16681-eGFP, carrying an enhanced green fluorescent protein (eGFP) at the amino terminus of the capsid protein, was produced by transfection of \u003cem\u003ein vitro\u003c/em\u003e-transcribed RNA of plasmid pFK-DV-G2A into Huh7 cells \u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. The DENV subgenomic reporter replicon (sgDVs-RLuc) consist out of a plasmid (denoted pFK, sgDVsR2A) which contains the non-structural genes NS1-NS5 of the DENV-2/16681 strain and the Renilla luciferase (RLuc) reporter gene. The sgDVs-RLuc was used to perform a transient DENV replicon assay \u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCompounds\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eA compound library at Johnson \u0026amp; Johnson consisting of 197,135 small-molecule antivirals was used for HCI-based screening. Compound JNJ-3644 and compounds of the same chemical class were synthesized in house. Reference compounds such as, Compound 24 \u003csup\u003e66\u003c/sup\u003e, Ribavirin \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e, NITD-008 \u003csup\u003e50\u003c/sup\u003e, 2-CMC \u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e, JNJ-1A \u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e, and Brequinar\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e were synthesized in-house. All compounds were \u0026gt;\u0026thinsp;95% pure, which was confirmed using liquid chromatography-mass spectrometry and proton nuclear magnetic resonance.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eHigh content imaging (HCI)\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eFor the primary HTS, 1,100 A549 cells/well were seeded in barcoded 384-well carrier plates (Cellcarrier-384, PerkinElmer), the cells were left to adhere at 37\u0026deg;C for 24 hours. Prior to infection, DENV-2/16681-eGFP virus was added to pre-spotted compound 384-well proxiplates (PerkinElmer). After 2 hours, the virus (MOI 0.15) preincubated with compound (25 \u0026micro;M) was transferred to the carrier plates. After 72 hours of incubation, cells were used for HCI assays. First, live staining was performed by incubating the cells with 50 nM MitoTracker Orange (Thermo Fischer Scientific) for 45 minutes at 37\u0026deg;C. Next, cells were fixed with formaldehyde (2% final concentration; Polyscience) at room temperature for 20 minutes and washed. Plates were then subjected to permeabilization (0.1% Triton-X100). For cell demarcation, nuclei were stained by Hoechst (3.5 \u0026micro;g/mL Hoechst 33258, Invitrogen) and entire cells by HCS CellMask\u003csup\u003e\u0026trade;\u003c/sup\u003e Deep Red (1 \u0026micro;g/mL CellMask\u003csup\u003e\u0026trade;\u003c/sup\u003e Deep red, Invitrogen) (Supplementary Fig.\u0026nbsp;1A). After staining, plates were imaged on the Cell Voyager 7000 (Yokogawa) confocal microscope, followed by data analysis. HCI data was analyzed with Phaedra HCI analysis software \u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e. eGFP positive cells were determined based on the fluorescent signal compared with background signal from the non-infected control cells. The percentage of infected cells was calculated by taking the ratio of infected cells to the total number of cells determined by cell segmentation (Supplementary Fig.\u0026nbsp;1B). Assay quality was further assessed using the Z prime for the percentage of GFP fluorescent cells (Supplementary Fig.\u0026nbsp;1B). The assay was validated using reference compounds with established \u003cem\u003ein vitro\u003c/em\u003e activity against DENV-2, including compound 24 \u003csup\u003e66\u003c/sup\u003e, Ribavirin \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e, NITD-008 \u003csup\u003e50\u003c/sup\u003e, 2-CMC \u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e, and JNJ-1A \u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e(Supplementary Fig.\u0026nbsp;1C).