key: cord-0888417-ewn0ggzk authors: Saul, Sirle; Karim, Marwah; Huang, Pei Tzu; Ghita, Luca; Chiu, Winston; Kumar, Sathish; Bhalla, Nishank; Leyssen, Pieter; Cohen, Courtney; Huie, Kathleen; Tindle, Courtney; Sahoo, Malaya; Sibai, Mamdouh; Pinsky, Benjamin A.; Das, Soumita; Ghosh, Pradipta; Dye, John; Solow-Cordero, David; Jin, Jing; Jochmans, Dirk; Neyts, Johan; Narayanan, Aarthi; Jonghe, Steven De; Einav, Shirit title: Pan-ErbB inhibition protects from SARS-CoV-2 replication, inflammation, and injury date: 2021-05-16 journal: bioRxiv DOI: 10.1101/2021.05.15.444128 sha: 7fd692ba2341c45c863267971cca272f38ee4e97 doc_id: 888417 cord_uid: ewn0ggzk Effective therapies are needed to combat emerging viruses. Seventeen candidates that rescue cells from SARS-CoV-2-induced lethality and target diverse functions emerged in a screen of 4,413 compounds. Among the hits was lapatinib, an approved inhibitor of the ErbB family of receptor tyrosine kinases. Lapatinib and other pan-ErbB inhibitors suppress replication of SARS-CoV-2 and unrelated viruses with a high barrier to resistance. ErbB4, but not lapatinib’s cancer targets ErbB1 and ErbB2, is required for SARS-CoV-2 entry and Venezuelan equine encephalitis virus infection and is a molecular target mediating lapatinib’s antiviral effect. In human lung organoids, lapatinib protects from SARS-CoV-2-induced activation of pathways implicated in acute and chronic lung injury downstream of ErbBs (p38-MAPK, MEK/ERK, and AKT/mTOR), pro-inflammatory cytokine production, and epithelial barrier injury. These findings reveal regulation of viral infection, inflammation, and tissue injury via ErbBs and propose approved candidates to counteract these effects with implications for coronaviruses and unrelated viruses. The recently emerging severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has Lapatinib has a broad-spectrum antiviral activity and a high genetic barrier to resistance We focused on defining the therapeutic potential of lapatinib, an already approved pan-ErbB 208 inhibitor that emerged in the screen (Figure 2A) (Figures 2G-2I ). To determine whether viruses can escape treatment with lapatinib, we focused on VEEV (TC- These results point to lapatinib as a potential broad-spectrum antiviral agent with a higher 240 relative barrier to resistance than a direct-acting antiviral, and support that lapatinib suppresses 241 viral infection by targeting a cellular function. To understand lapatinib's target(s) and mechanism of antiviral action, we first tested the 246 hypothesis that by targeting receptor tyrosine kinases, it inhibits the entry of vesicular stomatitis ErbB4, but not other lapatinib's targets, is essential for SARS-CoV-2 entry and VEEV (TC-257 83) infection The ErbB family is composed of four members (ErbB1-4), of which three are catalytically active: (Figures 3G and 3H) . RIPK2 depletion mildly reduced VEEV infection, but not rVSV-SARS- CoV-2-S infection (Figures 3F and 3H) . Depletion of the remaining 5 targets, including ErbB1 271 and ErbB2, had no effect on infection of both viruses (Figures 3F and 3H) . None of these 272 siRNA pools impacted cellular viability (Figures 3F and 3H ). To further probe the functional relevance of ErbB4, we silenced ErbB4 expression in Vero and and 3M-3O). ErbB4 depletion did not impact cell viability (Figures 3J, 3L and 3N ) and largely 282 correlated with the observed phenotype. These findings confirm a role for ErbB4 in viral entry; a 283 step of the viral life cycle, which is also inhibited by lapatinib. Lapatinib was shown to bind the ATP binding site of ErbB4 in a comparable manner to ErbB1 To further probe the requirement for ErbBs in SARS-CoV-2 infection, we evaluated the antiviral 291 effect of three other compounds with anti-ErbB activity. Tyrphostin AG 879, an experimental 292 compound that emerged in the HTS (Levitzki and Gazit, 1995) , dose-dependently inhibited 293 SARS-CoV-2 and rVSV-SARS-CoV-2-S infections in Calu-3 cells, with EC50 of 0.5-1.1 µM and 294 CC50>20 µM (Figures 3P and S3D ). Yet, its activity on ErbB2 and 4 could not be confirmed via 295 the in vitro kinase assay (Figures 3Q and S3C) , suggesting that another target may be Collectively, these results provide evidence that ErbB4, but not ErbB1 or 2, is required for 306 