DENV NS4B protein, which mediates critical binding with viral NS3 helicase/protease to assemble replication complex;
The compound blocks de novo NS3–NS4B protein complex formation, the EC50 of disrupting wild-type NS3/NS4B interaction is 0.006 μM; mutant NS4B V91A raises this EC50 to 0.2 μM (42-fold resistance), mutant T108I raises EC50 to 0.05 μM (9-fold resistance)[1].
The primary target of (+)-JNJ-A07 is the interaction between two viral proteins, NS3 and NS4B, which is essential for the formation of the dengue virus replication complex. By blocking this interaction, the compound prevents the formation of the viral replication complex and inhibits viral replication. (+)-JNJ-A07 exhibits a high barrier to resistance and demonstrates potent activity against all four dengue virus serotypes.

(+)- JNJ-A07 possesses a high resistance barrier that stops two viral proteins (NS3 and NS4B) from interacting to create viral replication complexes [1].

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1. Pan-dengue serotype antiviral assay on Vero E6 cells: JNJ-A07 exerts picomolar to low nanomolar EC50 against 21 clinical isolates covering all 4 DENV serotypes and multiple genotypes. For DENV-2 strain 16681 in Vero cells, EC50 = 0.0001 μM, CC50 =13 μM, selectivity index (SI)=130000; no strong cross-antiviral activity against Zika, JEV, WNV, yellow fever or other RNA/DNA viruses was observed[1]
2. Multi-cell-line anti-DENV-2 activity test: Active on Huh-7, THP-1 DC-SIGN+ cells, human immature dendritic cells and C6/36 mosquito cells; EC50 on Huh-7 cells =0.0008 μM, EC50 on immature dendritic cells =0.002 μM, EC50 on C6/36 cells =0.003 μM[1]
3. Time-of-drug-addition assay: If added before intracellular viral RNA synthesis initiation (10 h post-infection), JNJ-A07 fully suppresses DENV RNA production; inhibitory effect gradually weakens once viral replication starts[1]
4. In vitro resistance selection assay: Long-term serial DENV passaging under compound pressure (up to 40 weeks) is required to generate low-susceptible variants; resistant strains carry multiple NS4B mutations (L94F, T108I, T216N etc.) and show severely impaired replication in Aedes albopictus C6/36 mosquito cells[1]
5. NS3-NS4B co-immunoprecipitation western blot assay: 35 nM JNJ-A07 reduces co-precipitated wild-type NS3 protein by 95%; resistant NS4B mutants largely evade this blocking effect; compound slows proteolytic cleavage of NS4A-2K-NS4B precursor protein[1]
6. Subgenomic replicon fitness assay: Single NS4B resistance mutations lead to varied viral replication defects; L94F mutation confers highest resistance (~950-fold) while maintaining robust replication in mammalian cells[1]

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In vitro, (+)-JNJ-A07 demonstrates potent antiviral activity against dengue virus with an EC₅₀ of 0.1 nM in the DENV-2 strain (16681) in Vero cells. It exerts nanomolar to picomolar activity against 21 clinical isolates that represent the natural genetic diversity of known genotypes and serotypes. The compound prevents the formation of the viral replication complex by blocking the interaction between two viral proteins (NS3 and NS4B).

1. Prophylactic AG129 mouse lethal DENV challenge model (10⁶ PFU DENV-2 RL): Twice-daily oral JNJ-A07 (1/3/10/30 mg/kg) initiated 1 h pre-infection dose-dependently lowers plasma viral load on day 3; 30 mg/kg reduces viral RNA by 3.8 log10 copies/mL; 90% animal survival at 30 mg/kg vs nearly 100% mortality in vehicle group[1]
2. Non-lethal DENV kinetic mouse model (10² PFU DENV-2 RL): Oral twice-daily 3–30 mg/kg JNJ-A07 suppress plasma viral load to near lower limit of quantification; viral load AUC of 30/10 mg/kg groups is 0% of vehicle control[1]
3. Delayed therapeutic treatment mouse model: Starting treatment at day 4 (peak viremia) clears plasma virus within 72 h; even treatment initiation at day 5/6 (viral peak) produces immediate viral load reduction, though AUC inhibition declines to 52% at day 6 initiation[1]
4. Tissue viral load detection: Oral JNJ-A07 significantly decreases DENV RNA copies in mouse spleen, kidney and liver in a dose-dependent manner[1]
5. Cytokine detection in infected mouse plasma: Compound treatment normalizes elevated pro-inflammatory cytokines IL-6, IL-18, IFNγ and TNF induced by dengue infection[1]
6. Head-to-head comparison with NS4B inhibitor NITD-688: JNJ-A07 achieves far stronger viral suppression at equivalent oral doses in AG129 mice[1]

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In vivo, (+)-JNJ-A07 is orally active and demonstrates significant efficacy against dengue virus infection in mouse infection models. It has favorable pharmacokinetic properties that support its oral bioavailability and in vivo efficacy. The compound has been shown to reduce viral load and improve survival in animal models of dengue virus infection.

