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An Influenza Virus Entry Inhibitor Targets Class II PI3 Kinase and Synergizes with Oseltamivir Ryan O'Hanlon, Victor Leyva-Grado, Marion Sourisseau, Matthew Evans, and Megan Shaw ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.9b00230 • Publication Date (Web): 26 Aug 2019 Downloaded from pubs.acs.org on August 27, 2019

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ACS Infectious Diseases

An Influenza Virus Entry Inhibitor Targets Class II PI3 Kinase and Synergizes with Oseltamivir Ryan O’Hanlon1,2, Victor H. Leyva-Grado1, Marion Sourisseau1, Matthew J. Evans1, Megan L. Shaw1#* 1Department

of Microbiology, 2Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA #Present

address: Department of Medical Biosciences, University of the Western Cape, Robert Sobukwe Rd, Bellville 7535, South Africa *Corresponding

author: [email protected]

Two classes of antivirals targeting the viral neuraminidase (NA) and endonuclease are currently the only clinically useful drugs for the treatment of influenza. However, resistance to both antivirals has been observed in clinical isolates, and there was widespread resistance to oseltamivir (an NA inhibitor) amongst H1N1 viruses prior to 2009. This potential for resistance and lack of diversity for antiviral targets highlights the need for new influenza antivirals with a higher barrier to resistance. In this study we identified an antiviral compound, M85, that targets host kinases, EGFR and PIK3C2β, and is not susceptible to resistance by viral mutations. M85 blocks endocytosis of influenza viruses, and inhibits a broad-spectrum of viruses with minimal cytotoxicity. In vitro, we found that combinations of M85 and oseltamivir have strong synergism. In the mouse model for influenza, treatment with the combination therapy was more protective against a lethal viral challenge than oseltamivir alone, indicating that development of M85 could lead to combination therapies for influenza. Finally, through this discovery of M85 and its antiviral mechanism, we present the first description of PIK3C2β as a necessary host factor for influenza virus entry. Keywords Antivirals, Influenza A Virus, Oseltamivir, Virus Entry, Antiviral Drug Resistance, Kinases 1 ACS Paragon Plus Environment

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Introduction Despite the availability of vaccines and antiviral drugs, influenza virus has a significant negative impact on worldwide health and is a heavy economic burden every year1, 2. Influenza vaccines are limited in their ability to provide adequate protection because of frequent mismatches to circulating strains, the 5-month production time, and low coverage worldwide3, 4. Antiviral drugs are an effective alternative for the treatment of influenza, but the emergence of resistant strains threatens to make them irrelevant5. Currently, FDA-approved antivirals for influenza include inhibitors of the viral M2 ion channel (rimantadine and amantadine), viral neuraminidase, or NA, (oseltamivir, zanamivir, peramivir), and viral endonuclease, or PA, (baloxavir marboxil)6. However, only the NA and PA inhibitors are used clinically due to widespread resistance to the M2 inhibitors amongst circulating influenza A virus strains7, 8. Resistance to oseltamivir can be conferred by single residue mutations in NA and has already been observed in seasonal strains of influenza virus9,

10.

Furthermore, during the 2008-2009 influenza season, 95% of isolated

influenza A (H1N1) strains worldwide were resistant to oseltamivir11.

Although widespread

resistance to zanamivir or peramivir has not been observed, mutations in NA conferring resistance to these drugs have been found in clinical isolates of influenza A and B viruses12. This potential for resistance and the lack of diversity in antiviral targets prompted the search for influenza antivirals with novel mechanisms of action, which led to the development of baloxavir marboxil, which was approved first in Japan and then in the USA in 20186 . It targets the highly conserved PA subunit of influenza virus RNA-dependent RNA polymerase, but already resistance mutations in PA (I38T and I38M) have been reported from patients treated with baloxavir marboxil13. It has yet to be seen if these mutations will have a clinical impact, but this highlights the need for new influenza antivirals with a higher barrier to resistance. Antivirals targeting viral factors inherently carry the risk for selection of resistant viruses due to the error prone nature of RNA virus polymerases. As an alternative, host factors essential for virus replication can be considered as novel antiviral targets with the advantage that they are 2 ACS Paragon Plus Environment

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less susceptible to the emergence of resistance and have the potential for broad-spectrum activity. This has been demonstrated by two host-directed influenza antivirals currently in clinical trials. Nitazoxanide, originally approved as an antiprotozoal, targets host factors involved with the maturation and trafficking of de novo influenza virus hemagglutinin, or HA14. In vitro, serial passaging of influenza A virus or hepatitis C virus in nitazoxanide treated cells fails to select for resistance15, 16, and influenza virus collected from treated subjects in a phase III clinical trial did not have reduced susceptibility to nitazoxanide17. In addition, nitazoxanide inhibits a broad range of respiratory viruses, non-respiratory viruses, and bacteria18. The other host-directed antiviral, DAS181 (Fludase) is a recombinant sialidase fusion protein designed to remove the receptor of influenza virus, sialic acid, from respiratory epithelium19.

It is broadly effective

against influenza A and B viruses, as well as other respiratory viruses, including parainfluenza viruses and metapneumoviruses20,

21.

Serial passaging of influenza virus in DAS181 treated

cells did select for resistance mutations in HA, but these mutations were unstable and resulted in attenuated viral growth22. It is also possible to raise the barrier to resistance by combining antiviral drugs with different mechanisms of action, as has been done successfully with HIV and HCV therapies. Studies with amantadine + oseltamivir, ribavirin + oseltamivir, and favipiravir + oseltamivir demonstrate that these combinations are synergistic, allowing for higher potency at lower doses23-25. In addition, oseltamivir and amantadine combinations have been shown to reduce the emergence of resistant influenza A virus in vitro26.

The triple combination of amantadine, ribavirin, and

oseltamivir also works synergistically against influenza A viruses resistant to amantadine or oseltamivir, supporting the idea that combination therapies can treat infections caused by resistant viruses27. In this study we report the identification of a novel antiviral compound, M85, as an entry inhibitor of influenza A virus. We show that M85 targets EGFR and phosphoinositide 3 class II β (or PIK3C2β) kinases, which control early stages of endocytosis, thereby forming the basis for the 3 ACS Paragon Plus Environment


inhibitory action of M85 on virus entry28,

29.

Antiviral activity is also observed for influenza B

virus, hepatitis C virus, and human rhinovirus, designating M85 as a broad-spectrum antiviral. The inability to select for M85 resistance, underscores the potential for M85 to be a paninfluenza virus inhibitor with a high barrier to resistance. M85 displays strong synergism with oseltamivir in vitro and we show that a combination of M85 and oseltamivir is able to protect mice from a lethal influenza infection.