SARS-CoV-2 entry and VEEV (TC-83) infection, thereby validating it as a druggable antiviral 307 8 target. Its role as an entry co-factor, a step of the life cycle that is inhibited by lapatinib, supports 308 a hypothesis that inhibition of ErbB4 mediates the antiviral effect of lapatinib. ErbB4 is a molecular target mediating the antiviral effect of lapatinib To determine whether lapatinib exerts its antiviral effect by inhibiting phosphorylation of the 313 catalytically active ErbBs, lysates derived from SARS-CoV-2-infected Calu-3 cells treated with 314 lapatinib or DMSO were subject to Western blot analysis. Lapatinib treatment dose-dependently 315 suppressed the ratio of phosphorylated to total ErbB1, 2, and 4 levels at 24 hours post-infection 316 with EC50 values lower than 0.1 µM that correlated with reduced expression of the SARS-CoV-2 317 nucleocapsid protein (Figures 4A and 4B) . Similar findings were observed at 1.5 hour post-318 infection (Figures S4A and S4B) . These results provide evidence that drug exposure and the 319 antiviral effect of lapatinib in these cells are correlated with functional inhibition of ErbBs' 320 activity. To confirm that inhibition of ErbB4 is a mechanism underlying the antiviral effect of lapatinib, we 323 conducted gain-of-function assays. Ectopic expression of WT ErbB4, but not catalytically These results provide evidence that lapatinib inhibits activation of signaling pathways Next, we tested the hypothesis that by inhibiting this ErbB-regulated signaling in pathways (Figures 6H, 6I, and S5D ). In contrast, 36 hours following SARS- CoV-2 infection and DMSO treatment, claudin 7 stained as speckles or short segments that Most antiviral strategies target viral enzymes, thereby typically providing a "one drug, one bug" 449 approach that is prone to the emergence of viral resistance and is not easily scalable to meet Lapatinib similarly suppressed replication of flaviviruses, alphaviruses and filoviruses, indicating 461 its broad-spectrum potential and exhibited a higher relative barrier to resistance than a classical 462 direct-acting antiviral. Using lapatinib as a pharmacological tool, we discovered ErbB4, the least studied ErbB, as a 465 candidate antiviral target. We then showed that ErbB4 is a co-factor of SARS-CoV-2 entry that 466 is also required for VEEV (TC-83) infection, and that its kinase function is required for both 467 infections. Unlike other ErbBs, to the best of our knowledge, ErbB4 has not been previously by targeting all three catalytically active ErbBs, we predict that lapatinib will achieve pan-475 coronavirus coverage. We provide multiple lines of evidence to support modulation of ErbB4 activity as an important 478 mechanism of antiviral action of lapatinib in SARS-CoV-2 infection. We demonstrate that 479 lapatinib inhibits viral entry, analogous to the phenotype seen with RNAi-mediated suppression 480 of ErbB4. We establish that lapatinib's antiviral activity correlates with reduced phospho-ErbB4 12 levels in vitro. In accordance with this finding, we show that WT but not a kinase dead ErbB4 ErbB4-mediated antiviral effect is unique (Figure 7) . In agreement with studies in COVID-19 patients (Del Valle et al., 2020), we provide evidence 539 that SARS-CoV-2 infection increases production of the pro-inflammatory cytokines IL-1β, TNF-α 540 and IL-6 in human lung organoids. Importantly, lapatinib treatment dose-dependently inhibited 541 the production of these cytokines and concurrently increased the level of MCP-1, suggesting 542 that it may also augment innate immune responses (Sokol and Luster, 2015) . Our IF data 543 provides evidence for the potential utility of laptinib in maintaining lung epithelial barrier integrity. We predict that the anti-inflammatory and tissue protective effects of lapatinib are beyond its Collectively, these findings provide insight into the mechanisms underlying the antiviral, anti-565 inflammatory, and tissue protective effects of lapatinib. The repurposed approaches being 566 studied for COVID-19 to date typically target one of these processes, but not all three. Although toxicity is a concern when targeting host functions, finding a safe therapeutic window 593 may be feasible. Lapatinib has a favorable safety profile, particularly when used as a 