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eSIM2NIC analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe similarity to non-infected control (SIM2NIC) analysis quantifies the extent to which the morphology of infected, compound-treated cells resembles the morphology of non-infected cells. A high similarity, with a SIM2NIC close to 1, indicates a clean antiviral. First, 600 features capturing intensity, shape and texture properties at the single-cell level were extracted using all 4 fluorescent channels (Hoechst, HCS CellMask\u0026trade; Deep Red, MitoTracker Orange, eGFP reporter virus) and averaged across all cells per well using a custom-written Acapella (PerkinElmer) image analysis script. Then, features were normalized as z-scores relative to the infected controls/plate. Feature selection was done by data-driven minimum redundancy maximum relevance algorithm as described in Cox \u003cem\u003eet al.\u003c/em\u003e (2020) \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e on all tested compounds with \u0026ge;\u0026thinsp;2 replicates and \u0026ge;\u0026thinsp;50% virus inhibition (at any concentration) and resulted in 30 reproducible and non-redundant features, which define the \u0026ldquo;morphological profile\u0026rdquo; of each treatment (compound at concentration) and control well. Finally, each morphological profile was compared to the median profile of the non-infected control wells on the same plate by Pearson correlation, resulting in SIM2NIC score. To summarize SIM2NIC at the compound level, the maximum SIM2NIC score overall concentrations at which the compound achieved\u0026thinsp;\u0026ge;\u0026thinsp;50% virus inhibition and retained\u0026thinsp;\u0026ge;\u0026thinsp;30% cell count (relative to infected control) was computed and plotted. Compounds with SIM2NIC score\u0026thinsp;\u0026gt;\u0026thinsp;0.8 were considered for further screening.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAntiviral assays\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eAfter hit identification, the antiviral activity was determined against DENV-2/16681-eGFP on three different cell types (Vero, Huh7, and THP-1/DC-SIGN). In brief, 2,500 Vero or Huh7 cells or 7,500 THP-1/ DC-SIGN cells were seeded in 384-well black view plates (Corning, Sigma Aldrich) containing 200 nL of compounds in a 9-fold serially dilution. For Vero and Huh7 cells, the seeded plates were first incubated for 24 h at 37\u0026deg;C, before being infected with DENV-2/16681-eGFP at a multiplicity of infection (MOI) of 0.5 (Vero) or 5 (Huh7). THP-1/DC-SIGN cells were infected immediately after seeding of the cells with DENV-2/16681-eGFP (MOI 0.5). After three days of incubation at 37\u0026deg;C, viral replication was quantified by measuring eGFP fluorescence using the acumen Cellista (TtpLabtech). The 50% effective concentration (EC\u003csub\u003e50\u003c/sub\u003e) was calculated using dose-dependent inhibition curves. The cytotoxic effect was determined in the same plates after the eGFP-based readout, except for THP1 cells where toxicity is measured in a non-infected plate. Cytotoxicity was measured using an ATPlite\u003csup\u003e\u0026trade;\u003c/sup\u003e cell viability luminescence assay (PerkinElmer), and the luminescence signal was detected with the ViewLux\u003csup\u003e\u0026trade;\u003c/sup\u003e imaging system (PerkinElmer). The 50% cytotoxic concentration (CC\u003csub\u003e50\u003c/sub\u003e) was calculated using dose-dependent inhibition curves. The selective index (SI) was calculated as the ratio of CC\u003csub\u003e50\u003c/sub\u003e/EC\u003csub\u003e50\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eThe antiviral assay with JEV, WNV and ZIKV were conducted in a similar way. VeroE6 cells were seeded in a 96-well plate at a density of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well. The next day, a 3-fold (for ZIKV) or 5-fold serial dilution of the compounds was added to the plates. Lastly, the virus was added to the plates (JEV, MOI:0.1; WNV, MOI:0.1; ZIKV, MOI:0.2). After 1 week of incubation in a humified incubator at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e, virus-induced cytopathic effect (CPE) was determined by means of the MTS readout method (Promega), as described previously \u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. In the JEV antiviral assay, however, virus-induced CPE was determined using ATPlite\u003csup\u003e\u0026trade;\u003c/sup\u003e, according to the manufacturer\u0026rsquo;s protocol. The protocol of the YFV antiviral assay was essentially the same as for ZIKV with some differences: cells (Huh7) were seeded at a density of 5,500 cells/well and virus-induced CPE was determined (using MTS) on day 4 post-infection.