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SIM imaging resolves endocytosis of SARS-CoV-2 spike RBD in living cells

Miao et al., Cell Chemical Biology, doi:10.1016/j.chembiol.2023.02.001

Mar 2023

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HCQ for COVID-19

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No treatment is 100% effective. Protocols combine complementary and synergistic treatments. ^* >10% efficacy in meta analysis with ≥3 clinical studies.

4,100+ studies for 60+ treatments. c19hcq.org

In Vitro study showing that the antiviral compounds bafilomycin a1 (BafA1), ammonium chloride (NH4Cl), chloroquine (CQ), hydroxychloroquine (HCQ), and dynasore trap SARS-CoV-2 on the cell surface, preventing viral entry through the endocytic pathway. Using live cell imaging with organic dye probes attached to the host receptor ACE2 and viral spike protein, authors track the viral entry pathway from the cell surface to the late endosome. Treatment with 100 nM BafA1 trapped the virus on the cell surface, with almost no virus detected in late endosomes. Other compounds including HCQ also significantly reduced the endosomal to surface virus ratio, suggesting they block endocytic viral entry.

Antiviral compounds like BafA1, NH4Cl, CQ, and HCQ were thought to trap SARS-CoV-2 in late endosomes through alkalinization mechanisms that block a critical proteolytic step needed for viral entry. However, these results suggest that alkalinization of the endosome may not be the only or primary mechanism. It is possible that both of these mechanims play a role:

- Blocking ACE2 endocytosis: the imaging data shows that compounds like HCQ can trap the virus on the cell surface by preventing the internalization of the ACE2 receptor. This suggests that inhibiting viral entry at an early stage is an important mechanism of action for these drugs.

- Endosomal alkalinization: prior studies have shown that compounds like BafA1, NH4Cl, CQ, and HCQ can raise the pH of endosomes, which is thought to inhibit the proteolytic processing needed for SARS-CoV-2 to fuse with the endosomal membrane and release its contents into the cell. The imaging data suggests this may not be the primary mechanism, however it could still play a role in inhibiting any virus particles that manage to enter endosomes, or in different cells/environments where the previous mechanism is less effective.

Note that authors did not perform dose response analysis and the single dose tested for HCQ/CQ is relatively high. Only a small percentage of patients in Ruiz et al. had ELF concentrations exceeding this dose for 400mg HCQ daily. The new mechanism of action here may require higher concentrations.

31 preclinical studies support the efficacy of HCQ for COVID-19:

8 In Silico studies Alkafaas, Baildya, González-Paz, Hussein, Navya, Noureddine, Tarek, Yadav

2 In Vivo animal studies Shu-Han Lin, Wen

Important missing comments or errors? Let us know.

1. Alkafaas et al., A study on the effect of natural products against the transmission of B.1.1.529 Omicron, Virology Journal, doi:10.1186/s12985-023-02160-6.biomedcentral.com, doi.org.

2. Alsmadi et al., The In Vitro, In Vivo, and PBPK Evaluation of a Novel Lung-Targeted Cardiac-Safe Hydroxychloroquine Inhalation Aerogel, AAPS PharmSciTech, doi:10.1208/s12249-023-02627-3.springer.com, doi.org.

3. Andreani et al., In vitro testing of combined hydroxychloroquine and azithromycin on SARS-CoV-2 shows synergistic effect, Microbial Pathogenesis, doi:/10.1016/j.micpath.2020.104228.sciencedirect.com, doi.org.

4. Baildya et al., Inhibitory capacity of Chloroquine against SARS-COV-2 by effective binding with Angiotensin converting enzyme-2 receptor: An insight from molecular docking and MD-simulation studies, Journal of Molecular Structure, doi:10.1016/j.molstruc.2021.129891.sciencedirect.com, doi.org.

5. Clementi et al., Combined Prophylactic and Therapeutic Use Maximizes Hydroxychloroquine Anti-SARS-CoV-2 Effects in vitro, Front. Microbiol., 10 July 2020, doi:10.3389/fmicb.2020.01704.frontiersin.org, doi.org.