Results Antiviral Screening Identifies M85 as a Pan-Influenza Inhibitor In the search for compounds with pan-influenza antiviral activity, a previously described high throughput screen of over 900,000 compounds was performed using a cell-based assay that monitors influenza virus replication30. From the 744 primary screen hits, 151 were selected as good candidates for drug development, based on antiviral potency (IC50 < 2µM) and structural characteristics showing no obvious liabilities. This group of compounds was re-purchased and re-tested for antiviral activity against influenza A/WSN/1933 (H1N1) virus (WSN) and an influenza B/Yamagata/16/88 reporter virus31 to identify those with pan-influenza antiviral activity. The secondary screening assay involved immunofluorescence detection of NP as a marker for virus replication, which has previously been used to determine IC50 values32. M85 was among the secondary screen hits that showed potent activity against both influenza A (IC50: 2.05 µM) and B (IC50: 0.4 µM) viruses (Table 1, Figures S1A and B) and a selective index (SI) greater than 10. Treatment with 10µM M85 also corresponded to a 2-log reduction in viral titers of WSN (data not shown). To verify the breadth of anti-influenza virus activity, M85 was also tested against representative influenza A viruses of the H1N1, H3N2 and H5N1 subtypes, and all were found to be susceptible to M85 (Table 1). Moreover, M85 was active in human, canine, and murine cell lines. Based on this information, it was hypothesized that M85 could either be

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Table 1: M85 has broad-spectrum antiviral activity

targeting a conserved influenza virus protein or a host factor potentially utilized by a broader spectrum of viruses. We tested the breadth of antiviral activity for M85 using a diverse set of non-influenza RNA viruses (Table 1). Interestingly, M85 showed potent activity against human rhinovirus 14 (HRV-14) (IC50: 0.05 µM) and hepatitis C virus (HCV) (IC50: 0.76 µM) (Figure S1C and S1D), suggesting that it targets a host factor and not a viral protein. However, vesicular stomatitis virus, Zika virus, mumps virus, Newcastle disease virus, encephalomyocarditis virus, respiratory syncytial virus, and human parainfluenza virus were not susceptible to M85. This provides confidence that the observed inhibition of influenza viruses, HCV and HRV is not due to non-specific cytotoxicity. M85 has a High Barrier to Resistance We next assessed the ability to select for M85-resistant virus to investigate the barrier to resistance and supply further information for a viral or host target. Influenza virus (WSN) was serially passaged in A549 cells treated with M85 (5 µM), DMSO, or oseltamivir carboxylate (1 5 ACS Paragon Plus Environment


µM) for 9 passages. The concentrations of the compounds had similar potencies, each inhibiting viral replication by 1-1.5 logs (plaque assay readout; data not shown). After the 9th passage the M85 and oseltamivir-passaged viruses were assessed for their susceptibility to M85 or oseltamivir, respectively, in comparison to DMSO-passaged virus. In response to oseltamivir treatment, virus passaged in oseltamivir showed a 33-fold higher IC50 compared to virus passaged in DMSO, indicating that it had acquired resistance within 9 passages (Table S1). However, the IC50 values for M85 were similar between the viruses passaged in M85 and DMSO, suggesting that the virus had not acquired resistance to M85. This indicates that M85 has a high barrier to resistance, or at least higher than the approved influenza drug, and further suggests that M85 targets a host factor. M85 Targets Viral Entry To determine which stage of the viral lifecycle M85 inhibits, we performed a time of addition assay (Figure 1A). M85 could only inhibit viral replication if added before or at the time of infection, suggesting that it acts very early in the replication cycle, either on viral entry or early viral RNA synthesis. To test if M85 had an effect on entry, we determined if it could block entry of influenza virus HA/NA pseudotyped lentiviral particles that express luciferase upon transduction (Figure 1B)33. As a control we assessed VSV-G pseudotyped particles (Figure 1C). M85 significantly blocked entry of WSN HA/NA and VSV-G pseudotyped particles, although the effects on VSV-G particles were moderate. In contrast, an influenza virus specific entry inhibitor targeting HA, S2030, inhibited entry of HA/NA particles but not VSV-G particles. These data suggest that M85 is an entry inhibitor, but that the activity is not specific for influenza virus. To address the possibility that M85 is an RNA synthesis inhibitor, it was tested in an influenza viral RNA synthesis assay34 alongside the positive control compound, VX-78735, which targets the viral PB2 protein. M85 did not inhibit viral RNA synthesis (Figure 1D), suggesting that M85 acts only on viral entry. 6 ACS Paragon Plus Environment

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Figure 1. M85 inhibits viral entry. A) A549 cells were treated with 10µM M85 at the indicated times pre- and post-infection with influenza A/WSN/33 virus (MOI 1). Titers were measured at 24h post infection. B) A549 cells were infected with influenza virus WSN HA/NA or C) VSV-G pseudotyped lentivirus particles in the presence of M85 or a known influenza A virus specific entry inhibitor, S2030. Luciferase activity measured 24h post infection. D) A549 cells were transfected with plasmids for the influenza virus minigenome system in the presence of M85 or a PB2 cap-snatch inhibitor, VX-787. -PA is a control that lacks the PA-encoding plasmid. Firefly luciferase values normalized to a constitutively expressed Renilla control were determined 24h post transfection and are expressed relative to the DMSO control. For all experiments, the concentration of DMSO is 0.5%. For these assays, to ensure that a sufficient amount of M85 was used, 10µM was selected because it is over the IC90 for influenza A viruses. Data are represented as the mean ± SD (bar graph) of at least 3 independent biological replicates (hollow circles). Statistical significance was determined with a one way ANOVA. **** p < 0.0001.

M85 Acts on Endocytosis Knowing that influenza viruses utilize receptor-mediated endocytosis for entry, we hypothesized that the target of M85 is involved in this process36. First, we investigated if M85 showed inhibitory activity in a system where influenza virus can bypass endocytosis by fusing at the 7 ACS Paragon Plus Environment


plasma membrane (Figure 2A)37. Fusion of influenza virus envelopes with host membranes is a pH-dependent process, typically occurring in the low pH environment of late endosomes. By binding influenza virus to A549 cells and then adjusting the pH to 5.0, this allows fusion to occur artificially at the plasma membrane, thereby bypassing the need for endocytosis. Addition of ammonium chloride (NH4Cl) following the pH shift blocks any virus still entering via endocytosis to ensure that any infection signal observed is due only to virus fusing at the cell surface. Under conditions where virus entered normally via endocytosis, M85 significantly reduced the number of infected cells compared to DMSO treatment (Figure 2B), as did PY10238, a neutralizing antibody that blocks influenza virus attachment. At pH 5.0, approximately 50% of cells were infected via fusion at the plasma membrane under DMSO treatment, and M85 did not cause a significant reduction (Figure 2B), whereas PY102 treatment resulted in significantly reduced infection. This demonstrates that M85 is not affecting attachment and is also not affecting any step downstream of fusion, such as uncoating and nuclear import of vRNA. This confirms that M85 acts on endocytosis and therefore is likely only effective against viruses that enter in this manner. Receptor-mediated endocytosis of influenza virus is a multi-step process involving many host factors. To successfully enter the cell, the virus must associate with growth factor receptors to induce endocytosis, find its way into an endosome, traffic to the late endosome, and fuse with the endosomal membrane. Inhibition of any of these steps would be sufficient to prevent viral entry. Endosome acidification inhibitors, like bafilomycin A1, are known to inhibit viruses entering via pH-dependent fusion by preventing acidification of endosomes39. To determine if M85 inhibits endosome acidification, a fluorescent dye (LysoLive pH-Sensor Green) that concentrates in acidified compartments was used to monitor acidification in A549 cells pretreated with DMSO, M85, or bafilomycin (Figure 2C)40. In bafilomycin treated cells, the dye does not concentrate 8 ACS Paragon Plus Environment

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A

B

C D

E

Figure 2. M85 inhibits endocytosis, not endosome acidification. A) Schematic for the endocytosis bypass assay using influenza A/PR/8 virus (PR8) (MOI 10). A549 cells and virus were pretreated with DMSO, M85, or PY102, and then allowed to bind at 4°C. One condition was shifted to 37°C for 16h to allow infection to occur normally (Infection Only). In other conditions, the pH was maintained at 7.4 or shifted to 5.0 for 2 minutes at 37°C. NH4Cl was added to prevent virus from fusing at the late endosome. NP positive infected cells were quantified using a Celigo S imaging cytometer. B) Data for each of the conditions in the endocytosis bypass assay is represented as the mean ± SD (bar graph) percentage of infected/total cells in the same well across 6 independent biological replicates (hollow circles). Treatments and their concentrations are indicated. PY102 is an HA neutralizing antibody that prevents virus attachment to cells. C) Fluorescent microscopy images (60X) of live A549 cells unstained (left panels) or stained with LysoLive pH-Sensor Green (LysoLive 488) and treated with the indicated compound or DMSO. A high concentration of M85 (20µM) ensured that a sufficient amount was used to treat the cells while avoiding potential cytotoxicity. Cells were treated with compounds for 1hr, followed by addition of LysoLive, and imaged immediately afterward. D) Quantification of the (total number of clusters of acidified endosomes) / (total cell count) normalized to the DMSO control. Data are represented as the mean ± SD (bar graph) for 6 independent biological replicate wells (hollow circles). E) The average intensity of LysoLive staining per cell of at least 4000 cells per well with 6 independent biological replicate wells (hollow circles). The bar graph represents the average of the means and SDs across replicates. For all experiments, statistical significance was determined with a one way ANOVA. **** p < 0.0001, n.s.-not significant. Bar, 50µm.



