Identification of Anti-Influenza A Compounds Inhibiting the Viral Non-Structural Protein 1 (NS1) Using a Type I Interferon-Driven Screening Strategy

Abstract

There is an urgent need to identify efficient antiviral compounds to combat existing and emerging RNA virus infections, particularly those related to seasonal and pandemic influenza outbreaks. While inhibitors of the influenza viral integral membrane proton channel protein (M2), neuraminidase (NA), and cap-dependent endonuclease are available, circulating influenza viruses acquire resistance over time. Thus, the need for the development of additional anti-influenza drugs with novel mechanisms of action exists. In the present study, a cell-based screening assay and a small molecule library were used to screen for activities that antagonized influenza A non-structural protein 1 (NS1), a highly conserved, multifunctional accessory protein that inhibits the type I interferon response against influenza. Two potential anti-influenza agents, compounds 157 and 164, were identified with anti-NS1 activity, resulting in the reduction of A/PR/8/34(H1N1) influenza A virus replication and the restoration of IFN-β expression in human lung epithelial A549 cells. A 3D pharmacophore modeling study of the active compounds provided a glimpse of the structural motifs that may contribute to anti-influenza virus activity. This screening approach is amenable to a broader analysis of small molecule compounds to inhibit other viral targets.

Keywords: NS1; diverse compound library; influenza A viruses; luciferase reporter assay; pharmacophore modeling; small molecule screening; type I IFN.

Figure 1

Cell-based assay to evaluate the anti-IFN-I activity of NS1. (A) Schematic representation of plasmids, small molecules, and experimental points used for the cellular luciferase assay. The pBS IFN-β promoter-Luciferase reporter construct, the pUC57 nt shRNA (IFN-inducer), and the pFlag CMV2 NS1 (H1N1 Pdm 09) are shown. RNA Pol II promoter regions of IFN-β and the CMV virus promoters are shown as tick blue and violet arrows, respectively. RNA Pol III 7SK promoter is shown as a tick light blue arrow. Coding regions for luciferase and NS1 are shown as tick green and red rectangles, respectively. The forward and reverse sequences belonging to the nt shRNA IFN inducer are shown as petroleum and magenta rectangles, respectively. Compound x, able to act as an NS1 inhibitor, is shown as a purple-filled circle. RNA Pol II PolyA sequences and RNA Pol III terminator region are shown as mustard and red rectangles, respectively. The nt shRNA IFN inducer is shown as a 35 bp long red hairpin structure; pBS = pBluescript; 7SK p. = 7SK promoter; H1N1Pdm 09 = A(H1N1)pdm09 influenza virus; compound (Cpd). (B) Schematic representation of the expected outcome of transfection with different plasmid combinations and effective compound Cpd x treatment. The basal and induced luminescence signals of the IFN-β promoter luciferase construct, transfected in IFN-I competent cells alone or together with the nt shRNA (IFN inducer) expressing vector, are shown. The inhibition of luciferase signal after NS1 expressing vector co-transfection and the restoration of luciferase expression by the pretreatment with an x compound are also shown schematically. (A,B) were created with BioRender.com (accessed on 5 June 2023). (C) Evaluation of different IFN-inducer construct amounts to obtain an optimal IFN-β stimulation suitable for the assay. HEK 293 cells were transiently transfected with 25 ng of IFN-β promoter luciferase reporter construct alone (IFN-β p. L.) or together with increasing amounts of shRNA plasmid expressing the IFN-inducer (IFN-i). (D) Evaluation of the optimal NS1 expressing construct to be used in the assay. HEK 293 cells were transiently transfected with increasing amounts of pFLAG CMV2 expression A(H1N1) pdm09 NS1, together or not with the same amounts of IFN-β p. L. and IFN-I constructs used in (A). The green bars in both panels C and D indicate the optimal induction of the luciferase signal by the IFN-inducer (IFN-i) expression, while the red bars in panel D indicate the optimal inhibition of luciferase signal by the co-transfection of NS1 expression vector. IFN-β p. L. = IFN-β promoter Luciferase. FOI = Fold of Induction. (E) Expression of NS1 after transfection by Western blotting using anti-FLAG (NS1) and anti-actin (as a loading control) Antibodies. HEK 293 cells were transiently transfected with 150 ng of pFLAG CMV2 NS1 and WCE subjected to Western blotting with αFLAG monoclonal Ab, using actin as loading control, detected with αActin polyclonal Ab. Results shown in the bar graph are expressed as luciferase FOI with respect to cells transfected with the empty vector. Mean and standard deviation are shown. Statistical analysis was performed using the “two tailed unpaired t test” ((A), left panel). * p < 0.05; ** p < 0.01; *** p < 0.001; n.s. not significant.