594 monotherapy and for short durations, as those required to treat acute infections. Notably, the 595 largest safety database based on which the toxicity profile in lapatinib's package insert was 596 determined is based on data from over 12,000 patients with advanced cancer who received 597 lapatinib in combination with capecitabine or trastuzumab plus an aromatase inhibitor and for 598 long durations (Novartis, 2018) . In pivotal trials, the most commonly reported adverse events 599 reported more frequently with combination therapy including lapatinib compared with 600 capecitabine or trastuzumab monotherapy were diarrhea, palmar-plantar erythrodysesthesia, Notably, unlike the other receptor tyrosine kinase inhibitors erlotinib and gefitinib, lapatinib 619 monotherapy has not been associated with pneumonitis, interstitial lung disease or lung fibrosis An important consideration with lapatinib is, however, its potential for drug-drug interactions. Since metabolized by CYP3A4, concurrent use of suppressors of CYP3A4 should be avoided to 641 reduce risk of QT prolongation. Concurrent treatment with strong inducers of CYP3A4 should 642 also be avoided, as this can reduce lapatinib's levels to sub-therapeutic. Of particular relevance 643 is dexamethasone, standard of care for moderate COVID-19 patients, which induces CYP3A4. Since other steroids do not induce CYP3A4, lapatinib could be studied in combination with 645 hydrocortisone or prednisone, which have been shown to achieve a comparable protective Another kinase inhibitor that emerged in the HTS was sunitinib, an approved anticancer multi-649 kinase inhibitor that we have previously shown to protect mice from DENV and EBOV ErbB activity, the precise target(s) mediating the antiviral effect remain to be elucidated. Beyond kinases, diverse cellular functions emerged in our HTS as candidate targets for anti-660 SARS-CoV-2 approaches. One example is ion transport across cell membranes. Among the hits The emergence of gedunin, a natural product that inhibits HSP90 and has anti-inflammatory approaches. Moreover, they underscore the potential utility of natural products as broad-702 spectrum antivirals, yet limited scalability typically challenges the use of these products. In summary, our study reveals candidate broad-spectrum antiviral therapies that target a variety Dinaciclib and ribociclib were a gift from Dr. Mardo Koivomaegi (Stanford University). High-throughput screening (HTS) of compound libraries. Compounds from the libraries 756 listed above were plated in a total of 29 assay-ready 384-well plates (Greiner #7810192). Dispensing of 6 µl of the compound solutions was achieved using an automated Agilent Bravo The rest of the experiments were done using the USA-WA1/2020 strain. SARS-CoV-2 whole-839 genome amplicon-based sequencing was conducted by adapting an existing whole genome 840 sequencing pipeline for poliovirus genotyping (Sahoo et al., 2017) . Dose response curves with 841 lapatinib in Vero and Calu-3 cells (Figures 1E and 2B , respectively) were performed with a P3 In vitro kinase assays. ErbB2 and ErbB4 in vitro kinase assays were performed on the 970 LabChip platform (Nanosyn) or radiometric HotSpot TM kinase assay platform (Reaction Biology). Signaling pathway analysis. Following a 2-hour starvation under serum-low or -free Cells were blocked for 1h at RT with 3% BSA and 0.1 % Triton X in PBS. Cells were incubated 990 with mouse mAb SARS-CoV-2 nucleocapsid antibody (SinoBiological, 1:100) and rabbit Claudin 991 7 polyclonal antibody (ThermoFisher, 1:200) overnight at 4°C, followed by 1 hour incubation at 992 room temperature with goat anti-mouse AF488 (ThermoFisher, 1:400), goat anti-rabbit AF647 993 (ThermoFisher, 1:400), and counterstaining with DAPI (4', 6-diamidino-2-phenylindole, ThermoFisher, 1:10000) and phalloidin (ThermoFisher, 1:400). Images were taken on an SP8 995 microscope (Leica). Adjustment for brightness, contrast and color balance were done using Fiji 996 software. Quantification and Statistical Analysis. All data were analyzed with GraphPad Prism 999 software. Fifty percent effective concentrations (EC50) and 50% cytotoxic concentration (CC50) 1000 were measured by fitting of data to a 3-parameter logistic curve. 