Discovery of Small Molecule Influenza Virus NS1 Antagonist

Patnaik S, Basu D, Dehdashti S, et al.

Publication Details

The Non Structural 1 (NS1) is a 230–237 amino acid relatively well-conserved viral protein that is usually expressed only during infection [1]. It contains an RNA binding domain (RBD) and an effector domain (ED) that is connected by a variable linker [2]. The NS1 protein has been shown to be a key player that counters the host interferon (IFN) response to viral infection in strain-specific ways [3]. NS1 inhibits the function of OAS (2′–5′-oligoadenylate synthetase) and PKR (protein kinase R) [4] and block IFN-β synthesis at the post-transcriptional level by inhibiting the pre-mRNA splicing and blocking the export of poly(A)-RNAs from the nucleus to the cytoplasm [5]. It also hampers the host’s defense pathways by interfering with the host RNAi pathway, adaptive immune response, and the apoptotic response [6]. NS1 is also a key component in the temporal regulation of viral RNA synthesis, its splicing, and translation [7]. Thus, NS1 enhances the virulence of infection, posing as a compelling target for influenza treatment. These compounds will enable researchers to test how NS1 inhibition impacts the infection cycle and how antagonists can be leveraged alone or in combination with existing agents in the next generation of treatments for influenza infection. We developed and conducted a high-throughput screen using a novel yeast-based phenotypic assay to identify compounds which specifically inhibit NS1 function. Here we report a potent NS1 antagonist chemical series, represented by ML303. The SAR (structure activity relationship) around this chemical series was further developed by measuring the compound’s ability to inhibit replication of the influenza A/PR/8/34 virus in MDCK cells. The ML303 compound series specifically restored the NS1-inhibited expression of the interferon mRNA, inhibited the NS1 function in mechanistic assay and exhibited a potent antiviral activity in culture.

Assigned Assay Grant #: R03-MH085680

Version #: 1

Date Submitted: 4/13/2012

Screening Center Name & PI: NIH Chemical Genomics Center, Christopher P. Austin

Chemistry Center Name & PI: NIH Chemical Genomics Center, Christopher P. Austin

Assay Submitter & Institution: Daniel A. Engel, University of Virginia

PubChem Summary Bioassay Identifier (AID): 2336

Probe Structure & Characteristics

ML303.

ML303

1. Recommendations for Scientific Use of the Probe

ML303 exemplifies a pyrazolopyridine chemical series, where several compounds show potent reduction of viral titer in MDCK cells infected with the Influenza A/PR/8/34 strain. This antiviral small molecule addresses the weak activity limitations of the prior art compounds as it showed improved potency of IC90 of 155nM without overt cytotoxicity to the host cells. ML303 can therefore be used by virologists as a tool to inhibit the function of the influenza NS1 viral protein to study its role in the antagonism of the host interferon system and its impacts on the viral infection cycle. Given the reasonable pharmacokinetics and ADME profile of ML303, this probe can also enabling research clinicians to test how it can be leveraged (alone or in combination) with existing agents in the next generation of treatments for influenza and other viral infections where the NS1 protein is crucial.

2. Materials and Methods

Mammalian cells and viruses. All media and sera were from Invitrogen. MDCK cells were maintained in Iscove medium supplemented with 10% fetal bovine serum (FBS) and 2 mM L-glutamine. For infections, viral stocks were diluted in growth medium supplemented with 0.3% bovine serum albumin (BSA), 0.22% sodium bicarbonate, and 0.25 units of tolylsulfonyl phenylalanyl chloromethyl ketone (TPCK) -trypsin per mL. Influenza viruses A/PR/8/34 (PR) were gifts from Adolfo Garcia-Sastre. The viruses were propagated in 10-day-old embryonated chicken eggs at 37 °C. Titers of influenza virus stocks were determined by hemagglutination and analyzed via 50% tissue culture infective dose (TCID50) [8].