into any compartment and appears as a fluorescent haze throughout the cells. However, in M85 and DMSO treated cells, the dye forms distinct puncta, demonstrating the presence of acidified compartments. Clusters of puncta representing acidified compartments were quantified using an imaging cytometer to corroborate these findings (Figure 2D). Also, the amount of dye on average that each cell retained was found to be comparable, regardless of treatment, demonstrating that acidification inhibitors do not prevent dye from entering the cell, but do change where the dye is localized (Figure 2E). These results clearly indicate that M85 is not an endosome acidification inhibitor. Another likely scenario is that M85 can affect endocytosis of viral particles by inhibiting endosome trafficking. To address this, we infected A549 cells with influenza virus, treated with M85 (50µM, to compensate for the high MOI infection) or DMSO, and used confocal microscopy to determine where the virus gets trapped. One hour post-infection, for both DMSO and M85 treated cells, the virus can be seen at the cell surface as judged from the co-localization of viral nucleoprotein (NP) with the surface marker wheat-germ agglutinin (WGA) (Figure 3A). Quantitatively, 63% and 62% of the detected NP co-localized with WGA for DMSO and M85 treated cells, respectively. At 4h for the DMSO treated cells, NP can be seen accumulating in the nucleus, indicating that vRNA has successfully entered the cell and expression of viral proteins is proceeding normally (Figure 3B). In contrast, with M85 treatment at 4h there is no accumulation of nuclear NP and 42% still co-localizes with WGA on the surface. Visually, the virus appears to be trapped at the cell surface or in compartments close to the surface, suggesting that M85 blocks viral entry at an early step in endocytosis or during endosome trafficking.


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Figure 3. M85 traps virus near the cell edge. A549 cells stained for viral NP, the cell surface via wheat-germ agglutinin (WGA), and nuclei (DAPI) A) 1h and B) 4h after infection with influenza A/PR/8 virus at MOI 200 in the presence of 50µM M85 or DMSO. White arrows indicate examples of NP signal that colocalizes with WGA in M85 treated cells 4h post-infection. Images collected by confocal microscopy (63X). Bar, 50µm. 11 ACS Paragon Plus Environment


In vitro Binding and Activity Assays Determine that M85 Targets EGFR and PIK3C2β Kinases To begin to look for targets for M85, we searched for compounds with a similar structure that had known targets. Using a hash key algorithm (in Schrödinger Canvas 2.7), the structure of M85 and every compound in DrugBank (www.drugbank.ca) was converted into a binary sequence. DrugBank is a public database of small molecules and peptides with known targets, confirmed by co-crystal structures in published articles41. The Tanimoto similarity (T= # features in common / # features not in common) was then calculated based on these sequences. Three compounds had T > 0.2 for M85 (Figure S2), two of which target kinases, VX242 and LKG (PDB:3CCN). Although the scores are low, structurally, the heterocyclic group attached to a benzyl group resembles a similar feature on M85. This warranted further investigation into the possibility that M85 was a kinase inhibitor. To address the possibility that M85 binds to kinases and identify potential targets, we used a commercial kinase binding panel service, KINOMEscan43(Eurofins DiscoverX). This assayed for competitive binding to 97 representative members from all kinase families. From a KINOMEscan using a high concentration of M85 (10µM), 7 hits (defined as % Max Signal < 35%) and 2 borderline negative hits were identified (Table S2). Among these nine targets, 5 were related to PI3K signaling (PIK3C2β, RIOK2, FLT3, TRKA, and PIK3Cγ) and 3 were ERBB family kinases (EGFR, EGFR (L858R), and ERBB2), suggesting that M85 selectively affects these pathways. Other kinases that have been implicated in the entry of influenza viruses (IGFR, VEGFR, PDGFR, c-Met, FAK, MEK1, ERK1/2)44-46 were negative for binding to M85, suggesting that it is specifically the PI3K and ERBB pathways that contribute to the antiviral mechanism for M85. To examine the selectivity of M85 among ERBB and PI kinase family members, the Kd of M85 with these kinases was determined with a dose response KINOMEscan assay (Table 2). For 12 ACS Paragon Plus Environment

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Table 2. M85 Binding Affinities and Activity IC for Kinases in the ErbB and PI3 Kinase Families

50

ERBB family members, M85 showed a Kd of 3500nM for EGFR kinase, which is much weaker than a known inhibitor of EGFR such as gefitinib (1nM). ERBB2 had even weaker binding (Kd=9300nM), and ERBB4 had no binding. Surprisingly, M85 displayed the best binding to ERBB3 (Kd=1300nM), but it is known that the kinase domain of this protein is inactive in cells47. Amongst active ERBB kinases, M85 was therefore the most selective for binding EGFR. For PI3 kinases, M85 showed a Kd of 610nm for PIK3C2β (versus 89nM for a wortmannin control) and did not bind to PIK3Cα (class I PI3K), PIK4Cβ (a PI4 kinase), or PIP5K1α (a PIP5 kinase). In parallel to the binding assays, the ability of M85 to inhibit the activity of these kinases was tested in phosphotransferase assays (Table 2). Despite having weaker binding to EGFR, M85 displayed a low IC50 (79nM) that was comparable to that of the positive control, staurosporine48 (68nM). Amongst PI kinases, M85 was the most selective for PIK3C2β (IC50= 430nM) compared with other PI3 kinases from class I, II, and III (all with IC50 > 2µM), and this activity against PIK3C2β was stronger than that of the positive control, LY29400249 (IC50= 910nM), a broad PI3K inhibitor. Based on these results, we concluded that M85 was selective for EGFR and PIK3C2β kinases.



M85 Antiviral Activity is Partially Conferred by Inhibition of PIK3C2β By manipulating the activity of kinases, viruses can take advantage of host processes to promote their own replication. After influenza virus attaches to the cells, it mediates its internalization via signaling through receptor tyrosine kinases (RTK) such as EGFR, IGFR, PDGFR, VEGFR, and c-Met44. Via the formation of lipid raft complexes with EGFR, influenza virus can activate PI3 kinases to induce endocytosis of the viral particle50. However, the specific PI3K kinases required for viral entry and their roles in the process have not been well described. Based on their known cellular functions, PIK3C2β and other class II kinases are directly involved in early endosome trafficking and maturation of clathrin-coated pits51. This is regulated by their production of phosphoinositide 3 phosphate, or PI(3)P, which in turn binds to factors on endosomes (Rab5 and EEA1) that are required for sorting into early endosomes52. Also, PIK3C2α, produces PI(3,4)P on clathrin-coated pits to recruit sorting nexin 9 and other factors required for membrane constriction53. Since EGFR and PIK3C2β kinases are involved in endocytosis, it is possible that M85 is affecting endosome trafficking of viruses by inhibiting these factors. However, a role for PIK3C2β in viral entry specifically has not been described, so we investigated if its inhibition contributes to the antiviral activity of M85 and if it is a required host factor for influenza virus. First, we investigated if a PIK3C2β specific inhibitor had antiviral activity, but unfortunately there are no commercially available PIK3C2β specific inhibitors, and only 2 such compounds described in the literature54. However, in a preliminary SAR study of M85 analogs (parent scaffold in Figure 4A) we were fortunate to discover that analog M85-12 (Figure 4B) selectively inhibits PIK3C2β and not EGFR (Figure 4C). We assessed whether M85-12 had retained its selectivity against PIK3C2β by testing it against a panel of PI3 kinases in a phosphotransferase assay (Figure 4C). M85-12 shows a 2-fold lower IC50 against PIK3C2β, 5-fold improved potency against PIK3C2γ, and a 6-fold loss in potency for PIK3C2α in comparison to M85. But 14 ACS Paragon Plus Environment