\u003c/p\u003e \u003cp\u003eVero-GFP cells (2,500 cells/well) were seeded in 384-well black view plates (Corning\u0026reg;) containing 200 nL of compounds in a 9-fold serially dilution and then placed at 37\u0026deg;C for 24 hours. Next, the cells were infected with the different flaviviruses and corresponding amount of virus (DENV-1/TC974 #666, MOI:1; DENV-2/16681, MOI: 0.5; DENV-3/H87, MOI:0.5; DENV-4/H241, MOI:0.5; WNV/Uganda B956, MOI:0.5; ZIKV/MP1751, MOI:0.16; YFV/17D, MOI:0.06). Three or five days in case of YFV post-infection, the eGFP signal was measured using the acumen Cellista (TtpLabtech). After the eGFP-based readout, the cytotoxic effect was determined in the same plates using ATPlite\u003csup\u003e\u0026trade;\u003c/sup\u003e as described above for Vero cells, except for ZIKV for which the cytotoxic effect was measured on a non-infected plate.\u003c/p\u003e \u003cp\u003eFor the testing of the clinical isolate (DENV-2/Eden3295), Huh7 cells were seeded in a 24-well plate at 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well. Cells were first infected with DENV-2/Eden3295 at a MOI of 0.3 for 1 hour. Virus inoculums were then removed and fresh medium containing the compounds at concentrations ranging from 0.0 1 \u0026micro;M to 50 \u0026micro;M were added. Cells were incubated for additional 48 hours at 37\u0026deg;C and the supernatants were collected. Virus titers in the supernatants were determined by standard plaque assay on BHK-21 cells. Standard plaque assay on BHK-21 was performed as previously described \u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e. EC\u003csub\u003e50\u003c/sub\u003e values were determined using a sigmoidal dose response (variable slope) non-linear regression model in GraphPad Prism software. For antibody-dependent enhanced (ADE) infection, DENV-2/38865Y10 infection at MOI 10 and humanized 4G2 (0.05 \u0026micro;g) were mixed and incubated on ice for 1 hour to allow the formation of immune complexes. THP-1 cells (1\u0026times;10\u003csup\u003e5\u003c/sup\u003e) were infected with the immune complexes for 2.5 hours at 37\u0026deg;C with shaking. Cells were then washed once with PBS before resuspending in RPMI-1640 medium containing the compound at concentrations ranging from 0.1 nM to 25 \u0026micro;M followed by a further incubation of 48 hours. After 48 hours, supernatants were harvested and subjected to virus titer determination by standard BHK-21 plaque assay.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eTetravalent duplex real-time quantitative polymerase chain reaction (RT-qPCR)\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe tetravalent antiviral RT-qPCR was based on as previously described protocol \u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e. In brief, Vero cells (10,000 cells/well) were seeded in 96-well plates containing a serial dilution of the test compound. 24 Hours after seeding, the cells were infected with DENV (DENV-1/TC974 #666, MOI:0.1; DENV-3/H87, MOI:0.025; DENV-4/H241, MOI:0.6), and incubated at 37\u0026deg;C for three days. Intracellular RNA was measured by washing the adherent cells of the plates without supernatant with cold PBS and plates were incubated at -80\u0026deg;C for at least 24 hours. After 24 hours the cells were lysed with Cells-to-CT Bulk Lysis Reagents kit (Thermo Fisher Scientific) and the cell lysates were used to prepare cDNA (using Expand Reverse Transcriptase) of the target sequences, the 3\u0026rsquo;-untranslated region (3\u0026rsquo;UTR) of DENV (Forward primer: 5\u0026prime;-GGCCAGGTCATCACCATT-3\u0026prime;, Reverse primer: 5\u0026prime;-GAGACAGCAGGATC TCTGGTC-3\u0026prime;, Probe: FAM-5\u0026prime;-AAGGACTAGAGGTTAGAGGAGACCCCCC-3\u0026prime;-BHQ1), and the cellular housekeeping reference gene β-actin (Forward primer: 5\u0026prime;-GGCCAGGTCATCACCATT-3\u0026prime;, Reverse primer: 5\u0026prime;-ATGTCCACGTCACACTTCATG-3\u0026prime;, Probe: HEX-5'-TTCCGCTGC(ZEN)CCTGA GGCTCTC-3IABkFQ). Subsequently, a duplex RT-qPCR was performed on a Lightcycler480 II instrument (Roche) at the following conditions: 10 minutes at 95\u0026deg;C, followed by 40 cycles of 10 seconds at 95\u0026deg;C, 1 minute at 60\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eTime-of-addition assay\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe time-of-addition (TOA) assay was performed as shown in the schematics of Fig.