A focus on inhibiting host proteases and targeting the conserved main protease to target SARS-CoV-2 VOCs

Posted on April 28, 2022
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Essay for MICB 308

Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2, causes coronavirus 19, COVID-19. COVID-19 has two phases; the first phase involves virus transmission in the respiratory and gastrointestinal tract1, and the second phase involves collateral tissue damage and systemic failure2. Thus, a broad spectrum therapeutic is needed to prevent the second phase of COVID-19.

An approach would be to use a multidrug regimen, which combines multiple therapeutics that target different parts of the viral life cycle. A multidrug regimen is advantageous over using a single drug because “drug-resistant mutants emerge soon after single-agent antiviral therapy”3.

I hypothesize a multidrug regimen consisting of two IAAS, N-0385, decanoyl-RVKR-chloromethylketone (CMK), as well as a DAA, Glecaprevir, will be a successful multidrug regimen for treatment of SARS-CoV-2 Delta infections. Since “targeting of host molecules minimizes the issue of drug resistance”4, N-0385 and CMK were chosen. Additionally, Glecaprevir binds to conserved residues in the main protease5.

The first drug is N-0385. N-0385 is an IAA, inhibiting TMPRSS2-like proteases with its ketobenzothiazole warhead which forms a covalent bond with the TMPRSS-2 catalytic triad Ser144, His296, Asp3456. TMPRSS-2 is an important host factor that is used by SARS-CoV-2 to cleave and prime the S protein, facilitating viral entry and the fusion event7. N-0385 also has low nanomolar potency, is broad-spectrum, and single daily intranasal use8, meaning less can be used to treat infection, and it can be easily administered.

The second drug is CMK, an IAA which targets furin9. Furin cleaves the S protein for fusogenicity and synthia formation, the latter causing systemic spread. CMK “suppresses cleavage of spikes and the syncytium”10; immunoblot analysis showed a “dramatic decrease in the levels of processed S protein fragments in VeroE6 cells” which contained wild type S protein, and microscopic observation showed “significant inhibition of syncytium formation”11. Treatment with CMK one hour before infection significantly decreased “virus titers of progeny viruses” after 24 and 48 hours12. CMK is effective at a dosage of 5μM and has a 50% cytotoxic concentration of 318.2 μM13.

The third drug is Glecaprevir, which targets conserved residues in the binding pocket of SARS-CoV-2 Mpro14. The “substrate-binding pocket of SARS-CoV-2 Mpro which is highly conserved among all the structures of SARS-CoV-2 Mpro”15, makes it an attractive target. Additionally, it plays an important role in replication and proteolytic processing. Glecaprevir binds in-between domain I and domain II in the substrate-binding pocket of Mpro with -9.6 affinity, forming three hydrogen bonds among Thr25, Cys145, Gln189, and two fluorine interactions with Thr26, Gly14316. Thus, Glecaprevir hinders substrate accessibility, inhibiting SARS-CoV-2 Mpro.

There will need to be more research done to determine proper scheduling of use; how late after infection are the drugs still effective, and in what order or schedule results in maximal inhibition of infection. Additionally, we would need to manipulate the drugs into forms that are easily administered. N-0385 can be administered as a spray, but CMK and Glecaprevir administration were not discussed. TMPRSS-2 is not a cell essential gene17, so inhibiting it would probably cause “negligible side effects”18. Similarly, since furin is a “ubiquitously expressed endoprotease”19, the same could be predicted, but more research needs to be done.

  1. Trougakos, I.P., Stamatelopoulos, K., Terpos, E. et al. Insights to SARS-CoV-2 life cycle, pathophysiology, and rationalized treatments that target COVID-19 clinical complications. J Biomed Sci 28, 9 (2021). https://doi.org/10.1186/s12929-020-00703-5↩︎

  2. Trougakos, I.P., Stamatelopoulos, K., Terpos, E. et al. Insights to SARS-CoV-2 life cycle, pathophysiology, and rationalized treatments that target COVID-19 clinical complications. J Biomed Sci 28, 9 (2021). https://doi.org/10.1186/s12929-020-00703-5↩︎

  3. Cheng YW, Chao TL, Li CL, Chiu MF, Kao HC, Wang SH, Pang YH, Lin CH, Tsai YM, Lee WH, Tao MH, Ho TC, Wu PY, Jang LT, Chen PJ, Chang SY, Yeh SH. Furin Inhibitors Block SARS-CoV-2 Spike Protein Cleavage to Suppress Virus Production and Cytopathic Effects. Cell Rep. 2020 Oct 13;33(2):108254. doi: 10.1016/j.celrep.2020.108254. Epub 2020 Sep 23. PMID: 33007239; PMCID: PMC7510585.↩︎

  4. Cheng YW, Chao TL, Li CL, Chiu MF, Kao HC, Wang SH, Pang YH, Lin CH, Tsai YM, Lee WH, Tao MH, Ho TC, Wu PY, Jang LT, Chen PJ, Chang SY, Yeh SH. Furin Inhibitors Block SARS-CoV-2 Spike Protein Cleavage to Suppress Virus Production and Cytopathic Effects. Cell Rep. 2020 Oct 13;33(2):108254. doi: 10.1016/j.celrep.2020.108254. Epub 2020 Sep 23. PMID: 33007239; PMCID: PMC7510585.↩︎

  5. Anas Shamsi, Taj Mohammad, Saleha Anwar, Mohamed F. AlAjmi, Afzal Hussain, Md. Tabish Rehman, et al. Glecaprevir and Maraviroc are high-affinity inhibitors of SARS-CoV-2 main protease: possible implication in COVID-19 therapy. Biosci Rep. 2020 26 June 2020;40(6): BSR20201256. doi: https://doi.org/10.1042/BSR20201256↩︎