6. Dang et al., Structural basis of anti-SARS-CoV-2 activity of hydroxychloroquine: specific binding to NTD/CTD and disruption of LLPS of N protein, bioRxiv, doi:10.1101/2021.03.16.435741.biorxiv.org, doi.org.

7. Delandre et al., Antiviral Activity of Repurposing Ivermectin against a Panel of 30 Clinical SARS-CoV-2 Strains Belonging to 14 Variants, Pharmaceuticals, doi:10.3390/ph15040445.mdpi.com, doi.org.

8. Faísca et al., Enhanced In Vitro Antiviral Activity of Hydroxychloroquine Ionic Liquids against SARS-CoV-2, Pharmaceutics, doi:10.3390/pharmaceutics14040877.mdpi.com, doi.org.

9. González-Paz et al., Biophysical Analysis of Potential Inhibitors of SARS-CoV-2 Cell Recognition and Their Effect on Viral Dynamics in Different Cell Types: A Computational Prediction from In Vitro Experimental Data, ACS Omega, doi:10.1021/acsomega.3c06968.acs.org, doi.org.

10. Hussein et al., Molecular Docking Identification for the efficacy of Some Zinc Complexes with Chloroquine and Hydroxychloroquine against Main Protease of COVID-19, Journal of Molecular Structure, doi:10.1016/j.molstruc.2021.129979.sciencedirect.com, doi.org.

11. Kamga Kapchoup et al., In vitro effect of hydroxychloroquine on pluripotent stem cells and their cardiomyocytes derivatives, Frontiers in Pharmacology, doi:10.3389/fphar.2023.1128382.frontiersin.org, doi.org.

12. Liu et al., Hydroxychloroquine, a less toxic derivative of chloroquine, is effective in inhibiting SARS-CoV-2 infection in vitro, Cell Discovery 6, 16 (2020), doi:10.1038/s41421-020-0156-0.nature.com, doi.org.

13. Miao et al., SIM imaging resolves endocytosis of SARS-CoV-2 spike RBD in living cells, Cell Chemical Biology, doi:10.1016/j.chembiol.2023.02.001.sciencedirect.com, doi.org.

14. Milan Bonotto et al., Cathepsin inhibitors nitroxoline and its derivatives inhibit SARS-CoV-2 infection, Antiviral Research, doi:10.1016/j.antiviral.2023.105655.sciencedirect.com, doi.org.

15. Mohd Abd Razak et al., In Vitro Anti-SARS-CoV-2 Activities of Curcumin and Selected Phenolic Compounds, Natural Product Communications, doi:10.1177/1934578X231188861.sagepub.com, doi.org.

16. Navya et al., A computational study on hydroxychloroquine binding to target proteins related to SARS-COV-2 infection, Informatics in Medicine Unlocked, doi:10.1016/j.imu.2021.100714.sciencedirect.com, doi.org.

17. Noureddine et al., Quantum chemical studies on molecular structure, AIM, ELF, RDG and antiviral activities of hybrid hydroxychloroquine in the treatment of COVID-19: molecular docking and DFT calculations, Journal of King Saud University - Science, doi:10.1016/j.jksus.2020.101334.sciencedirect.com, doi.org.

18. Ou et al., Hydroxychloroquine-mediated inhibition of SARS-CoV-2 entry is attenuated by TMPRSS2, PLOS Pathogens, doi:10.1371/journal.ppat.1009212.plos.org, doi.org.

19. Purwati et al., An in vitro study of dual drug combinations of anti-viral agents, antibiotics, and/or hydroxychloroquine against the SARS-CoV-2 virus isolated from hospitalized patients in Surabaya, Indonesia, PLOS One, doi:10.1371/journal.pone.0252302.plos.org, doi.org.

20. Ruiz et al., Hydroxychloroquine lung pharmacokinetics in critically ill patients infected with COVID-19, International Journal of Antimicrobial Agents, doi:10.1016/j.ijantimicag.2020.106247.sciencedirect.com, doi.org.