\u0026nbsp;3 \u003csup\u003e75\u003c/sup\u003e. Briefly, 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e Huh7 cells were infected with DENV-2/Eden3295 at MOI 1 for 1 hour followed by treatment with 5 \u0026micro;M JNJ-1953 at 1, 2, 4, 6, 10 and 19 h post-infection (post-treatment). For pre-treatment, the cells were exposed to 5 \u0026micro;M of the respective compounds for 2 hours prior to infection. For co-treatment, the cells were infected with the virus that was mixed with 5 \u0026micro;M of the compounds for 1 hour and subsequently replaced with media. The endpoint assessment is by quantifying the infectious virus production after 24 hours post-infection.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDENV-2/16681\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003eresistance selection\u003c/b\u003e\u003c/p\u003e \u003cp\u003eVero cells were seeded at a density of 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells/well in a 96 well plate. The next day, cells were infected (MOI of 0.1) with DENV-2/16681 and incubated for 96 hours at 37\u0026deg;C in the presence of a 2-fold serial dilution of JNJ-3644 (5-0.039 \u0026micro;M) for 4 days at 37\u0026deg;C. After 4 days, cells were microscopically checked for CPE, and the supernatant from two adjacent wells showing 30\u0026ndash;70% CPE was collected and pooled. The collected supernatant was subsequently used to infect freshly seeded cells. The remaining supernatant was stored at \u0026minus;\u0026thinsp;80\u0026deg;C until further analysis. Virus was passaged twice a week. During passaging of the virus, the start concentration of the compound was gradually increased. This procedure was repeated until the observed EC\u003csub\u003e50\u003c/sub\u003e value approached the cytostatic concentration of the compound. To check for spontaneous and/or tissue-culture-adapted mutations, part of the wells served as wild-type virus controls to which no compound was added. Wild-type DENV-2/16681 was passaged using Vero cells in a similar way to compound-treated virus. Further analysis includes next-generation sequencing of the viral RNA isolated from cell culture supernatant (140 \u0026micro;L) using a QIAamp Viral RNA Mini kit (Qiagen) per the manufacturer\u0026rsquo;s protocol. Viral RNA was amplified into double-stranded DNA using a NuGEN Trio RNA-Sequence kit per manufacturer\u0026rsquo;s protocol. Full DENV-2/16681 genome was sequenced using next-generation sequencing technology (Illumina). Sequences were filtered for viral content by aligning the reads to DENV-2/16681 viral genome (GenBank accession: NC_00174). A coverage cut-off value of 100 and a 15% read frequency cut-off were used for the reliable detection of amino acid variants.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eTransient DENV replication assay\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eMutant subgenomic DENV reporter replicons (sgDVs-RLuc) each containing a NS2A single, double or triple resistance mutation (Epoch Life Science) were used to determine the compound resistance imposed by each of the mutations. First, each resistance mutation was inserted separately into the sgDVs-RLuc replicon. The plasmid (denoted pFK-sgDVs-R2A) contains the non-structural genes \u003cem\u003eNS1-NS5\u003c/em\u003e of the DENV-2/16681 strain with cell-adaptive mutations in \u003cem\u003eNS3\u003c/em\u003e (A546V and H451P), \u003cem\u003eNS4A\u003c/em\u003e (I116M) and \u003cem\u003eNS5\u003c/em\u003e (E892K), and the \u003cem\u003eRenilla luciferase\u003c/em\u003e (RLuc) reporter gene \u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. The plasmids of both wild-type and mutant sgDVs-Rluc were used to produce \u003cem\u003ein vitro\u003c/em\u003e transcribed (IVT) DENV