In-Vitro Fluorescence Microscopy Studies Show Retention of Spike-Protein (SARS-Cov-2) on Cell Membrane in the Presence of Amodiaquin Dihydrochloride Dihydrate Drug
This article has been Reviewed by the following groups
Listed in
- Evaluated articles (ScreenIT)
Abstract
The ability of S-glycoprotein (S-protein) in SARS-Cov-2 to bind to the host cell receptor protein (angiotensin-converting enzyme 2 (ACE2)) leading to its entry in the cellular system determines its contagious index and global spread. Three available drugs (Riboflavin, Amodiaquin dihydrochloride dihydrate (ADD), and Remidesivir) were investigated to understand the kinetics of S-protein and its entry inside a cellular environment. Optical microscopy and fluorescence-based assays on 293T cells (transfected with ACE2 plasmid) were used as the preamble for assessing the behavior of S-protein in the presence of these drugs for the first 12 hours post-S-protein - ACE2 binding. Preliminary results suggest relatively long retention of S-protein on the cell membrane in the presence of ADD drug. Evident from the %-overlap and colocalization of S-protein with endosome studies, a significant fraction of S-protein entering the cell escape endosomal degradation process, suggesting S-protein takes non-endocytic mediated entry in the presence of ADD. In contrast, in the presence of Riboflavin, S-protein carries out a normal endocytic pathway, comparable to the control (no drug) group. Therefore, the present study indicates ADD potentially affects S-protein’s entry mechanism (endocytic pathway) in addition to its reported target action mechanism. Hence, ADD substantially interferes with S-protein cellular entrance mechanism. This is further strengthened by 24 hrs study. However, detailed studies at the molecular scale are necessary to clarify our understanding of exact intermediate molecular processes. The present study (based on limited data) reveals ADD could be a potential candidate to manage Covid-19 functions through the yet unknown molecular mechanism.
Article activity feed
-
-
SciScore for 10.1101/2021.01.05.424956: (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. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources The cells were then incubated with the primary mouse anti-LAMP1 antibody (Sigma-Aldrich), at a 50 nM concentration in blocking for 30 min at room temperature. anti-LAMP1suggested: NoneAfter washing-off primary antibody treatment, the cells were treated with the secondary antibody, Alexa Fluor 594 (emission at 620 nm) tagged donkey anti-mouse IgG antibody (Thermofisher) at 50 nM concentration in blocking buffer for 30 min at room temperature. anti-mouse IgGsu…SciScore for 10.1101/2021.01.05.424956: (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. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources The cells were then incubated with the primary mouse anti-LAMP1 antibody (Sigma-Aldrich), at a 50 nM concentration in blocking for 30 min at room temperature. anti-LAMP1suggested: NoneAfter washing-off primary antibody treatment, the cells were treated with the secondary antibody, Alexa Fluor 594 (emission at 620 nm) tagged donkey anti-mouse IgG antibody (Thermofisher) at 50 nM concentration in blocking buffer for 30 min at room temperature. anti-mouse IgGsuggested: NoneExperimental Models: Cell Lines Sentences Resources Since, HEK293 cells do not express any appreciable level of ACE2 and thus are not susceptible to SARS-CoV-2 infection. HEK293suggested: CLS Cat# 300192/p777_HEK293, RRID:CVCL_0045)S-Protein Extraction using 293T cells as Host: Healthy 293 cells were cultured for extracting S-protein. 293suggested: NCI-DTP Cat# NCI-293TT, RRID:CVCL_1D85)S-protein binding and drug treatment method: For the S-protein binding study, the ACE2 transfected HEK293T (293T) cells (Figure 5) were washed and incubated with expression medium containing GFP-S (i.e., GFP expressing S-protein) for 4 and 8 hrs at 37 degrees and 5% CO2. HEK293Tsuggested: None293Tsuggested: NoneResults 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: Please consider improving the rainbow (“jet”) colormap(s) used on page 5. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- No funding statement was detected.
- No protocol registration statement was detected.
-