Engineering defensin α‐helix to produce high‐affinity SARS‐CoV ‐2 spike protein binding ligands
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (ScreenIT)
Abstract
The binding of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) spike protein to the angiotensin‐converting enzyme 2 (ACE2) receptor expressed on the host cells is a critical initial step for viral infection. This interaction is blocked through competitive inhibition by soluble ACE2 protein. Therefore, developing high‐affinity and cost‐effective ACE2 mimetic ligands that disrupt this protein–protein interaction is a promising strategy for viral diagnostics and therapy. We employed human and plant defensins, a class of small (2–5 kDa) and highly stable proteins containing solvent‐exposed alpha‐helix, conformationally constrained by two disulfide bonds. Therefore, we engineered the amino acid residues on the constrained alpha‐helix of defensins to mimic the critical residues on the ACE2 helix 1 that interact with the SARS‐CoV‐2 spike protein. The engineered proteins (h‐deface2, p‐deface2, and p‐deface2‐MUT) were soluble and purified to homogeneity with a high yield from a bacterial expression system. The proteins demonstrated exceptional thermostability (Tm 70.7°C), high‐affinity binding to the spike protein with apparent K d values of 54.4 ± 11.3, 33.5 ± 8.2, and 14.4 ± 3.5 nM for h‐deface2, p‐deface2, and p‐deface2‐MUT, respectively, and were used in a diagnostic assay that detected SARS‐CoV‐2 neutralizing antibodies. This work addresses the challenge of developing helical ACE2 mimetics by demonstrating that defensins provide promising scaffolds to engineer alpha‐helices in a constrained form for designing of high‐affinity ligands.
Article activity feed
-
-
SciScore for 10.1101/2022.02.09.479781: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources The cells were washed once (500 μL FCM buffer) and resuspended in 100 μL of the FCM buffer with 5μL of the secondary Antibody (PE-anti human IgG Fc Recombinant, Biolegend, Catalogue 366903) and incubated for 30 minutes at 4°C., in the dark. PE-anti human IgGsuggested: NoneExperimental Models: Cell Lines Sentences Resources The parental HEK293 cells were used as a control for binding specificity. HEK293suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)Recombinant DNA Sentences Resources Al… SciScore for 10.1101/2022.02.09.479781: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources The cells were washed once (500 μL FCM buffer) and resuspended in 100 μL of the FCM buffer with 5μL of the secondary Antibody (PE-anti human IgG Fc Recombinant, Biolegend, Catalogue 366903) and incubated for 30 minutes at 4°C., in the dark. PE-anti human IgGsuggested: NoneExperimental Models: Cell Lines Sentences Resources The parental HEK293 cells were used as a control for binding specificity. HEK293suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)Recombinant DNA Sentences Resources All genes were purchased from Biomatik Corporation (Ontario, CA) and cloned into pET-32a (+) between the Nco I and Xho I restriction sites. pET-32asuggested: RRID:Addgene_120288)Software and Algorithms Sentences Resources Biolisa COVID-19 Anticorpo Neutralizante kit (Ref: K243-1) was kindly provided by Bioclin®. Bioclin®suggested: NoneThe cells were then washed twice with 500 μL FCM buffer, and the cell pellets were resuspended back in 500 μL of FCM buffer for analysis in Becton-Dickinson FACSCalibur. Becton-Dickinsonsuggested: NoneFACSCalibursuggested: NoneThe data were analyzed using FlowJo software (BD Biosciences). FlowJosuggested: (FlowJo, RRID:SCR_008520)Results from OddPub: Thank you for sharing your data.
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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
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.
Results from scite Reference Check: We found no unreliable references.
-