RNA as described previously and electroporated into Huh7 cells \u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. The RLuc activity was measured using the Renilla-Glo\u0026reg; Luciferase assay system (Promega) following the manufacturer\u0026rsquo;s instructions and detected using the ViewLux\u003csup\u003e\u0026trade;\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFull-length DENV-2/Eden3295 cDNA clone (GenBank accession: EU081177) used in this study has been previously described \u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e. Site-directed mutagenesis was used to generate the different NS2A mutations (single mutants E21G or A32V and the double mutant E21G A32V) on the full-length DENV-2/Eden3295 clone. IVT RNA was obtained from the full-length cDNA clone using T7 mMESSAGE mMACHINE kit (Ambion) and transfected into C6/36 using the previously described electroporation conditions \u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e. Supernatants from the transfected C6/36 cells were collected on day 7 after transfection (P0 virus) and passaged once in C6/36 to obtain the P1 virus stock for compound efficacy evaluation assays. The IVT RNAs of DENV-2 wild-type or the various NS2A mutants were transfected into BHK-21 cells as previously described \u003csup\u003e\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e to profile its replication kinetics over a course of 3 days. Supernatants were collected for infectious virus quantification by standard plaque assay while the transfected cells were washed once prior to lysing with RLT buffer (Qiagen) for intracellular viral RNA quantification by RT-qPCR.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eConstruction of DENV-2 prM and NS2A mammalian expression plasmids\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe mammalian expression plasmids of DENV-2/Eden3295 (EU081177) prM and NS2A were designed and constructed as described in \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e (see schematic in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Briefly, the signal peptide from \u003cem\u003eGaussia luciferase (Gluc)\u003c/em\u003e together with the last 16-amino acids (aa) of NS1 is fused to the N-terminus of NS2A to ensure correct processing and membrane topology of NS2A. This NS2A construct (wild-type, WT) is then cloned into a C-terminal Flag-tagged pcDNA3.1\u0026thinsp;+\u0026thinsp;vector (Genscript) using \u003cem\u003eNhe\u003c/em\u003eI and \u003cem\u003eXho\u003c/em\u003eI restriction sites. The WT NS2A plasmid was subjected to reverse-engineering of the E21G/A32V double mutations (NS2A\u003csup\u003eE21G/A32V\u003c/sup\u003e) using Quikchange II XL site-directed mutagenesis kit (Stratagene) according to manufacturer\u0026rsquo;s instructions. For the prM construct, the anchor C signal peptide (AnC-14aa) is retained at the N-terminus of prM to ensure correct targeting to the ER membrane. This prM construct is then cloned into a N-terminal Myc pcDNA3.1\u0026thinsp;+\u0026thinsp;vector (Genscript) with \u003cem\u003eHind\u003c/em\u003eIII and \u003cem\u003eXho\u003c/em\u003eI restriction sites.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eCo-immunoprecipitation, SDS-Page and Western blot\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe co-immunoprecipitation was performed as described in \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Briefly, single, or various combinations of expression plasmids carrying wild-type NS2A or NS2A\u003csup\u003eE21G/A32V\u003c/sup\u003e and prM (each at a concentration of 5 \u0026micro;g) were transfected into HEK293T cells (2.5 \u0026times; 10⁵ cells per 10-cm dish) using the Fugene 6 transfection reagent (Promega). At 6 hours post-transfection, the cells were treated with 10 \u0026micro;M of JNJ-1953 or its inactive enantiomer (JNJ-2005). Following a 44-hour incubation period, the cells were lysed in 0.5 mL immunoprecipitation (IP) buffer (20 mM Tris, pH 7.5, 100 mM NaCl, 0.5% DDM, and EDTA-free protease inhibitor cocktail [Roche]) with rotation at 4\u0026deg;C for one hour. The lysates were then clarified by centrifugation at 21,000 \u0026times; g and 4\u0026deg;C for 30 minutes. The