Ultrasensitive chemiluminescent neuraminidase probe for rapid screening and identification of small-molecules with antiviral activity against influenza A virus in mammalian cells

Influenza A virus is the most virulent influenza subtype and is associated with large-scale global pandemics characterized by high levels of morbidity and mortality. Developing simple and sensitive molecular methods for detecting influenza viruses is critical. Neuraminidase, an exo-glycosidase displayed on the surface of influenza virions, is responsible for the release of the virions and their spread in the infected host. Here, we present a new phenoxy-dioxetane chemiluminescent probe (CLNA) that can directly detect neuraminidase activity. The probe exhibits an effective turn-on response upon reaction with neuraminidase and produces a strong emission signal at 515 nm with an extremely high signal-to-noise ratio. Comparison measurements of our new probe with previously reported analogous neuraminidase optical probes showed superior detection capability in terms of response time and sensitivity. Thus, as far as we know, our probe is the most sensitive neuraminidase probe known to date. The chemiluminescence turn-on response produced by our neuraminidase probe enables rapid screening for small molecules that inhibit viral replication through different mechanisms as validated directly in influenza A-infected mammalian cells using the known inhibitors oseltamivir and amantadine. We expect that our new chemiluminescent neuraminidase probe will prove useful for various applications requiring neuraminidase detection including drug discovery assays against various influenza virus strains in mammalian cells.

N-acetylneuraminic acid methyl ester (1 gr, 3.1 mmol, 1 eq) was dissolved in 5 ml of pyridine and cooled to 0℃. Acetyl chloride (1.76 ml, 24 mmol, 8 eq) was added dropwise, and upon completion the reaction mixture was stirred over night at room temperature. Afterwards reaction mixture was diluted with EtOAc and washed with HCl 1M. The organic layer was separated, dried over Na2SO4, filtered and the solvent was evaporated under reduced pressure, to afford compound 1 (1.5 gr, 92% yield) as an off-white solid. DIPEA (327µL, 1.88 mmol, 3 eq) was added to Compound 2 (320mg, 0.62mmol, 1 eq) and 4-Hydroxybenzaldehyde (153 mg, 1.25 mmol, 2 eq) dissolved in ACN. The reaction mixture was stirred at room temperature and monitored by TLC (EtOAc). Upon completion, the reaction mixture was diluted with EtOAc, and washed with brine. The organic layer was separated, dried over Na2SO4, filtered and the solvent was evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel (EtOAc) to afford compound 3c (234 mg, 58% yield) as a white solid.

Compound 4
Compound 3 (140 mg, 0.23 mmol, 1 eq) and silica gel (43 mg, 0.7 mmol, 3 eq) were dissolved in 2 ml in a mixture of iPrOH and CHCl3 (2:3) and cooled to 0℃. NaBH4 (9 mg, 0.23 mmol, 1 eq) was add to the reaction mixture and the reaction was allowed warm up to room temperature. The reaction mixture was stirred at room temperature and monitored by TLC (EtOAc). Upon completion, the reaction mixture was diluted with EtOAc (100 mL) and washed with 0.1M HCl (50 mL) and brine (50 mL). The organic layer was separated, dried over Na2SO4 and evaporated under reduced pressure to afford compound 4 (130 mg, 93% yield) as a off-white solid. Compound 4 (86 mg, 0.14 mmol, 1 eq) was dissolved in 1 ml of ACN and cooled to 0 ℃. Sodium Iodide (64 mg, 0.43 mmol, 3 eq) was added followed by the rapid addition of TMS-Cl (54 μl, 0.43 mmol, 3 eq). The reaction was allowed to warm up to room temperature and monitored by TLC (EtOAc). Upon completion, the reaction mixture was diluted with EtOAc, and washed with saturated Na2S2O3 followed by brine. The organic layer was separated, dried over Na2SO4, filtered and the solvent was evaporated under reduced pressure, the crude product was purified by column chromatography on silica gel (EtOAc) to afford the compound 5 (70 mg, 65% yield) an off-white solid.

Compound 6
Phenol enol ether 8 2 (50 mg, 0.12 mmol, 1.2 eq) and K2CO3 (26 mg, 0.18 mmol, 2 eq) were dissolved in DMF (1 mL). The solution was stirred for 5 minutes before compound 5 (80 mg, 0.11 mmol, 1 eq) was added. The reaction mixture was stirred at room temperature and monitored by TLC (EtOAc). Upon completion, the reaction mixture was diluted with EtOAc (100 mL) and washed with 0.1M HCl (50 mL) and brine (50 mL). The organic layer was separated, dried over Na2SO4 and evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel (EtOAc

Compound 7
Compound 6 (72 mg, 0.07 mmol, 1 eq) and LiOH (17 mg, 0.7 mmol, 10 eq) were dissolved in 3 mL solution of 4:1 THF:H2O. Reaction mixture was stirred at 60 °C and monitored by RP-HPLC. Upon completion, the solvent was concentrated under reduced pressure and the product was purified by preparative RP-HPLC (gradient of ACN in water).

