key: cord-318499-uihof6k6 authors: Beddingfield, Brandon; Iwanaga, Naoki; Zheng, Wenshu; Roy, Chad J.; Hu, Tony Y.; Kolls, Jay; Bix, Gregory title: The Integrin Binding Peptide, ATN-161, as a Novel Therapy for SARS-CoV-2 Infection date: 2020-06-16 journal: bioRxiv DOI: 10.1101/2020.06.15.153387 sha: doc_id: 318499 cord_uid: uihof6k6 Many efforts to design and screen therapeutics for the current severe acute respiratory syndrome coronavirus (SARS-CoV-2) pandemic have focused on inhibiting viral host cell entry by disrupting ACE2 binding with the SARS-CoV-2 spike protein. This work focuses on the potential to inhibit SARS-CoV-2 entry through a hypothesized α5β1integrin-based mechanism, and indicates that inhibiting α5β1 integrin interaction with ACE2 and the spike protein using a novel molecule ATN-161 represents a promising approach to treat COVID-19. SARS-CoV-2 spike protein interaction with integrins, and specifically α5β1. We performed similar assays to investigate ACE2 binding to α5β1, using a mixture of ATN-161 and human ACE2 protein (hACE2). Clear inhibition of ACE2/α5β1 binding by ATN-161 was apparent and dose-dependent (Fig. 1c) . Application of ATN-161 also did not reduce binding of trimeric spike protein to hACE2, but did reduce binding of monomeric spike ( Figure S1 ). The in vitro assessment of ATN-161 and therapeutic potential was performed using a once-passaged Vero (E6) African green monkey (Chlorocebus atheiops) kidney cell line utilizing competent SARS-CoV-2. ATN-161 was effective at reducing viral loads after infection (Fig. 2a) , with an estimated IC50 of 3.16 µM. The EC50 value for ATN-161 approximates the value for remdesivir 8 . Measuring cellular viability and underlying cytotoxicity is another metric for antiviral therapeutic potential that we explored with ATN-161 26 . After 24 hours infection at a MOI of 0.01, cells were lysed with CellTiterGlo and luminescence values were taken to measure ATP production in each treatment. Pretreatment with ATN-161 increased ATP production in infected cells, indicating increased viability, and was consistent with viral PCR data at concentrations as low as 1µM ATN-161 (Fig. 2b) . Addition of 10 µM ATN-161 resulted in a decreased cytopathic effect (i.e. fewer apparent rounded, bright cells) when cells were visualized by phase contrast microscopy (Fig. 2c) . In summary, we show that SARS-CoV-2 spike protein binds to both α5β1 and α5β1/hACE2, and that this binding can be effectively inhibited by ATN-161, which also disrupts SARS-CoV-2 infection in vitro. Prophylatic treatment of ATN-161 increased cell viability in the presence of SARS-CoV-2 and decreased cytopathic effects associated with viral infection. Taken together, and in light of ATN-161's previously demonstrated in vivo therapeutic efficacy against a closely related beta-coronavirus (porcine hemagglutinating encephalomyelitis virus 23 ) and its successful use in human cancer clinical trials 27 , these results support the performance of in vivo studies to assess the potential efficacy of ATN-161 as an experimental therapeutic agent for COVID-19. VeroE6 cells (ATCC# CRL-1586) were cultured in complete DMEM containing 10% fetal bovine serum (FBS). SARS-CoV-2 stock from viral seed (SARS-CoV-2; 2019-nCoV/USA-WA1/2020 (BEI# NR-52281) was obtained by infecting nearly confluent monolayers of VeroE6 cells for one hour with a minimal amount of liquid in serum free DMEM. Once adsorption was complete, complete DMEM containing 2% FBS was added to the cells and the virus was allowed to propagate at 37℃ in 5% CO2. Upon the presence of CPE in the majority of the monolayer, the virus was harvested by clearing the supernatant at 1,000 xg for 15 minutes, aliquoting and freezing at -80℃. Sequencing confirmed consensus sequence was unchanged from the original isolate. Enzyme-Linked Immunosorbent Assay (ELISA) was utilized to determine the ability of ATN-161 to disrupt binding events essential to entry of SARS-CoV-2 into a host cell. For determination of inhibition of ACE2/ α5β1 integrin binding by ATN-161, α5β1 was coated on 96-well plates at 1 µg/mL for 2 hours at room temperature and blocked overnight with 2.5% BSA. Addition of 0.5 µg/mL of hACE2-Fc (Sino Biological, Cat# 10108-H02H) in differing concentrations of ATN-161 followed, incubating for 1 hour at 37℃. Incubation with an HRP labeled goat anti-human Fc secondary antibody at 1:5000 for 30 minutes at 37℃ was followed by detection by TMB substrate. In order to assess disruption of binding of α5β1 to SARS-CoV-2 Spike protein, 96-well plates were coated as before, but incubation with ATN-161 was performed in conjunction with 1µg/mL spike (produced under HHSN272201400008C and obtained through BEI Resources, NIAID, NIH: Spike Glycoprotein Receptor Binding Domain (RBD) from SARS-Related Coronavirus 2, Wuhan-Hu-1, Recombinant from HEK293 Cells, NR-52306) in the presence of 1mM MnCl2, followed by detection with an anti-spike antibody. The rest of the procedure was consistent with the previous. In order to determine the ability of ATN-161 to reduce the infection capability of SARS-CoV-2 in vitro, a cell-based assay was utilized. VeroE6 cells were plated at a density of 1.25 x 10 4 cells/well in a 96-well plate and incubated overnight at 37℃ in 5% CO2. The next day, cells were treated with dilutions of ATN-161 in complete DMEM with 2% FBS for one hour at 37℃ in 5% CO2, followed by viral infection at an MOI of 0.1. After 48 hours, virus and cells were lysed via Trizol LS and RNA was extracted using a Zymo Direct-zol 96 RNA Kit (#R2056) according to manufacturer's instructions. Experiments were performed under Biosafety Level 3 conditions in accordance with institutional guidelines. Viral load was quantified using a Reverse Transcriptase qPCR targeting the SARS-CoV-2 nucleocapsid gene. RNA isolated from cell cultures was plated in duplicate and analyzed in an Applied Biosystems 7300 using TaqPath supermix with the following program: i)50℃ for 15 min., ii) 95℃ for 2 min. and iii) 45 cycles of 95℃ for 3s and 55℃ for 30s. Inhibition of SARS-CoV-2 spike protein binding to human ACE2 by ATN-161. Plates were precoated with a, trimeric or b, monomeric spike protein and incubated with a mixture of hACE2 and various ATN-161 concentrations, followed by detection of bound hACE2 via HRPconjugated anti-ACE2 antibody. Data was normalized to a no-ATN vehicle control. 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We would also like to thank K Andersen at Scripps Research Institute for sequencing of viral stock. G.B. is supported by Tulane University startup funds. JKK was supported by the following NIH grant R35HL139930 for this work. This research was supported in part by grant OD0011104 to CJR from the National Center for Research Resources and the Office of Research Infrastructure Programs (ORIP), NIH. TYH was supported by Department of Defense grant W8IXWH1910926 and NIH grants R21EB026347, R01AI122932, R01AI113725, R01HD090927 and R21AI126361. G.B. is the inventor on a filed provisional patent with the USPTO related to this work. The remaining authors declare no competing interests.