Hydroxychloroquine-mediated inhibition of SARS-CoV-2 entry is attenuated by TMPRSS2

Ou et al., PLOS Pathogens, doi:10.1371/journal.ppat.1009212, Jul 2020 (preprint)

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https://c19hcq.org/ou.html

HCQ for COVID-19

1st treatment shown to reduce risk in March 2020, now with p < 0.00000000001 from 423 studies, used in 59 countries.

Lower risk for mortality, hospitalization, progression, recovery, cases, and viral clearance.

No treatment is 100% effective. Protocols combine treatments.

5,700+ studies for 169 treatments. c19hcq.org

In Vitro analysis showing that HCQ efficiently blocks viral entry mediated by cathepsin L, but not by TMPRSS2, and that a combination of HCQ and a TMPRSS2 inhibitor prevents SARS-CoV-2 infection more potently than either drug alone.

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

11 In Silico studies1-11

24 In Vitro studies1,12-34

3 In Vivo animal studies16,26,35

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1. 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.

2. 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.

3. Guimarães Silva et al., Are Non-Structural Proteins From SARS-CoV-2 the Target of Hydroxychloroquine? An in Silico Study, ACTA MEDICA IRANICA, doi:10.18502/acta.v61i2.12533.kne-publishing.com, doi.org.

4. Nguyen et al., The Potential of Ameliorating COVID-19 and Sequelae From Andrographis paniculata via Bioinformatics, Bioinformatics and Biology Insights, doi:10.1177/11779322221149622.sagepub.com, doi.org.

5. 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.

6. 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.

7. 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.

8. 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.

9. 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.

10. 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.

11. Rowland Yeo et al., Impact of Disease on Plasma and Lung Exposure of Chloroquine, Hydroxychloroquine and Azithromycin: Application of PBPK Modeling, Clinical Pharmacology & Therapeutics, doi:10.1002/cpt.1955.wiley.com, doi.org.

12. Hitti et al., Hydroxychloroquine attenuates double-stranded RNA-stimulated hyper-phosphorylation of tristetraprolin/ZFP36 and AU-rich mRNA stabilization, Immunology, doi:10.1111/imm.13835.wiley.com, doi.org.

13. Yan et al., Super-resolution imaging reveals the mechanism of endosomal acidification inhibitors against SARS-CoV-2 infection, ChemBioChem, doi:10.1002/cbic.202400404.wiley.com, doi.org.

14. 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.

15. 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.

16. 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.

17. 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.

18. 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.

19. 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.

20. 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.

21. 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.

22. 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.

23. 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.

24. Zhang et al., SARS-CoV-2 spike protein dictates syncytium-mediated lymphocyte elimination, Cell Death & Differentiation, doi:10.1038/s41418-021-00782-3.nature.com, doi.org.

25. 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.

26. 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.

27. 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.

28. 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.

29. 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.

30. 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.

31. 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.

32. 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.

33. 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.

34. Wang (B) et al., Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro, Cell Res. 30, 269–271, doi:10.1038/s41422-020-0282-0.nature.com, doi.org.

35. 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.

Ou et al., 22 Jul 2020, peer-reviewed, 6 authors.

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

This Paper HCQ All

Hydroxychloroquine-mediated inhibition of SARS-CoV-2 entry is attenuated by TMPRSS2

Tianling Ou, Huihui Mou, Lizhou Zhang, Amrita Ojha, Hyeryun Choe, Michael Farzan

PLOS Pathogens, doi:10.1371/journal.ppat.1009212

Hydroxychloroquine, used to treat malaria and some autoimmune disorders, potently inhibits viral infection of SARS coronavirus (SARS-CoV-1) and SARS-CoV-2 in cell-culture studies. However, human clinical trials of hydroxychloroquine failed to establish its usefulness as treatment for COVID-19. This compound is known to interfere with endosomal acidification necessary to the proteolytic activity of cathepsins. Following receptor binding and endocytosis, cathepsin L can cleave the SARS-CoV-1 and SARS-CoV-2 spike (S) proteins, thereby activating membrane fusion for cell entry. The plasma membrane-associated protease TMPRSS2 can similarly cleave these S proteins and activate viral entry at the cell surface. Here we show that the SARS-CoV-2 entry process is more dependent than that of SARS-CoV-1 on TMPRSS2 expression. This difference can be reversed when the furincleavage site of the SARS-CoV-2 S protein is ablated or when it is introduced into the SARS-CoV-1 S protein. We also show that hydroxychloroquine efficiently blocks viral entry mediated by cathepsin L, but not by TMPRSS2, and that a combination of hydroxychloroquine and a clinically-tested TMPRSS2 inhibitor prevents SARS-CoV-2 infection more potently than either drug alone. These studies identify functional differences between SARS-CoV-1 and -2 entry processes, and provide a mechanistic explanation for the limited in vivo utility of hydroxychloroquine as a treatment for COVID-19.

media containing 2% FBS. Same infection procedures were applied on other cell lines without the plate coating and transfection steps. Cell surface expression and S protein analysis To measure surface TMPRSS2 expression of 293T-ACE2 transiently transfected with TMPRSS2 and the stable cell line 293T/ACE2/TMPRSS2, cells were detached by 1mM EDTA in PBS and then stained by 2 ug/ml of anti-Flag M2 antibody (Sigma-Aldrich, F1804) and 2 μg/ml of goat anti-mouse IgG (H+L) conjugated with Alexa 647 (Jackson ImmunoResearch Laboratories, #115-606-146). Flow cytometry analysis was done using Accuri C6 (BD Biosciences). To measure the endogenous TMPRSS2 expression of Vero, H1299, H1975 and Calu-3 cells, cells were permeabilized with PBS including 0.5% Triton X-100 (Sigma-Aldrich) at room temperature for 10 min, and detected by 2 μg/ml monoclonal rabbit Anti-TMPRSS2 antibody [EPR3861] (Abcam, ab92323) and goat anti-rabbit IgG conjugated with HRP (Sigma-Aldrich, A0545). To determine the cleavage of S proteins, 293T cells were transfected with 2 μL of lipofectamine 2000 (Life Technologies) in complex with 1 μg plasmid expressing the indicated S protein variant. Cells were harvested for western blot analysis 48 hours post transfection. Cells were permeabilized with PBS including 0.5% Triton X-100 (Sigma-Aldrich) at room temperature for 10 min, and detected by 1 μg/ml anti-Flag M2 antibody (Sigma-Aldrich, F1804) and goat anti-mouse IgG (Fab only) conjugated with HRP (Sigma-Aldrich, A9917). ..

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