Yeast strains and growth. Strain 9526-6-2 (MATahis3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 pdr1::KanMX4 pdr3::KanMX4) was a gift from Dan Burke. It was derived by tetrad dissection from two parent strains that had been modified by one step gene replacements. The pdr1::KanMX4 was constructed in BY4741 (MATahis3Δ1 leu2Δ0 met15Δ0 ura3Δ0) and the pdr3::KanMX4 was constructed in BY4742 (MATαhis3Δ1 leu2Δ0 met15Δ0 ura3Δ0). PCR-mediated one-step gene replacements, matings and tetrad dissections were performed as described previously [9]. The strain 9526-6-2/pYES-NS1 was generated by transformation of 9526-6-2 with the plasmid pYES-NS1 and maintained on synthetic complete medium lacking uracil (SC). For growth experiments and library screening, a single transformed colony was grown overnight, and the cell number was determined by using a coulter counter (Beckman Coulter Corp). The cells were diluted to 5 × 105 cells/mL in SC and containing raffinose and 2% galactose. A portion of this culture was added to preplated test compounds such that the final dimethyl sulfoxide (DMSO) concentration was 1%. Optical density readings at 600 nm (OD600) were taken using a Thermomax microplate reader (Molecular Devices).

General Methods for Chemistry. The reagents and solvents used were commercial anhydrous grade and used without further purification. The column chromatography was carried out over silica gel (100–200 mesh). 1H NMR spectra were recorded with a Varian 400 MHz spectrometer from solutions in CDCl3 and DMSO-d6. Chemical shifts in 1H NMR spectra are reported in parts per million (ppm, δ) with solvent peaks as internal. Molecular weight confirmation was performed using an Agilent 6224 Time-Of-Flight Mass Spectrometer (TOF, Agilent Technologies, Santa Clara, CA). A 3 minute gradient from 5 to 100% Acetonitrile in water (0.03% formic acid) was used with a 5.1 minute run time at a flow rate of 0.4 mL/min. A Waters Atlantis T3 C18 column (1.8 micron, 2.1 × 50 mm) was used at a temperature of 25 °C. Confirmation of molecular formula was confirmed using electrospray ionization in the positive mode with the Agilent Masshunter software (version B.02) while stability of the probe was assess using the liquid chromatography – mass spectrometry (LC/MS/MS) system.

2.1. Assays

Quantitative High-troughput Screen (qHTS) for Antagonists of Influenza NS1 Protein Function (BioAssay AID 2326). The primary yeast assay was developed where the expression of NS1 induces a slow-growth phenotype that could be observed by measuring optical density of the yeast. One hundred milliliter of yeast culture medium was inoculated with 1 mL of frozen yeast aliquot and incubated overnight at 30 ºC, mixed at 250 RPM. The culture was harvested through spinning at 4000 RPM and the pellet was washed with 15 mL H2O three times. The washed pellet was re-suspended in 10 mL of fresh yeast assay medium. A 1:100 dilution of the re-suspended culture was prepared and 3.5 μL of this yeast suspension was dispensed into 1536 black, clear-bottom assay plates (columns 2–48). Compounds were transferred via the Kalypsys pintool robotics equipped with a 23 nL pinheads. Additional 3.5 μL of yeast assay medium was added and read using the ViewLux plate reader at OD600.

Influenza Virus Replication Assay in MDCK Cells (BioAssay AID 504647 and AID 602454). The replication assay was developed to test the compounds ability to slow growth of the PR virus in the MDCK mammalian cells. Titers of influenza virus stocks were determined by 50% TCID50 analysis on MDCK cells, maintained in Iscove medium supplemented with 10% FBS and 2 mM L-glutamine. Confluent MDCK monolayers were infected with the PR virus at a multiplicity of infection (MOI) of 0.1 for 48 hr in the presence or absence of 5 or 10 μM compounds. Compounds were added at the beginning of infection and were present throughout the infection. After 48 hr virus titers were determined using the by standard TCID50 described in details in Basu et al publication [10].