1. NS3-NS4B binding immunoprecipitation functional assay: Constructs encoding NS2B-NS3 protease-helicase complex and NS4A-2K-NS4B (wild or mutant NS4B with C-terminal HA tag) were expressed in engineered Huh7-T7 cells. Test compound was supplemented during transfection or medium exchange. Cell lysates were prepared with mild non-ionic detergent lysis buffer containing protease inhibitors. Anti-HA affinity resin captured NS4B-associated protein complexes. Captured protein was separated via SDS-PAGE, followed by western blot quantification of co-eluted NS3 and NS4B precursor band intensity to calculate NS3/NS4B binding ratio and compound inhibitory potency[1]
2. In vitro pre-formed NS3-NS4B complex stability assay: Cell lysates containing assembled NS3/NS4B complexes were incubated with test compound at 20 °C or 37 °C for 2 h, followed by HA pull-down and western blot detection to verify whether compound can disrupt already formed protein heterodimers[1]

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In vitro enzyme/receptor binding assays for (+)-JNJ-A07 typically involve studying its effects on the NS3-NS4B interaction. The compound is tested for its ability to disrupt the interaction between these two viral proteins using biochemical assays such as co-immunoprecipitation, surface plasmon resonance, or fluorescence polarization. The EC₅₀ value is calculated from the dose-response curve.

1. EGFP reporter DENV cytopathic inhibition assay: Adherent Vero/Huh7 suspension THP-1 DC-SIGN cells were seeded in multi-well plates with serial diluted JNJ-A07. Cells were infected with eGFP-tagged DENV-2 at fixed MOI, incubated for 72 h. Fluorescence signal reflecting viral replication was quantified via laser imaging; parallel cell viability luminescence test measured compound cytotoxicity to calculate EC50 and CC50[1]
2. Human immature dendritic cell flow cytometry antiviral assay: Monocytes isolated from human peripheral blood were induced into immature dendritic cells with interleukins. Cells were infected with wild-type DENV-2 with gradient compound treatment. After 48 h incubation, cells were fixed and stained with anti-prM viral antibody, flow cytometry counted percentage of virus-positive cells; separate viability staining measured compound cellular toxicity[1]
3. Mosquito C6/36 cell viral yield reduction assay: Aedes albopictus C6/36 cells were cultured at 28 °C, incubated with serially diluted compound and DENV-2 RL strain for 7 days. Cell supernatant was collected for viral RNA RT-qPCR quantification; parallel cell nuclear staining quantified live cell count for CC50 calculation[1]
4. DENV whole-genome sequencing for resistant mutant identification: Wild-type DENV was serially passaged in compound-treated Vero cells weekly for up to 43 passages. Viral RNA extracted from culture supernatant was reverse transcribed, amplified and sequenced on Illumina platform; variant frequency threshold 15% was set to identify NS4B resistance mutations[1]
5. DENV subgenomic replicon luciferase assay: In vitro transcribed mutant/wild DENV subgenomic replicon RNA was electroporated into Huh7 cells. Serial compound dilutions were added to cell culture medium. After 48–96 h incubation, cell lysis luciferase signal was read to evaluate viral replication fitness and compound resistance fold change[1]

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In vitro cellular assays for (+)-JNJ-A07 involve testing its antiviral activity against dengue virus. Vero cells or other susceptible cell lines are infected with dengue virus and treated with serial dilutions of (+)-JNJ-A07 (0.001-100 nM). Viral replication is measured by plaque assay, qRT-PCR, or immunofluorescence after 48-72 hours of infection. The compound demonstrates concentration-dependent antiviral activity with an EC₅₀ of 0.1 nM against DENV-2. Cytotoxicity is assessed in parallel using MTT or CCK-8 assays.