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Figure 4. Inhibition of PIK3C2β contributes to the antiviral activity of M85. Structures of A) parent M85 and B) analog M85-12. C) Using the same kinase activity assay from Table 2, M85 and M85-12 were compared for their ability to inhibit EGFR and class I-III PI3 kinases. DH) Antiviral activity (in black, left y-axis) of D) M85-12, E) M85 combined with 10µM gefitinib, F) gefitinib, G) gefitinib combined with 10µM M85, and H) M85 against influenza A/WSN/33 (H1N1) virus (MOI 0.001) in A549 cells. The number of NP positive cells were quantified 48 hours post infection. Cytotoxicity was determined with the live/dead nuclei staining assay (blue, right yaxis). Oseltamivir carboxylate (10 µM) was added as a positive control (red). I) Titers of WSN virus in the supernatants of infected WT or PIK3C2βD1212A/D1212A MEF cells at 8, 24, and 48 hours post-infection. MOI= 0.001. All data are represented as the mean ± SD of at least 3 independent biological replicates. 15 ACS Paragon Plus Environment


overall it still maintains selectivity toward class II PI3 kinases. M85-12 showed weak antiviral activity (IC50= 28µM) against influenza virus (Figure 4D), suggesting that the antiviral potency of M85 is a product of inhibiting PIK3C2β in combination with other factors. To assess this, we tested an EGFR specific inhibitor, gefitinib, alone and in combination with M85-12 in the antiviral assay to determine if these could recapitulate the potency of M85. Gefitinib alone also had weak antiviral potency (IC50= 23µM) (Figure 4F). When combined, M85-12 and gefitinib show an improvement in potency (IC50: 17uM or 11uM) (Figure 4E and G), but this was not close to the potency of M85 (IC50= 0.35µM) (Figure 4H). This suggests that inhibition of EGFR and PIK3C2β alone may not completely account for the antiviral activity of M85, and other factors may play a role. Our hypothesis is that in addition to PIK3C2β, some degree of activity against PIK3C2α (which is absent for M85-12 but present for M85) is required, potentially because these can play redundant roles in the process of endocytosis. To directly investigate the effects of PIK3C2β on viral replication, we obtained PIK3C2βD1212A/D1212A kinase-dead knock-in (C2β KID) MEFs 29. These MEFs were isolated from C2β KID mice that still express a kinase-dead version of PIK3C2β. Therefore, these cells can be used to specifically determine the effect of the kinase activity on viral replication, independent of other possible roles for PIK3C2β e.g. scaffolding protein. We infected WT and C2β KID MEF cells with WSN influenza virus (MOI 0.001) and measured the viral titers in the supernatant at 8, 24, and 48 hours post infection (Figure 4I). In comparison to WT MEFs, growth of WSN in the C2β KID MEFs showed at least a 1 log reduction in viral titers at 48 hours. This suggests that PIK3C2β kinase activity is a necessary host factor for efficient influenza virus growth. M85 and Oseltamivir Combinations are Synergistic in vitro and Protect Mice better than Oseltamivir Alone


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There are several advantages to using antivirals in combination including reduced emergence of resistance and the possibility of synergistic activity. We therefore investigated the interaction of M85 and oseltamivir carboxylate in a drug combination study. We generated dose-response curves by titrating one drug against a static concentration of the other (Figure S3A) and were able to determine nine combination IC50s. The combination index (CI) for each of these was calculated using the Chou-Talalay method55, yielding an average CI value of 0.27, indicating that M85 and oseltamivir have strong synergism56. This is also clearly demonstrated by plotting the combination IC50s on an isobologram (Figure 5A). The impact of drug combinations on toxicity is always a concern, however we measured less than 5% cytotoxicity for every combination (Figure S3B), giving confidence that cell viability was not affected. Next, we investigated whether M85 could protect mice from a lethal challenge with influenza A/Netherlands/602/2009 (H1N1) pdm09 virus, and because M85 was shown to act synergistically with oseltamivir in cell culture, combinations of M85 and oseltamivir were tested in vivo. First, the pharmacokinetic properties of M85 were measured following a single intraperitoneal administration of mice at 30mg/kg: AUC, 1.2 hr*µM; maximum concentration of drug in serum (Cmax), 1.74 µM; time to maximum concentration of drug in serum (Tmax), 0.083h; half-life (t1/2), 13.1h. Based on these data, groups of BALB/c mice were administered either 20 mg/kg of M85, 5 mg/kg oseltamivir carboxylate (a subprotective dose) or a combination of 20 mg/kg of M85 and 5 mg/kg oseltamivir carboxylate via intraperitoneal injection twice daily, starting either 1.5 days before infection (M85 and combination) or 2h before infection (oseltamivir carboxylate) and proceeding until day 5 post infection. Mice were infected with 5X LD50 of influenza A/Netherlands/602/2009 (H1N1) pdm09 virus intranasally. Four mice per group were used to determine the lung virus titers on day 7 post infection (Figure 5B). Mice given the combination of M85 and oseltamivir had the greatest reduction in viral titers, although mice with oseltamivir alone also had a significant reduction. Mice given M85 alone did not have



A

C

B

D

Figure 5. Combinations of M85 and oseltamivir carboxylate display synergistic antiviral activity in vitro and show efficacy in vivo. A) A549 treated with combinations of M85 and oseltamivir carboxylate were infected with influenza A/WSN/33 virus at MOI 0.001 for 48h. For each treatment, the percentage of NP positive cells was determined and IC50 values calculated. The combination index values (CI) were calculated from the combination IC50 values using the Chou-Talalay method and plotted on an IC50 isobologram. The average CI value is 0.27. B) Groups of 4 C-D) or 10 BALB/c mice were treated with M85 (20 mg/kg), oseltamivir carboxylate (5 mg/kg), or a combination of M85 (20 mg/kg) / oseltamivir carboxylate (5 mg/kg) and were infected with 5X LD50 of influenza A/Netherlands/602/2009 (H1N1) pdm09 virus administered intranasally. Compounds were administered IP twice per day, starting at 1.5 days before infection (M85 and combination) or 2h before infection (oseltamivir carboxylate), and ending on day 5 post infection. B) Lung titers (7 days post infection), C) body weights, and D) survival of the mice up to 20 days post infection with the indicated treatments of M85 and oseltamivir carboxylate (Os). Mice that survived did not lose more than 25% of their body weight. C-D) One group of mice was given 20mg/kg of M85 (M85 Tox Only) without infection to assess toxicity. Another group was given 20 mg/kg oseltamivir to demonstrate that mice could be completely protected, and that 5 mg/kg was a subprotective dose. Statistical significance in comparison to the vehicle + virus group in the lung titer experiment was determined with a one way ANOVA. Mouse weights were compared with a two-way ANOVA. The Kaplan-Meier curves were compared using the log rank (Mantel-Cox) test. * p < 0.05, ** p < 0.01, *** p < 0.001.

a significant reduction in lung viral titers, albeit a slight reduction was observed relative to vehicle-treated mice. In addition, 10 mice per group were monitored daily for weight loss and 18 ACS Paragon Plus Environment