Mammalian Cell Viability Assay (BioAssay AID 623997). This viability assay was developed to determine general toxicity of the identified inhibitors. Opaque-walled 96-well plates were seeded with 104 MDCK cells per well in 100 μL medium and incubated overnight at 37 ºC. The compound was added in different concentrations (in triplicate) and incubated 37 ºC. After 48 hours, the plates were equilibrated for 30 min at room temperature and 100 μL CellTiter Glo reagent (Promega®) was added according to the manufacturer’s protocol. The plates were shaken for 2 min and then incubated for 10 min at room temperature. Luminescence was recorded in an LMAX 384 reader (Molecular Devices) with 1 sec integration time.

TCID50 Assay (BioAssay AID 602450). This assay was used to determine the virus titer in the MDCK cell line. Cell monolayers were infected at an MOI of 0.1 for 48 hr in the presence or absence of the compound added at the beginning of infection. Virus titers were determined by standard TCID50 analysis.

RT-PCR based secondary assay to measure induction of interferon-beta mRNA (BioAssay AID 602456). This secondary assay was developed to confirm the antiviral activity of the identified probe by assessing the interferon beta (IFN-β) expression of the MDCK cells in response to virus infection. The cells were infected for 6 hr with PR virus at MOI of 0.1 in the presence or absence of drug. Total RNA was isolated using RNeasy (Qiagen). For first-strand cDNA synthesis, 2 μg of total RNA was primed with random nanomers (New England Biolabs) at a final concentration of 2 μM. Reverse transcription was performed with 10 U/μL of Moloney murine leukemia virus reverse transcriptase (New England Biolabs) in the presence of 1 U/μL of RNase inhibitor (New England Biolabs). Thereafter, 1/20 volume of cDNA was used as a template for PCR (30 cycles). IFN-β mRNA (accession number NM_001135787) expression levels were quantitated using the 5′CCAGTTCCAGAAGGAGGACA3′ and 5′CCTGTTGTCCCAGGTGAAGT3′ primer pair; and normalized to β-actin (XM_536230) expression levels quantitated using the primer pair 5′GGCATCCTGACCCT GAAGTA3′ and 5′GGGGTGTTGAAAGTCTCGAA3′.

2.2. Probe Chemical Characterization

Probe ML303 (CID 53316412).

Probe ML303 (CID 53316412)

*

Purity 100% as determined by LC/MS and 1H NMR analyses

2-Methoxy-4-(3-methyl-1-(4-(trifluoromethyl)phenyl)-1H-pyrazolo[3,4-b]pyridin-4-yl)phenol: LC/MS (Agilent system) Retention time t1 (long) = 6.97 min; 1H NMR (400 MHz, DMSO-d6) δ ppm 2.35 (s, 3 H), 3.85 (s, 3 H), 6.96 (m, 1 H), 7.02 (m, 1 H), 7.17 (d, J=2.3 Hz, 1 H), 7.28 (d, J=4.7 Hz, 1 H), 7.94 (d, J=9.0 Hz, 2 H), 8.63 (d, J=9.0 Hz, 2 H), 8.66 (d, J=4.7 Hz, 1 H), 9.44 (s, 1 H); HRMS (ESI) m/z calculated for C21H17F3N3O2 [M + H]+ 400.1267 found 400.1270.

Figure 2. Structures of the ML303 and five analogs (with corresponding CID) that have been submitted to the MLSMR compound repository.

Figure 2

Structures of the ML303 and five analogs (with corresponding CID) that have been submitted to the MLSMR compound repository.

Table 1. List of the NS1 inhibitor probe ML303 and related analogs and their corresponding identification numbers.

Table 1

List of the NS1 inhibitor probe ML303 and related analogs and their corresponding identification numbers. Their corresponding molecular structures are shown above in Figure 2.

2.3. Probe Preparation

Preparation of 2-methoxy-4-(3-methyl-1-(4-(trifluoromethyl)phenyl)-1H-pyrazolo[3,4-b]pyridin-4-yl)phenol (ML303) is a multi-step process with 6 compound intermediates illustrated above (Scheme 1) and summarized below.

Scheme 1. Synthetic route to ML303 is a multi-step process with 6 compound intermediates A – F.

Scheme 1

Synthetic route to ML303 is a multi-step process with 6 compound intermediates A – F.

