key: cord-0996000-gstxmrn4
authors: Beddingfield, Brandon J.; Iwanaga, Naoki; Chapagain, Prem P.; Zheng, Wenshu; Roy, Chad J.; Hu, Tony Y.; Kolls, Jay K.; Bix, Gregory J.
title: The Integrin Binding Peptide, ATN-161, as a Novel Therapy for SARS-CoV-2 Infection
date: 2020-10-16
journal: JACC Basic Transl Sci
DOI: 10.1016/j.jacbts.2020.10.003
sha: b443ec72de38f7a492892404edd6df87fc8602f6
doc_id: 996000
cord_uid: gstxmrn4

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 α5β1 integrin-based mechanism, and indicates that inhibiting the spike protein interaction with α5β1 integrin (+/- ACE2), and the interaction between α5β1 integrin and ACE2 using a novel molecule ATN-161 represents a promising approach to treat COVID-19.

AUTHOR CONTRIBUTIONS G.B. conceived the study. B.B. conducted all live SARS-CoV-2 studies. N.I. and W.Z. conducted all ELISA's. B.B., N.I. and W.Z. collected data and performed computational analysis. B.B., C.R., T.H., J.K. and G.B. interpreted data and wrote the manuscript with input from all of the authors. DECLARATION OF INTERESTS G.B. is the inventor on a filed provisional patent with the USPTO related to this work. The remaining authors declare no competing interests.

As of September 28, 2020, there have been 995,836 deaths out of a total 32,968,853 confirmed COVID-19 cases, for an estimated fatality rate of almost 3.1%

(https://www.who.int/emergencies/diseases/novel-coronavirus-2019). This viral outbreak began in China in late 2019 (2) , with a likely origin in bats, with selection resulting in efficient human-tohuman transmission occurring before or after transfer to the human host (3) . This follows the same epizoontic transmission events seen in other severe viral infections, including SARS-CoV (4) and Ebola (5) , and was predicted prior to this outbreak (6) . Interaction between the SARS-CoV-2 spike protein and the angiotensin-converting enzyme II (ACE2) receptor has been implicated in SARS-CoV-2 entry and replication (7) . Many therapeutic efforts spurred by the current pandemic have focused on disrupting an aspect of the viral replication process (8, 9) , including host entry (10) , often focusing on inhibition of ACE2/spike protein binding (11) .

Integrin binding has also been implicated in the SARS-COV-2 cell entry mechanism, as the spike protein contains an integrin binding motif (RGD) (12) (13) (14) (15) (16) . Integrins are extracellular matrix receptors expressed throughout the body, including in the respiratory tract (e.g. epithelial cells (17) ) and vasculature (e.g. endothelial cells (18) ), and the β1 family of integrins are closely associated (in proximity and functional regulation) with ACE2 (19, 20) . A non-RGD peptide derived from the extracellular matrix component fibronectin, referred to herein as ATN-161, can bind to and inhibit the activity of certain integrins, including α5β1 (21, 22) , and has been previously used to study viral replication (23) . ATN-161 binds outside the RGD-binding pocket, thus acting as a non-competitive inhibitor of integrin binding, especially for α5β1 (24) . Likewise, ACE2 binds to α5β1 in an RGD-independent fashion, although it possesses an RGD motif in a region inaccessible for protein-protein interaction. 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% CO 2 . 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. 

The structure of the ACE2-Spike protein receptor binding domain complex (7) was obtained from the protein data bank (PDB ID 6m0j). To get the orientation of the SARS-CoV-2 spike protein trimer relative to ACE2, the receptor binding domain (RBD) was aligned with the sprung out RBD of the prefusion conformation of the spike protein trimer (PDB ID 6vsb) (28) .