21. Shang et al., Inhibitors of endosomal acidification suppress SARS-CoV-2 replication and relieve viral pneumonia in hACE2 transgenic mice, Virology Journal, doi:10.1186/s12985-021-01515-1.biomedcentral.com, doi.org.

22. Sheaff, R., A New Model of SARS-CoV-2 Infection Based on (Hydroxy)Chloroquine Activity, bioRxiv, doi:10.1101/2020.08.02.232892.biorxiv.org, doi.org.

23. Shu-Han Lin et al., Inhalable Chitosan-Based Hydrogel as a Mucosal Adjuvant for Hydroxychloroquine in the Treatment for SARS-CoV-2 Infection in a Hamster Model, Journal of Microbiology, Immunology and Infection, doi:10.1016/j.jmii.2023.08.001.sciencedirect.com, doi.org.

24. Tarek et al., Pharmacokinetic Basis of the Hydroxychloroquine Response in COVID-19: Implications for Therapy and Prevention, European Journal of Drug Metabolism and Pharmacokinetics, doi:10.1007/s13318-020-00640-6.springer.com, doi.org.

25. Wang et al., Chloroquine and hydroxychloroquine as ACE2 blockers to inhibit viropexis of 2019-nCoV Spike pseudotyped virus, Phytomedicine, doi:10.1016/j.phymed.2020.153333.nih.gov, doi.org.

26. Wang (B) et al., Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro, Cell Res. 30, 269–271, doi:L10.1038/s41422-020-0282-0.nature.com, doi.org.

27. Wen et al., Cholinergic α7 nAChR signaling suppresses SARS-CoV-2 infection and inflammation in lung epithelial cells, Journal of Molecular Cell Biology, doi:10.1093/jmcb/mjad048.oup.com, doi.org.

28. Yadav et al., Repurposing the Combination Drug of Favipiravir, Hydroxychloroquine and Oseltamivir as a Potential Inhibitor Against SARS-CoV-2: A Computational Study, Research Square, doi:10.21203/rs.3.rs-628277/v1.researchsquare.com, doi.org.

29. Yao et al., In Vitro Antiviral Activity and Projection of Optimized Dosing Design of Hydroxychloroquine for the Treatment of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Clin. Infect. Dis., 2020 Mar 9, doi:10.1093/cid/ciaa237.oup.com, doi.org.

30. Yuan et al., Hydroxychloroquine blocks SARS-CoV-2 entry into the endocytic pathway in mammalian cell culture, Communications Biology, doi:10.1038/s42003-022-03841-8.nature.com, doi.org.

Miao et al., 31 Mar 2023, peer-reviewed, 7 authors. Contact: zcxu@dicp.ac.cn (corresponding author), zcxu@dicp.ac.cn (corresponding author).

In Vitro studies are an important part of preclinical research, however results may be very different in vivo.

This Paper HCQ All

SIM imaging resolves endocytosis of SARS-CoV-2 spike RBD in living cells

Lu Miao, Chunyu Yan, Yingzhu Chen, Wei Zhou, Xuelian Zhou, Qinglong Qiao, Zhaochao Xu

Cell Chemical Biology, doi:10.1016/j.chembiol.2023.02.001

Highlights d RBD endocytosis is resolved by imaging the location and ratio of ACE2/RBD fluorescence d Exploring the initiation and influencing factors of RBD internalization d The movement and maturation of ACE2/RBD co-localized vesicles are tracked by SIM imaging

enlightenment on the pathogenic mechanism of COVID-19 and the development of antiviral drugs, but the molecular mechanism remains to be further analyzed. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: AUTHOR CONTRIBUTIONS Concepts were conceived by Z.X. and L.M.; L.M. designed the experiments and analyzed the data; C.Y., X.Z., and L.M. performed live-cell experiments; W.Z. and Q.Q. performed compound synthesis; L.M. and Z.X. wrote the manuscript. DECLARATION OF INTERESTS The authors declare no competing interests. STAR+METHODS KEY RESOURCES

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