  1. A mixture of (E)-3-aminobut-2-enenitrile and (4-(trifluoromethyl)phenyl)hydrazine was stirred in ethanol (EtOH) and heated to reflux for 16 hr. The mixture was cooled and poured into water and extracted with ethyl acetate (EtOAc). The organic phase was washed with brine, dried over MgSO4, filtered and then concentrated to yield 6.7 g (99% yield) 3-methyl-1-(4-(trifluoromethyl)phenyl)-1H-pyrazol-5-amine (intermediate A), a dark red oil which was used in the next step without further purification.
  2. Intermediate A and diethyl(ethoxymethylene)malonate were taken in 1,4-dioxane and heated to 120 °C for 16 hr. Analysis by LCMS indicated completion of the reaction. The mixture was concentrated to give the intermediate B, dark oil which crystallized on standing.
  3. The crude material (B) was dissolved in phosphoryl chloride (POCl3) and heated at 120 °C for 16 hr. The reaction mixture was cooled, poured over ice, and extracted with EtOAc. The organic layer was washed carefully with sat. aq. sodium bicarbonate (NaHCO3), then brined, dried over magnesium sulfate (MgSO4), filtered, concentrated, and purified via flash silicon dioxide (SiO2) chromatography to provide 2.7 g (25% yield over 2 steps) of ethyl 4-chloro-3-methyl-1-(4-(trifluoromethyl)phenyl)-1H-pyrazolo[3,4-b]pyridine-5-carboxylate (intermediate C).
  4. Intermediate C was dissolved in 1,4-dioxane, treated with excess 2 N lithium hydroxide (LiOH) and heated at 100 °C for 36 hr until full conversion to intermediate D was observed by HPLC. The reaction was cooled and extracted with EtOAc. The pH of the aqueous phase was adjusted to ~5 and was extracted with EtOAc.
  5. The organic layers were dried over MgSO4, filtered, and concentrated to provide D which was dissolved in ethylene glycol, treated with solid sodium hydroxide (NaOH), and heated at 180 °C for 4 hr until complete conversion to intermediate E was observed by LCMS. The reaction was cooled to room temperature, diluted with water, and the pH was adjusted to 6.5. The mixture was extracted with EtOAc.
  6. The organic layers were combined, dried over MgSO4, filtered, concentrated, and purified via flash SiO2 chromatography to provide 1.84 g (90% yield over 2 steps) of E. Phenol E was dissolved in POCl3 and heated at 120 °C for 16 hr. The reaction mixture was cooled, poured over ice, and extracted with EtOAc.
  7. The organic layer was washed carefully with sat. aq. NaHCO3, then brine, dried over MgSO4, filtered, concentrated, and purified via flash SiO2 chromatography to provide an 86% yield of 4-chloro-3-methyl-1-(4-(trifluoromethyl)phenyl)-1H-pyrazolo[3,4-b]pyridine (intermediate F).
  8. Intermediate F, 4-hydroxy-3-methoxyphenylboronic acid, potassium phosphate (K3PO4), and bis(triphenylphosphine)palladium(II) dichloride (PdCl2(PPh3)2) were taken in dimethylformamide (DMF) and water. The mixture was heated at 100 °C for 10 hr, cooled, concentrated, diluted with dichloromethane (DCM), and filtered through a PL-Thiol MP Resin cartridge (Agilent). The filtrate was concentrated and purified by flash SiO2 chromatography to obtain 2-methoxy-4-(3-methyl-1-(4-(trifluoromethyl)phenyl)-1H-pyrazolo[3,4-b]pyridin-4-yl)phenol (probe ML303).

3. Results

3.1. Dose Response Curves for Probe

Figure 3. Dose response activity of ML303 on viral titer.

Figure 3Dose response activity of ML303 on viral titer

Triplicate cultures were infected with PR virus at MOI of 0.1% and treated with different concentrations of ML303. After 48 hour, the supernatants were collected and analyzed to determine the reduction in virus by assaying the supernatant infectivity. The data is presented as a percent decrease in titer (A) which is calculated from TCID50 where the probe IC90 is 155 nM and as the actual TCID50(B) where 3 log units of viral suppression are observed at 1.6 μM concentration (AID 602450).