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ErbB4, but not other lapatinib's targets, is essential for SARS-CoV-2 entry and 1535 VEEV (TC-83) infection A) Schematic of the experiments shown in panels B-D Dose response to lapatinib of rVSV-SARS-CoV-2-S infection (black) and cell viability 1538 (blue) in Calu-3 (B) and Vero (C) cells via luciferase and alamarBlue assays at 24 hours post-1539 infection with a standard inoculum Dose response to lapatinib of rVSV-SARS-CoV-2-S entry in Vero cells by RT-qPCR at 3 1541 hours post-infection with a high inoculum Schematic of the experiments shown in panels F F) rVSV-SARS-CoV-2-S infection by luciferase assays and cell viability by alamarBlue assays 1544 (blue) measured at 24 hours post-infection of Vero cells transfected with the indicated siRNA 1545 pools G) Schematic of the experiments shown in panels H VEEV (T-83) infection by luciferase assays and cell viability by alamarBlue assays (blue) 1548 measured at 24 hours post-infection of U-87 MG cells transfected with the Confirmation of gene expression knockdown by RT-qPCR in Vero (I) and U-87 MG (K) 1551 cells at 48 hours post-transfection -83) infection (MOI=0.1) (L) measured by luciferase 1553 assays at 24 hours post-infection of Vero or U-87 MG cells transfected with the indicated 1554 siRNAs, respectively SARS-CoV-2 infection at 24 hours post-infection of ErbB4-depleted Vero cells with SARS-1557 CoV-2 (USA-WA1/2020 strain; MOI=0.05) by plaque assays SARS-CoV-2 entry at 3 hours post-infection of Vero cells (MOI=1) by RT-qPCR Chemical structures and dose response to tyrphostin AG 879, ibrutinib and sapitinib of 1560 USA-WA1/2020 strain; MOI=0.05) by plaque assays and cell 1561 viability (blue) by alamarBlue assays at 24 hours post-infection of Calu-3 cells. 1562 (Q) Binding affinity (KD), enzymatic activity (IC50) or percent binding of control (% control) of the 1563 indicated kinase inhibitors on the 3 catalytic ErbBs, the source of kinome data Individual 1566 experiments had 3 biological replicates, means ± SD are shown. *P < 0.05, **P < 0.01, ***P < 1567 0.001 relative to DMSO (B-D, P) or to siNT (F-O) (one-way ANOVA followed by Dunnett Lapatinib's antiviral activity is correlated with functional inhibition of ErbB 1574 activity and is mediated by ErbB4 A) Schematic of the experiment shown in B B) ErbB1, 2 and 4 phosphorylation in Calu-3 cells that are uninfected (lane 1), infected and 1577 treated with DMSO (lane 2) or infected and treated with increasing concentrations of lapatinib 1578 (lanes 3-7) measured by Western blotting 24 hours post-infection with Shown are representative membranes blotted for phospho-and total 43 SARS-CoV-2 nucleocapsid and actin and quantitative phospho-to total protein 1581 ratio data relative to infected cells treated with DMSO Schematics of the experiments shown in D, E (C) and G, H (F) Level of ErbB4 and actin expression measured by Western blot following transfection of 1584 Vero (D) or U-87 MG (G) cells with control or ErbB4-expressing plasmids Rescue of rVSV-SARS-CoV-2-S infection (E) or VEEV (TC-83) infection (H) in the 1586 presence of lapatinib upon ectopic expression of the indicated plasmids measured by luciferase 1587 assays 24 hours after infection in Vero (E) or U-87 MG cells (H) Data in all panels are representative of 2 or more independent experiments. Shown in panels E 1589 and H are means±SD of results of two combined experiments conducted each with three (E) or 1590 five (H) replicates Tukey's multiple comparisons test at each lapatinib concentration. Ns, non-significant Lapatinib inhibits SARS-CoV-2-induced activation of ErbB-regulated 1598 inflammatory and tissue injury signals, 1599 (A) Schematic of the experiment shown in B 1601 infected and treated with DMSO (lane 2) or infected and treated with lapatinib (lanes 3 and 4) 1602 measured by Western blotting 1.5 hours (B) and 24 hours (C) post-infection with SARS-CoV-2 1603 (USA-WA1/2020 strain, MOI=1) Lapatinib inhibits SARS-CoV-2 infection, inflammation, and tissue injury ex vivo 1623 in human adult lung organoids (ALOs) ALOs were infected with 1625 virulent SARS-CoV-2 (USA-WA1/2020 strain, MOI=1) Dose response to lapatinib of SARS-CoV-2 infection (black) and cell viability (blue) in ALO 1627 supernatants via plaque and alamarBlue assays at 48 hours post-infection, respectively. 