1. Intravenous pharmacokinetic test in male CD-1 mice: JNJ-A07 formulated in mixed PEG400 and sodium-adjusted water solvent at concentration 0.5 mg/mL, single 2.5 mg/kg intravenous injection. Blood samples were collected via limb vein at 0.12, 0.33, 1, 2, 4, 7 h and terminal heart puncture at 24 h for plasma compound concentration LC-MS/MS detection[1]
2. Oral pharmacokinetic test in male CD-1 mice: Compound dissolved in identical PEG400-water solvent, single oral gavage at 1, 3, 10, 30 mg/kg. Blood sampling time points consistent with IV group, non-compartmental PK analysis calculated exposure parameters[1]
3. Rat oral and IV pharmacokinetics: Sprague Dawley male rats received 2.5 mg/kg IV injection or 10 mg/kg oral compound solution, plasma sampling and LC-MS/MS quantification performed same as mouse protocol[1]
4. Lethal dengue prophylaxis mouse model (AG129 female mice, 7–11 weeks): Intraperitoneal injection of 10⁶ PFU DENV-2 RL; 24 h before infection, single anti-flavivirus antibody injection to mimic antibody-dependent enhancement. JNJ-A07 oral gavage twice daily (1/3/10/30 mg/kg), administration started 1 h before viral challenge, treatment lasted 5 consecutive days. Mice were observed daily for weight loss, clinical signs up to day 25; terminal blood and organ tissue collected for viral load detection[1]
5. Delayed therapeutic dengue mouse model (AG129 female): Intraperitoneal injection of low-dose 10² PFU DENV-2 RL. Twice-daily oral 30 mg/kg JNJ-A07 treatment initiated at day 0/1/2/3/4/5/6 post infection, treatment duration fixed at 6 days. Animals were split into alternating blood collection subgroups to measure longitudinal plasma viral RNA levels over 14 days[1]
6. Rodent 15-day repeated-dose toxicity test: Male rats received daily oral gavage of JNJ-A07 up to 300 mg/kg for 15 consecutive days, clinical adverse reactions were monitored during dosing period[1]

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In vivo animal studies for (+)-JNJ-A07 involve efficacy testing in dengue virus infection models. Mice are infected with dengue virus and treated with (+)-JNJ-A07 via oral administration at various doses. Survival rates, body weight changes, and viral loads in tissues are monitored over the course of infection. The compound demonstrates significant efficacy, reducing viral load and improving survival in mouse infection models.

1. Mouse intravenous PK parameters (2.5 mg/kg IV): Plasma clearance CLp =4.1±0.4 mL/min/kg;
Steady-state volume of distribution Vdss=0.78±0.04 L/kg;
Terminal elimination half-life t1/2=3.0±0.04 h;
Total plasma exposure AUC0-last=10227±903 ng·h/mL[1]
2. Mouse oral PK parameters: Oral bioavailability F ranges 37%–59% across 1–30 mg/kg doses;
30 mg/kg oral group Cmax=12143±4500 ng/mL, Tmax=2.0±1.7 h, AUC0-last=71963±8550 ng·h/mL[1]
3. Rat intravenous PK (2.5 mg/kg): CLp=4.4±2.5 mL/min/kg, Vdss=1.0±0.4 L/kg, t1/2=3.1±0.3 h[1]
4. Rat oral PK (10 mg/kg): Oral bioavailability >100%, Cmax=4440±322 ng/mL, Tmax=5.0±1.7 h[1]
5. Tissue distribution in infected mice: Oral administered JNJ-A07 penetrates into spleen, kidney and liver tissue to exert local anti-dengue activity[1]
6. Linear exposure characteristic: Within tested oral dose range (1–30 mg/kg mouse), plasma AUC and Cmax increase proportionally with rising oral dose[1]

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(+)-JNJ-A07 has a molecular formula of C₂₈H₂₆ClF₃N₂O₆ and a molecular weight of 578.96. The compound is intended for research use only and has not been approved for clinical use. It is a highly potent, orally active pan-serotype dengue virus inhibitor. Detailed pharmacokinetic parameters such as half-life and oral bioavailability have been characterized in preclinical studies.