3.2. Cellular Activity

Figure 4. Compound-dependent restoration of yeast growth.

Figure 4Compound-dependent restoration of yeast growth

Growth of the control yeast strain pYES (without NS1) and the NS1 expressing stain NS1-PYES were measured as OD600 nm at 22, 42 and 60 hours. Results showed the slow growth phenotype exhibited by the NS1-pYes strain is reversed by the addition of 50 μM ML303 and after 60 hour the growth phenotype was comparable to that of the control treatments.

Figure 5. Compound-dependent restoration of IFN-β mRNA levels in MDCK cells.

Figure 5Compound-dependent restoration of IFN-β mRNA levels in MDCK cells

Cells were mock infected (lane 2) or infected with influenza strain A/PR/8 at an MOI of 0.1 (lanes 4–9). Infected cells were treated with negative control 1% DMSO or different compounds (20 μM) including the probe ML303 (lane 7). After 6 hour, cells were harvested for RT-PCR analysis of IFN-β and β-actin mRNA levels. Results showed treatment with these compound restored IFN-β levels comparable to the positive control treatment poly(I:C) which directly induces IFN-β (lane 3) (AID 602456).

3.3. Profiling Assays

Preliminary ADME profiling revealed that ML303 and relative analogs have good stability in mouse live microsomes (MLL) (45% remaining after 30 minutes) and good permeability with low efflux in Caco-2 monolayer of cells (Table 2). ML303 and members of this series showed low solubility (< 50 μM), but this didn’t translate to poor in vivo pharmacokinetics as ML303 exhibited high exposure in plasma and lung after a single 30 mg/kg intra-peritoneal (IP) dose in mice (Table 3) and after 48 hours of probe treatment (Figure 6). It is well known that low solubility impacts permeability in Caco-2 assays, but the use of an appropriate formulation often results in reasonable in vivo bioavailability. Additionally the compound showed good stability in mouse plasma and PBS buffer (Figure 1).

Table 2. In vitro ADME data for ML303 and relative active analogs.

Table 2

In vitro ADME data for ML303 and relative active analogs.

Table 3. Pharmacokinetics (PK) data showing the mean plasma, live, and lung concentration of ML303 in IP treated male C57BL/6 mice (n=3).

Table 3

Pharmacokinetics (PK) data showing the mean plasma, live, and lung concentration of ML303 in IP treated male C57BL/6 mice (n=3).

Figure 6. Time course PK plot of the ML303 concentration in plasma, liver and lung of IP treated mice.

Figure 6

Time course PK plot of the ML303 concentration in plasma, liver and lung of IP treated mice. Each point represents the mean +/− SD of n=3.

Figure 1. Solubility and stability data of probe ML303 in different media as evaluated by LC/MS/MS.

Figure 1

Solubility and stability data of probe ML303 in different media as evaluated by LC/MS/MS. (A) Dot plot of the stability of the probe in D-PBS pH 7.4 at room temperature in a 24 hour period. (B) Dot plot of the stability of the probe in mouse plasma at (more...)

4. Discussion

4.1. Comparison to Existing Art and How the New Probe is an Improvement

Influenza continues to pose a global public health problem. The development of novel antivirals is imperative due to the ability of the virus to develop drug resistance. A patent disclosing eight chemical series of NS1 influenza protein inhibitors was revealed in 2009 but their chemical structures were not disclosed to public [11]. The Engle lab also identified the small molecule JJ3297 that indirectly perturb NS1with potency of 5μM. However, the precise mechanism of action remains to be elucidated. Moreover, treatment with JJ329 is limited to infections at low multiplicity since it showed no effect on virus spread or medium titer during infections at high multiplicity [12]. ML303 is a new NS1 inhibitor that exhibits improved antiviral activity. Compared to the prior art, ML303 has better potency of 155nM (IC90) against the NS1 without an overt cytotoxicity to the host cells. Furthermore, ML303 showed good stability and in vivo bioavailability. Hence the ML303 probe poses as a tool compound for evaluation of NS1 antagonism in advance in vitro assay and animal studies as a therapeutic strategy against influenza.

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