supernatants (200 \u0026micro;L) were combined with IP buffer (20 mM Tris [pH 7.5], 0.5% DDM), and NaCl was added to achieve a final concentration of 400 mM. 2 \u0026micro;g of rabbit anti-c-myc antibody (Sigma) were then added to the mixture, followed by agitation overnight at 4\u0026deg;C (end-to-end shaker) to form immune complexes. The immune complexes were captured by the addition of 30 \u0026micro;L of Pierce\u0026trade; Protein A/G Plus Agarose (Thermo Scientific), and the mixtures were tumbled further for 1\u0026ndash;2 hours. Thereafter, the beads-bound immune complexes were collected by centrifuging at 900 \u0026times; g at 4\u0026deg;C for 3 min and washed five times with PBS containing 0.1% Tween 20 (PBS-T). The beads-bound immune complexes were eluted by boiling in 5\u0026times; sodium dodecyl sulfate (SDS) (Bio Basic Asia Pacific) sample buffer supplemented with 50 mM dithiothreitol (DTT) at 100\u0026deg;C for 10 min. The tubes were briefly vortexed and then subjected to centrifugation at 10,000 \u0026times; g for one minute. A total of 40 \u0026micro;L of the sample was loaded onto a 4\u0026ndash;20% SDS-PAGE gel (Bio-Rad, Cat # 4561094). Subsequently, the proteins were resolved and transferred onto a nitrocellulose membrane (Bio-Rad) using the Bio-Rad Blotting System. To prevent the light chain (size 26 kDa) from obscuring the prM or NS2A protein bands (size approx. 24 kDa), the protein blot was stained with Ponceau as previously described \u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e and trimmed precisely at 25 kDa. Subsequently, the blot was incubated for 1 h in a blocking buffer containing 5% skim milk in PBS-T. The blot was washed twice with PBS-T and incubated with a primary antibody, either rabbit anti-Myc (Sigma) or mouse anti-Flag (Sigma), and an anti-GAPDH antibody (Thermo Fisher), for approximately 16 hours at 4\u0026deg;C while shaking. Following three washes with PBS-T buffer, the protein blot was incubated with horseradish peroxidase (HRP)-conjugated rabbit or mouse antibody for one hour at room temperature on a shaker. To demonstrate equal expression and loading, the protein blot with inputs (10%) was probed with an anti-Myc, anti-Flag, or anti-GAPDH antibody. Subsequently, the blots were subjected to three comprehensive washes with PBS-T buffer. Thereafter, the ECL substrates (Advansta Inc.) were applied in accordance with the instructions provided. Subsequently, the chemiluminescence signals were detected using the ChemiDoc system (Bio-Rad).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eDensitometric analysis of the band intensities of the CoIP were determined for 4 individual experiments, combined and compared using unpaired Student\u0026rsquo;s t-test (p-value\u0026thinsp;=\u0026thinsp;0.096; degrees of freedom\u0026thinsp;=\u0026thinsp;6).\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information. The uncropped images of the western blot shown in Fig. 5 are presented in Supplementary Fig. 2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe natural occurrence of the mutations was retrieved from the Bacterial and Viral Bioinformatics Research center (BV-BRC) database. Graphs and figures were generated using Microsoft PowerPoint or GraphPad Prism (v.9.0.0 and v.7.04); the software was made available by Johnson \u0026amp; Johnson.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Natalene Hui Shan Yuen, Sze Woei Ng, Nanthini Ramanathan at the center of global health discovery for technical assistance; Caroline Collard and Elke Maas for technical assistance at the KU Leuven; Edgar Jacoby for the Alphafold model of NS2A. We also thank Drs Lee Ching Ng and Judith Wong (National Environmental Agency, Singapore) for providing the DENV2/38865Y10 strain used in this study. We received funding from the Flanders Agency Innovation \u0026amp; Entrepreneurship (VLAIO O\u0026amp;O grants HBC.2021.1131 and HBC. 2017.0947. This work was further supported by the Center of Global Health Discovery (CGHD) fund 2022-1723 administrated by Johnson \u0026amp; Johnson as well as the IAF-ICP I2301E0019 by Agency for Science, Technology and Research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eD.B, O.G