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survival. An uninfected group receiving 20 mg/kg M85 twice daily for 5 days was included to assess potential toxicity, but no weight loss was observed in this group (Figure 5C). Another group was given 20mg/kg oseltamivir carboxylate to demonstrate complete protection verses the sub-protective dose of 5 mg/kg. Mice given M85 or oseltamivir (5mg/kg) alone showed 10% and 30% survival, respectively, but this was significantly improved to 70% survival in mice receiving the M85/oseltamivir combination (Figure 5D). In a similar experiment where mice were infected with a lower virus dose (3X LD50), we also observed a significant increase in survival with mice receiving the combination (80%) versus either alone (40%) (Figure S4). These data provide evidence of in vivo efficacy for M85 and suggest that combinations with oseltamivir provide enhanced protection from both sub-lethal and lethal influenza infections. Discussion As long as viral mutations can confer reduced susceptibility to the FDA approved drugs for influenza, there will be concerns about resistance. To address this issue, we searched for host directed antivirals that limit the ability of the virus to escape via mutation, creating a high barrier to resistance. We have identified one such compound, M85, which did not generate virus with reduced susceptibility after serial passaging. M85 inhibits the endocytosis of viral particles and is particularly effective against a broad spectrum of viruses (influenza virus, HRV, HCV) that utilize this process for entry36, 57, 58. The ability of M85 to halt virus entry post attachment, but near the cell surface potentially suggests that M85 blocks the formation of endocytotic pits, endocytotic vesicles, or endosome trafficking. Further studies will be required to delineate the precise location where virus is being sequestered. However, we identified two host targets for M85, EGFR and PIK3C2β kinases, which have functions that relate to the antiviral mechanism of action for M85. Also, these kinases are highly conserved across mammals59, 60. EGFR kinase activity has been previously implicated in the entry of many viruses, including clathrinmediated endocytosis for influenza viruses61. On the other hand, PIK3C2β is not known to



participate in viral entry, but has been shown to play an important role in endosome trafficking. Overall, we explored the potential for M85 as an antiviral and elucidated a role of PIK3C2β in viral entry. M85 displays many benefits of a host-targeting antiviral that could make it a promising drug. Resistance would have to develop from mutations in the virus that change how it interacts with the host target, in this case EGFR and PIK3C2β, to rescue its pro-viral functions. However, during early endocytosis, the virus never has direct contact with either EGFR kinase or PIK3C2β in the cytoplasm, making it difficult for viral mutations to have an effect. M85 is also a broadspectrum inhibitor that works in human, mouse, and canine cell lines, suggesting that it blocks a highly conserved virus-host interaction. This broadens the potential use of M85 for many viral diseases and possibly ones without any available treatment. However, M85 may not be able to inhibit the endocytosis of viruses that do not strongly rely on EGFR signaling for entry or are able to utilize other growth factor receptors. For instance, M85 was a surprisingly weak inhibitor of VSV and Zika virus, although they utilize clathrin-mediated endocytosis. VSV is thought to mediate attachment and internalization via LDLR family receptors, although it is reported that the virus is still able to enter fibroblasts deficient in this receptor, albeit with reduced efficiency62. Given that M85 shows a very high IC50 (43µM) against VSV, it may only partially rely on EGFR and PIK3C2β for entry. Likewise, Zika virus does not require these factors as it relies on C-type lectin receptors (such as DC-SIGN) for attachment and internalization63. At this time, there are less than a handful of viruses with a described role for EGFR in virus entry and trafficking, but for many viruses the details of the surface receptors required for internalization signals is poorly understood, leaving the possibility that dependencies on EGFR for more viruses may be elucidated. Inhibiting endocytosis in general would be an effective way to block entry, but it would be reasonable to assume that such an inhibitor would display some toxicity. Remarkably, M85


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does not have toxicity at the highest concentrations tested in vitro (50µM), nor was there any toxicity or signs that a 20mg/kg dose was not well tolerated in mice after almost a week of treatment. This might be explained by the fact that EGFR is not the only inducer of PI3K and MAPK, and that Vps34 (class III PI3K) also contributes to PI(3)P formation. Given that M85 does not inhibit Vps34, nor bind to other RTKs implicated in internalization of influenza virus, it is unlikely M85 can completely shut down these activities to levels harmful to the cell. This would imply that M85 does not generally block endocytosis, but does block a virus-induced event that results in internalization. In the literature, it has been reported that influenza virus may trigger endocytic pathways differently from growth factors such as EGF. In one study, depletion of Epsin-1 inhibited clathrin-mediated endocytosis of influenza virus, but did not prevent endocytosis of transferrin and other ligands64. We postulate that influenza virus prefers to activate endocytic pathways through EGFR and class II PI3 kinases which is why it is susceptible to the actions of M85. In this regard, it is also possible that M85 acts by preventing the virus induced upregulation of EGFR effectors and PI(3)P production, but overall cellular levels of these activities are normal (explaining the lack of toxicity). Last, but not least, it may be difficult to overcome the fear of developing kinase inhibitors as antivirals as they are known to have significant side effects in cancer patients65, 66. However, almost all the anti-cancer kinase inhibitors developed to date target class I PI3K, which regulates cell growth, survival, and apoptosis. It has yet to be seen if a drug specifically targeting class II PI3K, which does not regulate these functions, has the same side effects. Collectively, there is no reason to suspect that M85, or any therapeutic targeting class II PI3K, would have high toxicity. The data from this study should also encourage further investigation into the role of class II PI3K in viral replication. For many viruses, including influenza virus, it is not known which effectors are causing internalization after association with signaling receptors (growth factors, GPCRs, and ion channels). We have implicated PIK3C2β as a necessary host factor for influenza virus,



but given the broad-spectrum activity of M85, it is likely important for other viruses, too. This is supported by the evidence that PIK3C2β activity is required for proper endosomal trafficking in general, and that depleting cells of this activity results in sequestration of cargo at the very early endosome stage29. Furthermore, since the sequestration phenotype has similarities to the antiviral mechanism of action of its inhibitor, M85, we postulate that PIK3C2β is a critical host factor during an early step in viral entry. Further investigations will determine if the incoming virus specifically upregulates PIK3C2β activity, and if inhibiting it independently of EGFR stimulation is sufficient to recapitulate the mechanism of action of M85. Fortunately, we discovered an analog, M85-12, that is selective for PIK3C2β, and could be used as a tool to investigate the roles of this kinase pharmacologically. Currently, there are less than a handful of inhibitors like this reported in literature, but unlike M85-12, they are not publicly available54. The inability to recapitulate the full antiviral potency of M85 by inhibiting EGFR and PIK3C2β alone suggest that it affects another important factor, and there is a strong possibility that PIK3C2α contributes to the antiviral activity of M85. M85-12 showed weaker antiviral activity than expected given its potency against PIK3C2β in the in vitro assays. However, this analog completely lost its activity against PIK3C2α, which functionally has a similar role to PIK3C2β in PI(3)P production and localization during EGF stimulation51. It is possible that there is redundancy in this regard, and inhibiting PIK3C2β alone may not be enough to deplete PI(3)P pools. In addition, since there are functional differences between both isoforms, it may be possible that they have distinct roles as required host factors. PIK3C2α, but not PIK3C2β, has been implicated as a regulator of the maturation of clathrin-coated pits67, and depletion of PIK3C2α also downregulates autophagy (also a PI(3)P dependent process), which is not the case for PIK3C2β68. Also, in contrast to PIK3C2β, attempts to generate PIK3C2α knockout mice results in embryonic lethality69, suggesting that it is more critical for survival. This also suggests that a strong inhibitor against PIK3C2α could have high toxicity, which would 22 ACS Paragon Plus Environment