Figure 2

(A,B) Screening of a diverse library of compounds in the IFN-β luciferase system (treatment with compounds from 153 to 192 is shown in panel (A), while treatment with compounds from 193 to 236 is shown in panel (B)). (A) HEK 293 cells were transiently transfected with the IFN-β promoter luciferase reporter construct alone, together with the IFN-inducer shRNA plasmid alone, or together with the pFLAG CMV2 A(H1N1)pdm09 NS1 construct. Cpds 153 to 192 of the library were added to cells at a final concentration of 50 μM in fresh medium 6 h post-transfection, and cells were harvested 42 h later (48 h after transfection) and subjected to luciferase assay. (B). HEK 293 cells were transiently transfected as in A, and cpds 193 to 236 of the library, were added to cells as in (A). The reference cpd JJ3297 was used at 5 μM. The empty vector pFLAG CMV2 was used to normalize the amount of transfected DNA in each experimental point. Results shown in the bar graph are expressed as luciferase fold of induction (FOI) with respect to cells transfected with the empty vector. Green and red bars indicate the upregulation and downregulation of luciferase activity, respectively. Blue bars underline >50% restoration obtained with the effective compounds. All experimental points were transfected with the IFN-β promoter reporter construct. The green bars in both panels indicate the induction of the luciferase signal by the IFN-inducer (IFN-i) expression (in panel (B), the far-right green bar also indicates the treatment with positive control JJ3297). DMSO (Dimethyl sulfoxide) presence is indicated in both panels.

Figure 3

Characterization of inhibitory compounds (A,B). (A) Confirmation experiments of compounds identified as effective in the screening. (B) Specificity test of the same compounds analysed in A in the absence of NS1 expressing plasmid; compound = Cpd. DMSO (Dimethyl sulfoxide) presence is indicated in both panels. H1N1Pdm 09 = A(H1N1)pdm09 influenza virus. (A,B) HEK 293 cells were transiently transfected with the IFN-β promoter luciferase reporter construct alone, together with the IFN-inducer nt shRNA plasmid alone, or together with the pFLAG CMV2 A(H1N1)pdm09 NS1 construct. Compounds testing positive by luciferase analysis were added to cells at a final concentration of 50 μM in fresh medium 6 h post-transfection, and cells were harvested 42 h later (48 h after transfection) and subjected to luciferase assay. Results shown in the bar graph are expressed as luciferase fold of induction (FOI) relative to cells transfected with the empty vector. Green and red bars indicate the upregulation and downregulation of luciferase activity, respectively. Blue bars underline restoration obtained with the effective compounds. Mean and standard deviation are shown. Statistical analysis was performed using the “two tailed paired t test”. * p < 0.05, ** p < 0.01, n.s. not significant.

Figure 4

Compounds 157 and 164 inhibit influenza virus replication. (A) Compounds 157 and 164 inhibit influenza virus replication. A549 cells were seeded at a density of 2 × 10⁵/mL in 24 multiwell plates for 24 h. Then, cells were treated with cpds 164 and 157 for 3 h and infected with influenza virus A/PR/8/34(H1N1) at an MOI of 0.1. After 24 h of infection, viral titer was evaluated in the supernatant of infected cells through hemagglutination assay. Results shown in the bar graph are expressed as % viral yield (HAU/mL). Means ± standard deviations from three separate experiments are shown. Statistical analysis was performed using the “one-way analysis of variance” (ANOVA), followed by the Newman–Keuls post hoc test. ** p < 0.01 vs. untreated cells. Cpd = compound. DMSO (Dimethyl sulfoxide) presence is indicated. (B) IC₅₀, CC₅₀, and therapeutic index of tested compounds are indicated in the A549 cellular system. Dose–response curves for both IC₅₀ and CC₅₀ are also shown in the upper and lower graphs of panel (B).

Figure 5

(A) IFN-β, (B) RNase L, (C) OAS, and (D) PKR ISGs expression in the presence of compounds 164 and 157 in influenza A/PR/8/34(H1N1)-infected cells upon normalization for HAU (hemagglutinating unit). A549 cells were seeded at a density of 2 × 10⁵/mL in 24 multiwell plates for 24 h. Then, cells were treated with compounds 164 and 157 for 3h and infected with influenza virus A/PR/8/34(H1N1) at an MOI of 0.1. At 24 h post-infection, total RNA was extracted and subjected to quantitative RT PCR to detect the accumulation of IFN-β (A), RNase L (B), OAS (C), and PKR (D) mRNA. Results are expressed as FOI (fold of induction) upon normalization with GAPDH mRNA and also with HAU (hemagglutination units). Means ± standard deviations from three separate experiments are shown (A,B). DMSO (Dimethyl sulfoxide) presence is indicated in all panels. Statistical analysis was performed using the “two tailed paired t test” ((A), left panel). * p < 0.05.

Figure 6

Similarities in the chemical functionalities of compounds JJ3297, A22, 157, and 164. (A) 2D structure of the active compounds. (B) The pharmacophore model was obtained by aligning all four compounds. The pharmacophore model obtained by aligning the four compounds in the 3D space accounts for two aromatic portions (orange rings), the amide NH as H-bond donor (cyan arrow) and the amide CO as H-bond acceptor (red arrows). The four compounds are represented as green (JJ3297), purple (A22), cyan (157), and yellow (164) sticks.