1. Rodent repeated-dose toxicity: Daily oral dosing up to 300 mg/kg in rats for 15 consecutive days induced no observable adverse toxic signs[1]
2. In vitro cellular cytotoxicity: High selectivity index against DENV-infected cells; CC50 in Vero cells =13 μM, far higher than antiviral EC50 (0.0001 μM); CC50 >25 μM in Huh7 cells, minimal cytotoxicity at effective antiviral concentrations[1]
3. Mosquito cell toxicity: CC50 of JNJ-A07 on C6/36 cells =18 μM, no significant cell damage at working antiviral concentrations[1]
4. Off-target viral toxicity: No meaningful inhibitory activity against other flaviviruses (ZIKV, JEV, WNV, YFV) or unrelated RNA/DNA viruses at therapeutic concentrations, indicating low off-target virotoxic risk[1]
5. Resistant virus transmissibility toxicity: NS4B mutant resistant viruses replicate poorly in mosquito C6/36 cells, even if resistance emerges in human hosts, transmission via mosquito vectors is greatly restricted[1]

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The toxicity profile of (+)-JNJ-A07 has not been extensively characterized. As a research compound, standard toxicity studies would include assessment of acute oral toxicity, repeated-dose toxicity, and genotoxicity in animal models. The compound is intended for research use only and is not approved for clinical use. Further toxicological studies would be needed to fully establish its safety profile for therapeutic applications.

[1]. A pan-serotype dengue virus inhibitor targeting the NS3-NS4B interaction. Nature. 2021 Oct;598(7881):504-509.

1. Mechanism of action core: JNJ-A07 only inhibits de novo formation of NS3–NS4B protein complexes, cannot dissociate pre-assembled NS3/NS4B heterodimers; compound slows proteolytic cleavage of NS4A-2K-NS4B polyprotein precursor to further block replication complex assembly[1]
2. Resistance barrier feature: High genetic barrier requires multiple simultaneous NS4B point mutations to generate high-level drug resistance; single mutation only produces mild to moderate susceptibility reduction; naturally circulating clinical dengue isolates almost carry zero high-risk NS4B resistance substitutions[1]
3. Clinical translational value: Effective in both prophylactic and delayed therapeutic mouse dengue models; capable of rapidly lowering high peak viremia, which matches clinical dengue patient viral dynamic characteristics, supporting human therapeutic and preventive application potential[1]
4. Compound development background: Derived from indole scaffold high-throughput screening, total ~2000 analogues synthesized for structural optimization; synthetic routes recorded in multiple global patent publications, one close structural analogue entered follow-up preclinical evaluation[1]
5. Comparative advantage over existing NS4B inhibitor NITD-688: Distinct NS4B binding mode and resistance mutation profile; JNJ-A07 shows markedly stronger in vivo antiviral potency at equivalent oral doses in dengue mouse models[1]
6. Indication orientation: Developed as pan-serotype oral small-molecule therapeutic and prophylactic agent for dengue fever and severe dengue infection, filling the blank of approved dengue oral antiviral drugs[1]

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(+)-JNJ-A07 (CAS 2135640-93-4) is a highly potent, orally active pan-serotype dengue virus inhibitor targeting the NS3-NS4B interaction. It has a molecular formula of C₂₈H₂₆ClF₃N₂O₆ and a molecular weight of 578.96. The compound exerts nanomolar to picomolar activity against 21 clinical isolates and has an EC₅₀ of 0.1 nM against DENV-2. It demonstrates significant efficacy in mouse infection models. It is intended for research use only and is not approved for clinical use.

C28H26CLF3N2O6

578.96

578.143

C, 58.09; H, 4.53; Cl, 6.12; F, 9.84; N, 4.84; O, 16.58

2135640-93-4

(-)-JNJ-A07;2135640-92-3

131964318

White to light brown solid powder

6.3

2

10

11

40

843

0

N1(CCC2=C1C=C(C=C2)OC(F)(F)F)C(C(C1=CC=C(C=C1)Cl)NC1=CC(OCCCC(O)=O)=CC(=C1)OC)=O

WUBVXTOLZKVNEG-UHFFFAOYSA-N

InChI=1S/C28H26ClF3N2O6/c1-38-22-13-20(14-23(15-22)39-12-2-3-25(35)36)33-26(18-4-7-19(29)8-5-18)27(37)34-11-10-17-6-9-21(16-24(17)34)40-28(30,31)32/h4-9,13-16,26,33H,2-3,10-12H2,1H3,(H,35,36)

4-[3-[[1-(4-chlorophenyl)-2-oxo-2-[6-(trifluoromethoxy)-2,3-dihydroindol-1-yl]ethyl]amino]-5-methoxyphenoxy]butanoic acid

JNJ-A07; (+)-JNJ-A07; (+)-JNJ A07; SCHEMBL25852911;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : 100 mg/mL (172.72 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.32 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.32 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)