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complicate efforts to design an antiviral against this target. The strong antiviral potency and low toxicity features of M85 could be a product of the right balance of potencies against both isoforms. Its weak activity against PIK3C2α (activity assay IC50= 3270 nM) may confer partial inhibition of its pro-viral functions, but avoids a potentially greater cytotoxicity. However, M85 may be able to compensate for this partial inhibition by combining it with strong activity against PIK3C2β (activity assay IC50= 430 nM). Host-directed antivirals for influenza have been criticized for lacking a good balance of in vivo efficacy and low toxicity. This can be addressed by developing synergistic combinations of host-directed antivirals with direct-acting antivirals. For influenza, combinations of host-directed antivirals with NAIs may display increased potency and a higher barrier to resistance, and this is a likely future direction for influenza therapies because monotherapy with direct-acting antivirals can encourage the development of resistance. In regards to this trend, M85 and oseltamivir have the potential to work as a combination therapy. In a drug combination study M85 and oseltamivir carboxylate demonstrated strong synergism in vitro and had potent antiviral effect at concentrations well below their individual IC50s. At this time, we have yet to determine the mechanism for the observed synergism. It is unknown if the M85 target may interact with the viral NA protein or, more likely, that it is simply due to targeting two distinct stages of the viral life-cycle – entry and release. Future investigations will explore if this synergism is recapitulated in vivo, however the data indicating that a combination of M85 and oseltamivir is significantly better at protecting mice than oseltamivir or M85 alone suggests that it is at least additive if not synergistic. We are also optimistic that combinations of M85 and oseltamivir could decrease the incidence of resistance to oseltamivir given that we were not able to select for resistance against M85 in the same timeframe in which oseltamivir resistance arose. We believe that it will be possible to optimize M85 via extensive medicinal chemistry to further improve its potency



and/or bioavailability and perhaps allow it to be effective when given as a monotherapy as well as combination therapy. Conclusion While characterizing the targets of M85 and deciphering its mechanism of action, we have described a novel required host factor for influenza virus entry, PIK3C2β. By implicating this factor as the downstream component of virus-induced RTK signaling and an effector for events in early endocytosis, this research identifies a new antiviral target and lays the groundwork for elucidating the poorly understood process of how viruses induce these events. Further study will be required to resolve the details of this mechanism and determine if this process is important for other viruses using RTKs and PI3K pathways for entry.

Methods Mice For in vivo efficacy studies, 6-8 week old male or female BALB/c mice (Jackson Laboratories) were randomly assigned to experimental groups of 10 mice. All procedures and handlings are in compliance with the current standards specified in the Guide for the Care and Use of Laboratory Animals provided by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Mice are bred and housed in a BSL2 barrier facility in accordance with the institutional guidelines. All experiments in mice were conducted on an IACUC-approved protocol and performed in a research facility registered with the USDA. Cell Lines A549, MEF, MDCK, Vero, PIK3C2β knock-in-dead MEF, and Huh 7.5 cells were maintained at 37°C and 5% CO2 in Dulbecco’s modified Eagle’s medium (Gibco BRL Life Technologies , Gaithersburg, MD) supplemented with 10% fetal bovine serum (Laboratory Disposable 24 ACS Paragon Plus Environment

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Products, Inc., Wayne, NJ, USA). The PIK3C2βD1212A/D1212A kinase-dead knock-in MEFs were obtained from Bart Vanhaesebroeck (University College London). Viruses Influenza A/WSN/33 (H1N1), vesicular stomatitis virus, mumps virus, Newcastle disease virus, encephalomyocarditis virus were propagated in A549 cells with complete DMEM (10% FBS, 1% Penicillin-Streptomycin) for 48h at 37°C. Influenza A/Vietnam/1203/04 (H5N1) HALo70 and influenza A/Panama/2007/99 (H3N2) were propagated for 48h at 37°C or at 33°C for influenza B/Yamagata/16/88 (PB1-mNeonGreen)31 in the chorioallantoic cavity of 10-day-old embryonated chicken eggs. Influenza A/California/07/2009 (H1N1) was propagated for 48h at 37°C in 8-day-old eggs. Zika virus (MR766) was propagated in low passage Vero cells with complete DMEM (10% FBS, 1% antibiotic-antimycotic) for 72 hours at 37 degrees Celsius with 5% CO2. For the aforementioned viruses, their titer in culture supernatants was determined by a plaque assay. Jc1 HCV cell culture (HCVcc) virus was produced as previously described71. Briefly, supernatants from Huh-7.5 cells transfected by electroporation with in vitro transcribed HCV genomic RNA were collected at 2, 3, and 4 days post transfection, filtered (0.45μm pore size), and used for subsequent infections. Jc1 HCV plasmid, provided by Charles Rice (Rockefeller University), encoding the structural proteins from the HC-J6 isolate and the nonstructural proteins from the genotype 2a JFH-1 isolate, was of bicistronic configuration with the HCV IRES directing translation of Gaussia luciferase (GLuc), and the EMCV IRES driving translation of the chimeric HCV genome. Compounds M85 (N-[3-(3-pyridin-3-yl-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)phenyl]-2-thiophen-2-ylacetamide) and M85-12 (5-ethyl-N-[3-(3-pyridin-3-yl-[1,2,4]triazolo[4,3-b]pyridazin-6-yl)phenyl]thiophene-2sulfonamide) were synthesized at Life Chemicals (Kyiv, Ukraine) and were determined to be >99% and >95% pure by LC-MS. S20 (1-N',2-N'-bis(2,5-dimethylphenyl)-1-N,2-N25 ACS Paragon Plus Environment


dihydroxyethanediimidamide) was synthesized at Chembridge (San Diego, CA, USA) and was determined to be >95% pure by LC-MS. Oseltamivir carboxylate, gefitinib, and bafilomycin A1 were purchased from Sigma-Aldrich (New York, NY,USA). VX-787 was purchased from Roche (Branchburg, NJ, USA). M85, S20, VX-787, and bafilomycin A1 were dissolved in 100% DMSO, but never exceeded a concentration of 0.5% in the culture medium. Virus Infections and IC50 Calculations For the following dose response antiviral assays listed in Table 1, an immunofluorescence protocol was adapted from assays previously described32, 72. This was designed to be a highthroughput alternative to plaque assays for measuring inhibition of viral replication and spread. Cells (from Table 1) were first plated 24 hours before infection. Cells were then treated with complete DMEM containing seven concentrations of compounds (three-fold dilutions starting from 50µM), or DMSO vehicle control, 2 two hours prior to infection. The pre-treatment media was removed, cells were washed in phosphate-buffered saline (PBS), and then cells were infected for 1 hour. Virus inoculum was then removed and replaced with media containing 0.5µg/mL TPCK treated trypsin and the same dilutions of compounds or vehicle. All antiviral assays with influenza viruses, vesicular stomatitis virus, mumps virus, and Newcastle disease virus were performed in a 96-well format. Cells were seeded at density of 25,000 per well, and infected with an MOI of 1 (Newcastle disease virus, mumps virus, influenza A/California/07/2009 (H1N1), and Influenza A/Vietnam/1203/04 (H5N1) HALo), 0.1 (influenza A/Panama/2007/99 (H3N2)), or 0.001 (influenza A/WSN/33 (H1N1), influenza B/Yamagata/16/88 (PB1mNeonGreen), and vesicular stomatitis virus). 24 (vesicular stomatitis virus, mumps virus, and Newcastle disease virus) or 48 (all influenza viruses) hours post-infection, cells were fixed in 4% PFA for 1 hour, and permeabilized with HCS Reagent (Cellomics) for 15 minutes. Assays with influenza A viruses were then blocked with 1% BSA/PBS, incubated with monoclonal mouse anti-NP antibody, HT10373 (Mount Sinai, New York, NY, USA) for one hour and then incubated 26 ACS Paragon Plus Environment

