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LL-37 fights SARS-CoV-2: The Vitamin D-Inducible Peptide LL-37 Inhibits Binding of SARS-CoV-2 Spike Protein to its Cellular Receptor Angiotensin Converting Enzyme 2 In Vitro

Annika Roth, Steffen Lütke, Denise Meinberger, Gabriele Hermes, Gerhard Sengle, Manuel Koch, Thomas Streichert, Andreas R. Klatt
doi: https://doi.org/10.1101/2020.12.02.408153
Annika Roth
1Institute for Clinical Chemistry, Medical Faculty, University of Cologne, 50937 Cologne, Germany
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Steffen Lütke
2Institute for Biochemistry II, Medical Faculty, University of Cologne, 50931 Cologne, Germany
3Clinic for Pediatric and Adolescent Medicine, Medical Faculty, University of Cologne, 50937 Cologne, Germany
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Denise Meinberger
1Institute for Clinical Chemistry, Medical Faculty, University of Cologne, 50937 Cologne, Germany
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Gabriele Hermes
1Institute for Clinical Chemistry, Medical Faculty, University of Cologne, 50937 Cologne, Germany
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Gerhard Sengle
2Institute for Biochemistry II, Medical Faculty, University of Cologne, 50931 Cologne, Germany
3Clinic for Pediatric and Adolescent Medicine, Medical Faculty, University of Cologne, 50937 Cologne, Germany
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Manuel Koch
2Institute for Biochemistry II, Medical Faculty, University of Cologne, 50931 Cologne, Germany
4Institute for Dental Research and Oral Musculoskeletal Biology, Medical Faculty, University of Cologne, 50931 Cologne, Germany
5Center for Molecular Medicine Cologne, University of Cologne, 50931 Cologne, Germany
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Thomas Streichert
1Institute for Clinical Chemistry, Medical Faculty, University of Cologne, 50937 Cologne, Germany
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Andreas R. Klatt
1Institute for Clinical Chemistry, Medical Faculty, University of Cologne, 50937 Cologne, Germany
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  • For correspondence: andreas.klatt@uk-koeln.de
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Abstract

Objective Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the pathogen accountable for the coronavirus disease 2019 (COVID-19) pandemic. Viral entry via binding of the receptor binding domain (RBD) located within the S1 subunit of the SARS-CoV-2 Spike (S) protein to its target receptor angiotensin converting enzyme (ACE) 2 is a key step in cell infection. The efficient transition of the virus is linked to a unique protein called open reading frame (ORF) 8. As SARS-CoV-2 infections can develop into life-threatening lower respiratory syndromes, effective therapy options are urgently needed. Several publications propose vitamin D treatment, although its mode of action against COVID-19 is not fully elucidated. It is speculated that vitamin D’s beneficial effects are mediated by up-regulating LL-37, a well-known antimicrobial peptide with antiviral effects.

Methods Recombinantly expressed SARS-CoV-2 S protein, the extended S1 subunit (S1e), the S2 subunit (S2), the receptor binding domain (RBD), and ORF8 were used for surface plasmon resonance (SPR) studies to investigate LL-37’s ability to bind to SARS-CoV-2 proteins and to localize its binding site within the S protein. Binding competition studies were conducted to confirm an inhibitory action of LL-37 on the attachment of SARS-CoV-2 S protein to its entry receptor ACE2.

Results We could show that LL-37 binds to SARS-CoV-2 S protein (LL-37/SStrep KD = 410 nM, LL-37/SHis KD = 410 nM) with the same affinity, as SARS-CoV-2 binds to hACE2 (hACE2/SStrep KD = 370 nM, hACE2/SHis KD = 370 nM). The binding is not restricted to the RBD of the S protein, but rather distributed along the entire length of the protein. Interaction between LL-37 and ORF8 was detected with a KD of 290 nM. Further, inhibition of the binding of SStrep (IC50 = 740 nM), S1e (IC50 = 170 nM), and RBD (IC50 = 130 nM) to hACE2 by LL-37 was demonstrated.

Conclusions We have revealed a biochemical link between vitamin D, LL-37, and COVID-19 severity. SPR analysis demonstrated that LL-37 binds to SARS-CoV-2 S protein and inhibits binding to its receptor hACE2, and most likely viral entry into the cell. This study supports the prophylactic use of vitamin D to induce LL-37 that protects from SARS-CoV-2 infection, and the therapeutic administration of vitamin D for the treatment of COVID-19 patients. Further, our results provide evidence that the direct use of LL-37 by inhalation and systemic application may reduce the severity of COVID-19.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • ↵# These authors share the first authorship

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted December 04, 2020.
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LL-37 fights SARS-CoV-2: The Vitamin D-Inducible Peptide LL-37 Inhibits Binding of SARS-CoV-2 Spike Protein to its Cellular Receptor Angiotensin Converting Enzyme 2 In Vitro
Annika Roth, Steffen Lütke, Denise Meinberger, Gabriele Hermes, Gerhard Sengle, Manuel Koch, Thomas Streichert, Andreas R. Klatt
bioRxiv 2020.12.02.408153; doi: https://doi.org/10.1101/2020.12.02.408153
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LL-37 fights SARS-CoV-2: The Vitamin D-Inducible Peptide LL-37 Inhibits Binding of SARS-CoV-2 Spike Protein to its Cellular Receptor Angiotensin Converting Enzyme 2 In Vitro
Annika Roth, Steffen Lütke, Denise Meinberger, Gabriele Hermes, Gerhard Sengle, Manuel Koch, Thomas Streichert, Andreas R. Klatt
bioRxiv 2020.12.02.408153; doi: https://doi.org/10.1101/2020.12.02.408153

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