1628 (C) Dose response to lapatinib of SARS-CoV-2 nucleocapsid (N) copy number in ALO lysates 1629 by RT-qPCR assays at 48 hours post-infection SARS-CoV-2 nucleocapsid (green) and 1631 DAPI (blue) in naïve and SARS-CoV-2-infected ALOs pre-treated with DMSO or 10 µM lapatinib 1632 24 hours post-infection (E) and AKT, ERK and 1634 p38 MAPK (F) phosphorylation in ALOs that are uninfected (lane 1), SARS-CoV-2-infected and 1635 treated with DMSO (lane 2) or infected and treated with lapatinib (lanes 3-5) measured by 1636 Western blotting 48 hours post-infection. Shown are representative membranes blotted for 1637 phospho-and total kinases and actin and quantitative phospho-to total kinase ratio data relative 1638 to Heat map showing the concentration of cytokines (pg/mL) in the supernatants of ALOs 1640 under the indicated conditions at 48 hours post-infection with SARS-CoV-2 measured by 1641 LEGENDplex (Biolegend) kit H) Schematic of the experiment shown in I Confocal IF microscopy images of Claudin 7 (gray) and DAPI (blue) in naïve or SARS-CoV ALOs treated at 4 hours post-infection either with DMSO or 10 µM lapatinib and imaged 1645 at 36 hours post-infection Data in all panels are representative of 2 or more independent experiments Individual experiments had 3 biological replicates, means ± SD are shown in panels B and C Representative merged images at 40x magnification are shown in panels D and I. Scale bars 1649 are 50 m Validation of ErbB4 as an antiviral target Confirmation of siRNA-mediated gene expression knockdown by Western blot in Vero cells 1726 at 48 hours after transfection. Notably, two anti-ErbB4 antibodies detected no signal of 1727 endogenous protein in Vero cells in two independent experiments Confirmation of siRNA-mediated (ON-TARGETplus SMARTpool siRNAs [Dharmacon]) gene 1729 expression knockdown by RT-qPCR in Vero cells. Shown is gene expression normalized to 1730 GAPDH and expressed relative to the respective gene level in the non-target Dose response to lapatinib, tyrphostin AG 879 and sapitinib of ErbB2 and/or ErbB4 kinase 1733 activity in vitro (Nanosyn) Dose response to tyrphostin AG 879 (D) and ibrutinib (E) of rVSV-SARS-CoV-2-S 1735 infection (black) by luciferase assays and cell viability (blue) by alamarBlue assays at 24 hours 1736 post Dose response to sapitinib of SARS-CoV-2 infection (black) by plaque assay and cell 1738 viability (blue) by alamarBlue assay 24 hours post-infection of Vero cells Data in all panels are representative of 2 or more independent experiments. Individual 1740 experiments had 3 biological replicates, means ± SD are shown. *P < 0.05, **P < 0.01, ***P < 1741 0.001 relative to siNT by one-way ANOVA followed by Dunnett's multiple comparisons test ErbB4 is modulated by lapatinib and is the molecular target mediating its antiviral effect. 1746 (A, B) ErbB2 and ErbB4 phosphorylation in Calu-3 cells that are uninfected (lane 1), infected 1747 and treated with DMSO (lane 2) or infected and treated with lapatinib (lanes 3 and 4) measured 1748 by Western blotting 1.5 hours (A) and 24 hours (B) post-infection Shown are representative membranes blotted for phospho-and total 1750 ErbB4, and actin and quantitative phospho-to total ErbB ratio data relative to infected 1751 cells treated with DMSO C) and U-87 MG (D) cell viability by alamarBlue assays 48 hours post-transfection 1753 of the indicated plasmids Data in panels C and D are representative of 2 or more independent experiments. Individual 1755 experiments had 3 biological replicates, means ± SD are shown Human ALOs for studying the antiviral and tissue protective effects of lapatinib. 1765 (A, B) Viral titer by plaque assays in culture supernatants (A) and viral nucleocapsid (N) copy 1766 number by RT-qPCR in lysates (B) from human lung organoids at 24, 48 and 72 hours post-1767 infection SARS-CoV-2 nucleocapsid (green) and 1769 DAPI (blue) in naïve and SARS-CoV-2-infected ALOs pre-treated with DMSO or 10 µM lapatinib 1770 24 hours post-infection DAPI (blue) in naïve or SARS-CoV-1772 2-infected ALOs treated at 4 hours post-infection either with DMSO or 10 µM lapatinib and 1773 imaged at 36 hours post-infection Representative merged images at 20x magnification are shown in panels C and D. Scale bars 1775 are 100 m