In-vivo Protection from SARS-CoV-2 infection by ATN-161 in k18-hACE2 transgenic mice

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an infectious disease that has spread worldwide. Current treatments are limited in both availability and efficacy, such that improving our understanding of the factors that facilitate infection is urgently needed to more effectively treat infected individuals and to curb the pandemic. We and others have previously demonstrated the significance of interactions between the SARS-CoV-2 spike protein, integrin alpha5beta1 and human ACE2 to facilitate viral entry into host cells in vitro. We previously found that inhibition of integrin alpha5beta1 by the clinically validated small peptide ATN-161 inhibits these spike protein interactions and cell infection in vitro. In continuation with our previous findings, here we have further evaluated the therapeutic potential of ATN-161 on SARS-CoV-2 infection in k18-hACE2 transgenic (SARS-CoV-2 susceptible) mice in vivo. We discovered that treatment with single- or repeated intravenous doses of ATN-161 (1 mg/kg) within 48 hours after intranasal inoculation with SARS-CoV-2 lead to a reduction of lung viral load, viral immunofluorescence and improved lung histology in a majority of mice 72 hours post-infection. Furthermore, ATN-161 reduced SARS-CoV-2-induced increased expression of lung integrin alpha 5 and alpha v (an alpha 5-related integrin that has also been implicated in SARS-CoV-2 interactions) as well as the C-X-C motif chemokine ligand 10 (Cxcl10), further supporting the potential involvement of these integrins, and the anti-inflammatory potential of ATN-161, respectively, in SARS-CoV-2 infection. To the best of our knowledge, this is the first study demonstrating the potential therapeutic efficacy of targeting integrin alpha5beta1 in SARS-CoV-2 infection in vivo and supports the development of ATN-161 as a novel SARS-CoV-2 therapy.

The spike protein of SARS-CoV-2 induces endothelial inflammation through integrin α5β1 and NF-κB

“…Inhibitors of integrin α5β1 have been developed as promising therapeutics. For example, volociximab, a chimeric anti-integrin α5β1 monoclonal antibody, is under clinical evaluation for the treatment of cancer 27 ; and preclinical studies have evaluated the efficacy of the integrin α5β1 binding peptide, ATN-161, to inhibit beta-coronavirus 28 and SARS-CoV-2 virus infections 11,15 . Here, we show that both volociximab and ATN-161 block the binding of spike and the spike receptor-binding domain to α5β1immovilized in ELISA plates (Figure 5a); and prevent the leukocyte adhesion to HUVEC (Figure 5b) and HUVEC hyperpermeability (Figure 5c) in response to spike, spike receptor-binding domain, and RGD tripeptide.…”

“…ACE2 is the best-established host receptor for spike [31][32][33] , although other spike cell surface receptors have been described, i.e., neuropilin-1 34 , toll-like receptos 9,35 , and RGD-binding integrins 11,14 . In particular, integrin a5b1 is an RGD-binding integrin that, upon spike binding, mediates SARS-CoV-2 entry and infection of epithelial cells and monocytes in vitro 11 and increases lung viral load and inflammation in vivo 15 . Ligation of integrin a5b1 by fibronectin RGD motif activates the expression of proinflammatory genes in EC 16 , but the binding of spike to a5b1 in EC and its impact on the EC inflammatory response has not been addressed.…”

“…Finally, we showed that two inhibitors of α5β1, volociximab and ATN-161, developed as promising therapeutics, blocked spike-induced leukocyte adhesion and hyperpermeability of EC. Indeed, ATN-161 has already been shown to inhibit SARS-CoV-2 virus infection in vivo 15 . Our findings, add EC as a target for these drugs to reduce vascular inflammation in COVID-19, suggest the therapeutic potential of agents commonly used to treat vascular diseases, and provide tools for guiding research into the molecular mechanisms mediating the pathophysiology of COVID-19.…”

“…The spike protein contains an integrin-binding RGD motif exposed on the surface of the receptorbinding domain 12,13 that binds to β1 integrins on pulmonary epithelial cells and monocytes 14 . In particular, blockage of the binding of the spike protein to the integrin ⍺5β1 inhibits SARS-CoV-2 infection in vitro 11 and in vivo 15 , demonstrating the therapeutic efficacy of targeting integrin ⍺5β1 in COVID-19. Furthermore, fibronectin ligation of integrin ⍺5β1 in EC activates the transcriptional factor NF-κB responsible for the expression of proteins involved in inflammation and angiogenesis 16 .…”

Receptor binding domain of SARS-CoV-2 is a functional αv-integrin agonist

“…Positive detection of S1-integrin binding via solid-phase ELISA assays has been reported by several independent groups for both α 5 β 1 (35,36) and α v β 3 (37) integrins. Viral infection studies further showed that cell-surface binding and viral uptake of SARS-CoV-2 can be inhibited by integrin antagonists, including the peptide inhibitors Cilengitide (37) and ATN-161 (35,38), as well as by cell-permeable inhibitors of inside-out integrin signaling (39). Thus, converging evidence suggests that S1-integrin interactions occur during SARS-CoV-2 infection, though the specificity and selectivity for specific integrins, as well as the implications for SARS-CoV-2 infection and disease remain to be elucidated.…”