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with Alexa Fluor 488 goat anti-mouse IgG (Life Technologies, Carlsbad, CA, USA) and 100ng/mL DAPI for one hour. Influenza B/Yamagata/16/88 (PB1-mNeonGreen), vesicular stomatitis virus, mumps virus, and Newcastle disease virus expressed GFP, and were only treated with 100ng/mL DAPI. Plates were then analyzed by a fluorescent plate cytometer, the Celigo S (Nexcelom, Lawrence, MA, USA), for number of distinct events in the GFP and DAPI channels. Cells, defined by the DAPI positive signal, which also GFP positive were considered infected. For the serial passaging of WSN in A549, 100,000 cells were plated in a 24-well 24 hours before infection. 2 two hours prior to infection cells were pre-treated with DMSO or compounds. The pre-treatment media was removed, cells were washed in phosphate-buffered saline, and then cells were infected with WSN at MOI 0.001 for 1 hour. Virus inoculum was then removed and replaced with media containing 0.5µg/mL TPCK treated trypsin and the same dilutions of compounds or vehicle. 48 hours post-infection, supernatants were collected and titers of virus were determined by a standard plaque assay. To propagate with serial infections, these supernatants were diluted in PBS to an MOI of 0.001. Zika virus (MOI 0.001) and encephalomyocarditis virus (EMCV) (MOI 1) assays were performed in a similar manner, but in a 24-well format with Vero and A549 cells, respectively. Supernatants were collected at 24h (EMCV) and 48h (Zika virus) post-infection and tittered with a plaque assay. Titers were determined by a standard plaque assay in A549 cells for EMCV, and in Vero cells for Zika virus as described previously74. The relative infection for each treatment was determined by dividing the average number of infected cells or titers in the treatment well by the amount in the vehicle control wells. Based on these values, a 4 parameter logistic nonlinear regression model was used to calculate the IC50 values in Graphpad Prism 6.



For hepatitis c virus infections, 104 cells were seeded on poly-L-lysine coated 96 well tissue culture plates 24 h prior to infection. Cells were pre-incubated for 1 hour with the indicated concentration of M85 prior infection with 100uL of HCVcc diluted 1:2 and containing the indicated concentration of M85. Twenty-four hours post infection, cells were washed 3 times with fresh media to remove Gluc protein present in the inoculum. Supernatants were harvested 48h post infection in 25 μl cell culture lysis buffer and the expression of the luciferase reporter was measured as previously75. The Icahn School of Medicine at Mount Sinai utilized the non-clinical and pre-clinical services program offered by the National Institute of Allergy and Infectious Diseases for the antiviral testing of compounds against human rhinovirus 14, respiratory syncytial virus, human parainfluenza virus in a cytopathic effect assay. Compound Toxicity and CC50 Calculation For the screen with influenza A and B (Figures 1B-C), compound toxicity was measured using the reagents and instructions provided by the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Madison, WI, USA). Briefly, A549 and MDCK cells were plated at a density of 1,250 per well in a 96-well plate for 24 hours. Cells were then treated with complete DMEM containing seven concentrations of compounds (three-fold dilutions starting from 50µM), or DMSO vehicle control, and incubated for 48 hours. CellTiter-Glo® Reagent was then added to the wells at a 1:2 final dilution, and incubated at room temperature for 10 minutes. The luciferase units for each well were measured with a GloMax®-Multi Detection System (Promega). The viability for each compound concentration was expressed as a percentage of the average luciferase units in the treatment (n=6) over the vehicle control wells (n=12). For all other antiviral assays, treatment of the cells with compounds was similar, but fluorescence microscopy of cells stained with Hoechst 33342 (Invitrogen, Carlsbad, CA, USA)


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and propidium iodide, or PI (Nexcelom), was used to determine viability. Cells were plated at a density of 25,000 cells per well and treated with compounds for 24 and 48 hours. A final concentration of 100µg/mL propidium iodide (PI) and 2.5µg/mL Hoechst in PBS was added to the cells for 20 minutes, and then measured for dead cells and total cells by the Celigo S. The viability was calculated by: 100% - (percentage dead (PI+) / total cells (Hoechst +)). Based on the dose-response curve of the viability values, a 4 parameter logistic nonlinear regression model was used to calculate the CC50 value in Graphpad Prism 6. Plaque assay MDCK cells were seeded in a six-well plate at 1x106/well overnight. 1:5-1:10 dilutions of virus were made in virus dilution buffer. Cells were washed in 1xPBS and then cells were infected for 1 hour. Virus inoculum was then removed and replaced with a 1% agar and L15 media (Gibco) containing 0.5µg/mL TPCK treated trypsin (Sigma). Viruses incubated for 48 hours at 37°C. Cells were then fixed in 4% PFA for 1 hour, washed in 1XPBS, then stained with 1% crystal violet for 30 minutes, and de-stained in dH2O for 30 minutes. Titers were determined from visual plaques counts. Pseudotyped Particle Production and Entry Assay Human 293T cells in 10-cm dishes were transfected with pGag/Pol (MuLV ), pV1-gLuc, and either pCAGGs WSN-HA and NA, pVSV-G, or pcDNA 3.1 using Lipofectamine LTX (Invitrogen) for 6 hours before replenishing the media. Supernatants were collected 48 hours later and filtered through a 0.45 μm filter. For the entry assay, A549 cells were seeded in a 96-well plate at 12,000 cells/well, overnight. Polybrene (4μg/mL final concentration) plus WSN HA/NA, VSV-G, and no envelope lentivirus was incubated for 1hr on A549 cells pre-treated with compounds or DMSO in complete DMEM for 2 hours before infection. The virus was aspirated, and cells were replenished with



compounds in complete DMEM. 24 hours later, the gaussia luciferase production in the supernatant was detected using a Renilla Luciferase Assay kit and measured with the GloMax (Promega). All values subtracted the average of the no envelope virus in the same treatment category before normalizing them to DMSO. Viral Polymerase Activity Assay Transfections were done with A549 cells in 96-well plates at a Lipofectamine LTX /DNA ratio of 3:1 (μL/μg). WSN pPol I NP-LUC reporter, pRLTK reporter, WSN pDZ-PB1, WSN pDZ-PB2, and WSN pDZ-NP, and WSN pDZ-PA expression plasmid (or empty vector for negative control) were co-transfected in 100 μL of Opti-MEM (Invitrogen). Incubation of Lipofectamine LTX and DNA was done at room temperature for 30 min prior to addition of the transfection complex directly to A549 cells pre-treated with compounds or DMSO in complete DMEM for 2 hours. Twenty-four hours post-transfection, cells were lysed and luciferase production was measured with the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s specifications. Acid-Induced Endocytosis Bypass Assay A549 cells were seeded at 25,000 cells per well in a 96-well in complete DMEM, overnight. A549 cells and influenza A/PR/8 (H1N1) virus (MOI 10) were separately pretreated with DMSO, M85, or PY102 (Mount Sinai, New York, NY, USA) for one hour and then mixed together at 4°C for one hour. Treatment with 50µM of M85 was required to compensate for the high MOI of virus in the assay. Wells were shifted to 37°C, either for 16h (Infection Only), or for 2 minutes for the pH shift, followed by a 16h incubation. For the latter, wells were shifted to pH 7.4 by adding PBS or to pH 5.0 by adding 50mM citrate buffer (0.35% citric acid and 0.93% sodium citrate, w/v). After 2 minutes, wells were washed in PBS and replaced with fresh complete media with 10mM NH4Cl. All cells were stained with an anti-NP antibody (infected cells) and


