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
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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/S Strep K D = 410 nM, LL-37/S His K D = 410 nM) with the same affinity, as SARS-CoV-2 binds to hACE2 (hACE2/S Strep K D = 370 nM, hACE2/S His K D = 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 K D of 290 nM. Further, inhibition of the binding of S Strep (IC 50 = 740 nM), S1e (IC 50 = 170 nM), and RBD (IC 50 = 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.
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SciScore for 10.1101/2020.12.02.408153: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
No key resources detected.
Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar …
SciScore for 10.1101/2020.12.02.408153: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
No key resources detected.
Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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SciScore for 10.1101/2020.12.02.408153: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization Entrenas Castillo, M. et al. “ Effect of calcifediol treatment and best available therapy versus best available therapy on intensive care unit admission and mortality among patients hospitalized for COVID-19: A pilot randomized clinical study”. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Software and Algorithms Sentences Resources Results Recombinant expression of SARS-CoV-2 proteins To study the interaction of LL-37 with SARS-CoV-2 we used recombinantly expressed SARS-CoV-2 SStrep (139 kDa), SHis (136 kDa) SARS-CoV-2…SciScore for 10.1101/2020.12.02.408153: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization Entrenas Castillo, M. et al. “ Effect of calcifediol treatment and best available therapy versus best available therapy on intensive care unit admission and mortality among patients hospitalized for COVID-19: A pilot randomized clinical study”. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Table 2: Resources
Software and Algorithms Sentences Resources Results Recombinant expression of SARS-CoV-2 proteins To study the interaction of LL-37 with SARS-CoV-2 we used recombinantly expressed SARS-CoV-2 SStrep (139 kDa), SHis (136 kDa) SARS-CoV-2suggested: (Active Motif Cat# 91345, RRID:AB_2847847)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.
Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
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