CLNA
Compound 7 (30 mg, 0.04 mmol) and a catalytic amount of methylene blue (~1 mg) were dissolved in 10 mL of DCM. Oxygen was bubbled through the solution while irradiating with yellow light. The reaction was monitored by RP-HPLC. Upon completion, the solvent was concentrated under reduced pressure and the product was purified by preparative RP-HPLC (gradient of ACN in water). CLNA was obtained as a white solid (8 mg, 26% yield After 72 hours, the supernatant was aliquoted and stored at -80°C. The titer of the virus was determined by plaque assay. First, by incubation of a confluent layer of MDCK cells in 6-well plates with 750 μl DMEM containing 10-fold dilutions of virus stocks, supplemented with 0.6 μg/ml T0303, which were incubated at 37° C 5% CO2 for 1 hour, with rocking every 15 minutes, followed by placement of an agarose overlay of DMEM (2 % FBS, PSN, 0.45% Seakem agarose) and incubation at 37° C 5% CO2 for 72 hours. Plaque formation indicated the presence of a single plaque-forming unit, and the amount of plaque-forming units per milliliter (PFU/ml) was subsequently calculated.

Plaque assay with antivirals
Confluent monolayers of MDCK cells in 6-well plates (in triplicates) were incubated with 750 μl DMEM (Gibco™) containing 0.6 μg/ml trypsin T0303 and ~100 PFU of virus for 1 hour at 37° C, 5% CO2, rocking the plate every 15 mins, followed by placement of an agarose overlay of DMEM (2 % FBS; 0.6 μg/ml T0303; 0.45% Seakem™ agarose) containing 10-fold dilutions of antiviral compound/s. After 72 hours, 1 ml of PFA was placed on each agarose overlay and allowed to fix the cells and plaques overnight at 4° C. para-Formaldehyde was then removed with a pipette, and the agarose plug was removed gently by vacuum. Fixed cells were washed with Dulbecco's™ phosphate-buffered saline (PBS) and a solution of 5% crystal violet containing 20% methanol was placed on top of the cells, followed by incubation at room temperature for ~30 mins. Stained cells were then washed with water and allowed to dry. Plaques were counted against a white backlight, and plaque reduction was calculated as % reduction in plaque number relative to the well that was untreated with the antiviral. Doseresponse curves were plotted using GraphPad Prism™.

Antivirals
Amantadine (Sigma™ Chemicals, St Louis MO), Arbidol, and Oseltamivir (Sigma™ Chemicals) were dissolved in DMSO to make 100-and 200-mM stock solutions. All stock solutions were stored at -20° C. All novel inhibitors described here were purchased from ChemBridge (ChemBridge™, San Diego, CA). and, based on their solubility, were dissolved in DMSO to make 100-or 200-mM stock solutions.

Statistical analysis
Statistical analysis was performed using GraphPad Prism software, with sigmoidal non-linear curves used to calculate the IC50 of inhibitors.

Channel Expression into Xenopus Oocytes
Oocytes were obtained by surgical removal of ovary pieces from Xenopus laevis frogs (Xenopus 1) anesthetized with 0.15% tricaine (Sigma-Aldrich). The procedures for surgery and maintenance of frogs were approved by the animal research ethics committee of Tel Aviv University and in accordance with the Guide for the Care and Use of Laboratory Animals (National Academy of Sciences  Figure S1.           The left cell-based assay was obtained following the general cell assay procedure, while the right cell-based assay was obtained following the general cell assay procedure with one exception; the media was replaced with a Phenol red-free MEM media (Gibco™), 2% trypsin supplemented with 2% FCS and 1.2 μg/ml trypsin T0303. *Phenol red is known to affect the light emission measured; therefore, phenol red-free media is necessary for the measurements. 1 Figure S22. IC50 values and sigmoidal fit of Amantadine activity against PR8T (left) and Fort Monmouth (right). Results were obtained after incubation of cells with NA-Star analog [10μM] for 30 minutes and the addition of 20% Emerald-II™ Enhancer. Total light emission was measured for 13 minutes upon the addition of the enhancer at 37C.      Figure S29. Representative current traces of M2 channel (left), exposed to 100 µM compound a (upper), 100 µM Amantadine (middle), or to DMSO (lower). The oocytes were held at a constant voltage of -20 mV. The experimental scheme (pH level and compound application) is indicated by the bars above the traces. Mean percentage inhibition analysis (right) of the small-molecule inhibitors tested, evaluated by a current amplitude ratio at 60 sec and 180 sec. The DMSO mean ratio was subtracted from all groups. (*** p < 0.001, **** p < 0.0001; oneway ANOVA; n=3 (compound a), n=6 (Amantadine), n=5 (DMSO).   Fluorescence ratio analysis (right). The change of mean ratio between 41°C and 30 °C fluorescence was used to indicate small-molecule interaction with the hemagglutinin. ns-not significant, unpaired t-test; n=12 (DMSO)).