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DAPI (total cells) as described above in “Virus Infections and IC50 Calculations” and were counted using a Celigo S imaging cytometer. Endosome Acidification Assay A549 cells were seeded at 25,000 cells per well in a 96-well in complete DMEM, overnight. Cells were pre-treated with M85, bafilomyicin, or DMSO for one hour, and 2.5µg/mL Hoechst for 20 minutes. Next, LysoLive pH-Sensor Green (Marker Gene Technologies, Eugene, OR, USA) was added directly to the cells at 200nM for 30 seconds. Cells were immediately washed in PBS and imaged on an EVOS FL (Advanced Microscopy Group, Mill Creek, WA, USA) at 60X (Figure 3C) or quantified on the Celigo S for cells with bright endosomes (Figure 3D-E). Confocal Microscopy A549 cells were seeded at 10,000 cells per well on top of glass coverslips in a 24-well in complete DMEM, overnight. Cells were pre-treated with M85 or DMSO for 2 hours, washed in PBS, and then incubated with influenza A/PR/8 (H1N1) virus at MOI 200 at 4°C for one hour to allow binding. Cells were then shifted to 37°C, and 30 minutes later the virus inoculum was replaced with fresh M85 or DMSO in complete DMEM, and incubated 1 or 4 hours. Treatment with 50µM of M85 was required to compensate for the high MOI of virus in the assay. After each time point, cells were treated with 10µg/mL wheat-germ agglutinin (WGA) conjugated with Alexa 488 (Invitrogen) for 1 minute, and washed four times before fixation with 4% PFA. Cells were permeabilized with 0.1% Triton-X 100 in PBS for 15 minutes, blocked with 1% BSA/PBS for one hour, incubated with HT103 overnight at 4°C, and finally incubated with Alexa Fluor 488 goat anti-mouse IgG (Life Technologies) and 100ng/mL DAPI for one hour. Coverslips were mounted onto glass slides using ProLong Gold antifade reagent (Life Technologies) overnight. Images of the cells 1µM above the bottom of the slide were taken on a LSM880 (Leica, Wetzlar, Germany) inverted microscope with a Plan-Apochromat 63x/1.4 oil objective using the ZEN 2.3



SP1 FP2 Black edition software. For the co-localization, 5 images were taken 0.5-3µM above the bottom of slide. Using ImageJ version 1.51j76, thresholds in the red and green channels were set to minimize the antibody background. The percentage co-localization of NP to WGA was then calculated by dividing the area of simultaneously red and green pixels by the area of all red pixels of these images (n=12). Chemoinformatics SMILES for M85 and all drugs listed in DrugBank (downloaded from their website: https://www.drugbank.ca/releases/latest) were imported into Canvas 2.7. Linear hashed fingerprints were created for all SMILES with the Binary Fingerprints application. Fingerprints were then processed through the Hierarchical Clustering application to group compounds with similar features. Finally, M85 was processed through a Similarity/Distance Screen application to identify the most structurally similar compounds. Compounds with a Tanimoto similarity score > 0.2 were considered hits. Publications with co-crystal structures of the hits were used to identify their targets, and the descriptions of the functions of the targets were sourced from GeneCards (www.genecards.org). In vitro KINOMEscan, kinase binding, and activity assays KINOMEscan and Kd determinations by KdELECT were outsourced to DiscoverX services (https://www.discoverx.com/services/drug-discovery-development-services/kinase-profiling). The phosphotransferase activity assays for PI kinases (Adapta) and EGFR (Z’Lyte) were outsourced to LifeTech SelectScreen services from ThermoFisher (https://www.thermofisher.com/us/en/home/products-and-services/services/customservices/screening-and-profiling-services/selectscreen-profiling-service/selectscreen-kinaseprofiling-service.html). Pharmacokinetics Study


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Pharmacokinetics analysis of M85 was performed by Bioduro, Inc. (Shanghai, China). Briefly, n=3 male CD1 mice received an intraperitoneal administration of 30mg/kg M85 and then were bled by a saphenous vein puncture for time points up to 24 hours. Plasma M85 concentrations were determined by liquid chromatography-mass spectrometry. In Vivo Infections For in vivo efficacy studies, 6-8 week old male or female BALB/c mice (Jackson Laboratories, Bar Harbor, ME, USA) were randomly assigned to experimental groups of 10 mice. Vehicle (10%DMSO / 50%PEG400 / 40%H2O), M85, and oseltamivir were administered intraperitoneally twice per day, starting at 1.5 days before infection (M85 and combination) or on the day of infection (oseltamivir), and ending on day 5 post infection. For virus challenges, mice were anesthetized by intraperitoneal injection of a mixture of ketamine (100 mg/kg of body weight) and xylazine (5 mg/kg) before intranasal administration of with 3X or 5X mLD50 of influenza A/Netherlands/602/2009 (H1N1) pdm09 in a volume of 30ul. Animals were monitored daily for clinical signs of illness, and body weights were recorded daily for 20 days. Upon reaching 75% of initial body weight, animals were humanely euthanized. On day 7 post infection, 4 additional mice per experimental group were euthanized, and the lungs were collected and homogenized (BeadBlaster 24; Benchmark Scientific) in 1 ml of sterile phosphatebuffered saline (PBS). The lung homogenates were spun at 16,000 × g for 10 min to pellet tissue debris, and the supernatants were collected. Samples were stored at −80°C until titration was performed by standard plaque assay in MDCK cells. Statistical analysis Quantitative data are presented as mean ± SD from at least three independent samples (unless indicated otherwise) and were compared by one-way analysis of variance (ANOVA) with a Bonferroni correction, or log rank test (Mantel-Cox) for the in vivo studies. The weights of mice



in the in vivo studies were compared by a two-way ANOVA with a Bonferroni correction. Z’ were calculated as described before77 using Z’=1-3(σ positive + σ negative) / |µpositive- µnegative|. Combination indices (CI) were calculated by the Chou-Talalay method (CI= (D1(C)/D1(A)) + (D2(C)/D2(A))), where DX(C) is the drug concentration at the combination IC50 and DX(A) is the IC50 of the drug alone. Supporting information Full dose response antiviral and cytotoxicity curves for M85 against an influenza A virus, influenza B virus, HRV-14, and HCVcc (Figure S1). Structures and features of top hits from the structural similarity chemoinformatics analysis (Figure S2). Data from the in vitro combination study (Figure S3). Weights and survival of mice in the in vivo study using 3X LD50 (Figure S4). Fold resistance data from WSN serially passaged in inhibitors and controls (Table S1). List of hits from the KINOMEscan with M85 (Table S2). Data availability The data that support the findings of this study are available from the corresponding author, M.L.S., upon reasonable request.

Acknowledgements We thank Ben Fulton, Lisa Miorin, Megan Schwarz, and Kristopher Azarm, for the influenza B/Yamagata (PB1-mNeonGreen) virus, EMCV, Zika Virus (MR766), and mumps-GFP virus, respectively. We thank Robert DeVita for analyzing the drug-like properties of M85 and Utah State University for HRV, RSV, and HPIV testing via the NIAID in vitro antiviral program. We thank Bart Vanhaesebroeck (University College London) for generously providing the PIK3C2βD1212A/D1212A kinase-dead knock-in MEFs. This work was supported in part by the NIH T32-Research Training Award AI07647 (to R.O) and U19AI135972, R56AI139015 to M.L.S.


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Author Contributions R.O., V.H.L.G., and M.L.S. conceived the experiments; R.O., M.S., and V.H.L.G. carried out the experiments; R.O. and M.L.S. wrote the original draft; All the authors edited and reviewed the paper; R.O. and M.L.S. provided the funding; M.S., V.H.L.G., M.J.E. and M.L.S. provided experimental resources; Supervision, M.L.S provided overall project supervision and administration.

Competing interests The authors declare no competing interests.

Additional Information Correspondence and requests for materials should be addressed to M.L.S.

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