and D.P. planned, coordinated and performed the experimental virology work at Johnson \u0026amp; Johnson. P.G, A.E.B, D.L, S.M, J.V.D.V, P.V performed the experimental virology work at Johnson \u0026amp; Johnson. K.W.K.C, M.M.C and S.G.V planned, coordinated and performed the experimental virology work at the center for global health discovery in Singapore. P.B. performed experimental virology work at the center for global health discovery in Singapore. S.J.F.K. and J.N. planned, coordinated and performed the experimental virology work at KU Leuven. S.V.B planned, coordinated and performed the medicinal chemistry work at Johnson \u0026amp; Johnson. B.S. planned and coordinated the pharmacokinetics and pharmacodynamics work at Janssen Johnson \u0026amp; Johnson. S.J. and D.P performed data analysis on the High-throughput screen at Johnson \u0026amp; Johnson. O.G. designed and initiated the project at Johnson \u0026amp; Johnson. A.K., O.G., D.B. and S.G.V secured funding from external organizations. D.B wrote the manuscripts with contributions from S.G.V, K.W.K.C, M.M.C, S.J, S.J.F.K, B.S and O.G and with comments from all the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eD.B., S.V.B., S.J, J.V.D.V, P.V., D.P., B.S., A.K. and G.O. are full-time employes of Johnson \u0026amp; Johnson and potential stockholders of Johnson and Johnson. The other authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eTabachnick, W. J. Climate Change and the Arboviruses: Lessons from the Evolution of the Dengue and Yellow Fever Viruses. \u003cem\u003eAnnu Rev Virol\u003c/em\u003e \u003cstrong\u003e3\u003c/strong\u003e, 125-145, doi:10.1146/annurev-virology-110615-035630 (2016).\u003c/li\u003e\n\u003cli\u003eDutta, S. K. \u0026amp; Langenburg, T. 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Ponceau S waste: Ponceau S staining for total protein normalization. \u003cem\u003eAnal Biochem\u003c/em\u003e\u003cstrong\u003e575\u003c/strong\u003e, 44-53, doi:10.1016/j.ab.2019.03.010 (2019).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5979312/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5979312/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFlaviviruses, such as dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV), Japanese encephalitis virus (JEV), and yellow fever virus (YFV), constitute a significant public health concern with billions of people at risk of infection. Climate change and the expanding geographical distribution of mosquito vectors transmitting flaviviruses have increased their potential to cause large-scale disease outbreaks. The frequency and severity of disease outbreaks highlights the urgent need for a broad-spectrum antiviral agent targeting flaviviruses. In this work, we conducted a comprehensive morphological profiling of approximately 200,000 small molecules through a fluorescence-based high-content imaging platform, which led to the identification of a singular small molecule exhibiting broad-spectrum activity against flaviviruses. Subsequent hit deconvolution against DENV serotype 2 (DENV-2) revealed NS2A as a novel therapeutic target and suggested a mechanism whereby the identified small molecule inhibits the interaction between NS2A and the prM protein, revealing a previously uncharacterized antiviral mechanism of action.\u003c/p\u003e","manuscriptTitle":"Identification of a broad-spectrum flavivirus inhibitor targeting NS2A, a previously unidentified target","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-21 16:23:43","doi":"10.21203/rs.3.rs-5979312/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ae81eaf6-8568-4af7-9f5e-d7914cf27c13","owner":[],"postedDate":"February 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":44517830,"name":"Biological sciences/Drug discovery/Pharmaceutics"},{"id":44517831,"name":"Biological sciences/Microbiology/Virology/Dengue virus"}],"tags":[],"updatedAt":"2025-12-16T15:39:39+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-21 16:23:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5979312","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5979312","identity":"rs-5979312","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}