Similarly, the integrin α5β1 ectodomain structure (29) (22) . To our knowledge, this is the first report of 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 ( Figure 1B) . Furthermore, application of ATN-161 reduced binding of trimeric spike protein to hACE2, either alone or in combination with α5β1, the latter of which trended to support greater spike binding than to hACE2 alone ( Figure 1C ). Application of ATN-161 also reduced binding of monomeric spike to hACE2 ( Figure S1 ). Fig 3A. One of these is at the interface between the ACE2 and the spike RBD. This may affect the binding of RBD with the ACE2. ATN-161 is also found to bind the integrin α5β1 ectodomain complex near the RGD motif binding site located at the interface between the α5 and β1 chain (29) , potentially affecting the binding of α5β1 with proteins containing the RGD motif. Although ACE2 contains the RGD sequence, it is inaccessible for binding under physiological conditions. Therefore, it is believed that another sequence KGD (residues 353, 354,355), which closely resembles the sequence RGD, may bind α5β1 via the RGD-binding site (37) . Figure 3B shows the ACE2-α5β1 complex obtained from protein-protein docking using Zdock with the ACE2 residues around KGD and the α5β1 residues J o u r n a l P r e -p r o o f around the RGD-binding site selected as preferred binding partners. This docking results in a complex with the desired orientations of the integrin chains (38) and ACE2 relative to the plasma membrane ( Fig 3A) . As shown in Fig. 3B , the binding of the α5β1 to ACE2 at this site masks the binding site for the spike RDB, potentially inhibiting the SARS-CoV-2 entry (37) . The binding of ATN-161 in the interface may disrupt the α5β1-ACE2 complex.

Separately, we performed docking of α5β1 to the spike protein RBD, which contains the RGD sequence that is accessible for binding. This results in a complex of the spike RBD and α5β1 as shown in Fig. 3C . For this binding to occur, the RGD-binding interface of integrin needs to be oriented differently than binding with ACE2, consistent with the active conformation of integrin (7) . ATN-161 binding near the RGD motif binding site of integrin may inhibit the α5β1spike RBD complex formation. We hypothesize that SARS-CoV-2 entry is facilitated by binding to ACE2-associated α5β1 integrin via its spike protein, and that ATN-161 treatment will inhibit infection by blocking this binding event and by disrupting the initial ACE2 and α5β1 interaction ( Figure 3D ). One potential limitation of our study is that ATN-161, although primarily characterized as an inhibitor of α5β1 integrin, can also bind to and inhibit αvβ3 integrin, a receptor that is present in VeroE6 cells and implicated as a viral co-receptor (23, 35) .

However, while this possible mechanism of action should be investigated in future studies, α5β1

integrin's known association with ACE2, that has not been demonstrated for αvβ3 integrin, makes this possibility less likely.

In summary, we show that SARS-CoV-2 spike protein binds to both α5β1 and J o u r n a l P r e -p r o o f Perspectives Competency in Medical Knowledge: Entry into host cells is one of the most essential functions of a virion, with a large amount of variation in approaches amongst species that are capable of infecting humans. Increasing knowledge of how these entries occur is crucial to understanding and applying future therapeutics targeted against these mechanisms.

Translational Outlook: Utilization of host proteins that are not typically considered targets for therapeutics blocking viral entry can expand the pool of novel antivirals. Future work directed at both in vivo efficacy of ATN-161 as well as other integrin-binding molecules is warranted. In addition, more work to elucidate integrin binding and entry in SARS-CoV-2 would facilitate the increase in potential antivirals. Inhibition of SARS-CoV-2 spike protein binding to human ACE2 by ATN-161. Plates were precoated with monomeric spike protein and incubated with a mixture of hACE2 and various ATN-161 concentrations, followed by detection of bound hACE2 via HRP-conjugated anti-ACE2 antibody. Data was normalized to a no-ATN vehicle control. Data represent mean ± SD, n=3, *

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We thank R Garry, Tulane University School of Medicine, for use of PCR reagents, N Maness at Tulane National Primate Research Center for viral stock, A Mazar, Monopar Therapeutics, for helpful discussions on the preparation and handling of ATN-161 for in vitro studies, and I Rutkai and A Narayanappa for collection of technical information regarding antibodies used in ELISA studies. 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.