  6. Tirosh Shapira, I. Abrrey Monreal, Sébastien P. Dion, Mason Jager, Antoine Désilets, Andrea D. Olmstead, Thierry Vandal, David W. Buchholz, Brian Imbiakha, Guang Gao, Aaleigha Chin, William D. Rees, Theodore Steiner, Ivan Robert Nabi, Eric Marsault, Julie Sahler, Avery August, Gerlinde Van de Walle, Gary R. Whittaker, Pierre-Luc Boudreault, Hector C. Aguilar, Richard Leduc, François Jean. A novel highly potent inhibitor of TMPRSS2-like proteases blocks SARS-CoV-2 variants of concern and is broadly protective against infection and mortality in mice. BioRxiv [Preprint]. 2021 [cited 2021 Dec 12]. Available from: https://www.biorxiv.org/content/10.1101/2021.05.03.442520v1↩︎

  7. Tirosh Shapira, I. Abrrey Monreal, Sébastien P. Dion, Mason Jager, Antoine Désilets, Andrea D. Olmstead, Thierry Vandal, David W. Buchholz, Brian Imbiakha, Guang Gao, Aaleigha Chin, William D. Rees, Theodore Steiner, Ivan Robert Nabi, Eric Marsault, Julie Sahler, Avery August, Gerlinde Van de Walle, Gary R. Whittaker, Pierre-Luc Boudreault, Hector C. Aguilar, Richard Leduc, François Jean. A novel highly potent inhibitor of TMPRSS2-like proteases blocks SARS-CoV-2 variants of concern and is broadly protective against infection and mortality in mice. BioRxiv [Preprint]. 2021 [cited 2021 Dec 12]. Available from: https://www.biorxiv.org/content/10.1101/2021.05.03.442520v1↩︎

  8. Tirosh Shapira, I. Abrrey Monreal, Sébastien P. Dion, Mason Jager, Antoine Désilets, Andrea D. Olmstead, Thierry Vandal, David W. Buchholz, Brian Imbiakha, Guang Gao, Aaleigha Chin, William D. Rees, Theodore Steiner, Ivan Robert Nabi, Eric Marsault, Julie Sahler, Avery August, Gerlinde Van de Walle, Gary R. Whittaker, Pierre-Luc Boudreault, Hector C. Aguilar, Richard Leduc, François Jean. A novel highly potent inhibitor of TMPRSS2-like proteases blocks SARS-CoV-2 variants of concern and is broadly protective against infection and mortality in mice. BioRxiv [Preprint]. 2021 [cited 2021 Dec 12]. Available from: https://www.biorxiv.org/content/10.1101/2021.05.03.442520v1↩︎

  9. Cheng YW, Chao TL, Li CL, Chiu MF, Kao HC, Wang SH, Pang YH, Lin CH, Tsai YM, Lee WH, Tao MH, Ho TC, Wu PY, Jang LT, Chen PJ, Chang SY, Yeh SH. Furin Inhibitors Block SARS-CoV-2 Spike Protein Cleavage to Suppress Virus Production and Cytopathic Effects. Cell Rep. 2020 Oct 13;33(2):108254. doi: 10.1016/j.celrep.2020.108254. Epub 2020 Sep 23. PMID: 33007239; PMCID: PMC7510585.↩︎

  10. Cheng YW, Chao TL, Li CL, Chiu MF, Kao HC, Wang SH, Pang YH, Lin CH, Tsai YM, Lee WH, Tao MH, Ho TC, Wu PY, Jang LT, Chen PJ, Chang SY, Yeh SH. Furin Inhibitors Block SARS-CoV-2 Spike Protein Cleavage to Suppress Virus Production and Cytopathic Effects. Cell Rep. 2020 Oct 13;33(2):108254. doi: 10.1016/j.celrep.2020.108254. Epub 2020 Sep 23. PMID: 33007239; PMCID: PMC7510585.↩︎

  11. Cheng YW, Chao TL, Li CL, Chiu MF, Kao HC, Wang SH, Pang YH, Lin CH, Tsai YM, Lee WH, Tao MH, Ho TC, Wu PY, Jang LT, Chen PJ, Chang SY, Yeh SH. Furin Inhibitors Block SARS-CoV-2 Spike Protein Cleavage to Suppress Virus Production and Cytopathic Effects. Cell Rep. 2020 Oct 13;33(2):108254. doi: 10.1016/j.celrep.2020.108254. Epub 2020 Sep 23. PMID: 33007239; PMCID: PMC7510585.↩︎

  12. Cheng YW, Chao TL, Li CL, Chiu MF, Kao HC, Wang SH, Pang YH, Lin CH, Tsai YM, Lee WH, Tao MH, Ho TC, Wu PY, Jang LT, Chen PJ, Chang SY, Yeh SH. Furin Inhibitors Block SARS-CoV-2 Spike Protein Cleavage to Suppress Virus Production and Cytopathic Effects. Cell Rep. 2020 Oct 13;33(2):108254. doi: 10.1016/j.celrep.2020.108254. Epub 2020 Sep 23. PMID: 33007239; PMCID: PMC7510585.↩︎

  13. Cheng YW, Chao TL, Li CL, Chiu MF, Kao HC, Wang SH, Pang YH, Lin CH, Tsai YM, Lee WH, Tao MH, Ho TC, Wu PY, Jang LT, Chen PJ, Chang SY, Yeh SH. Furin Inhibitors Block SARS-CoV-2 Spike Protein Cleavage to Suppress Virus Production and Cytopathic Effects. Cell Rep. 2020 Oct 13;33(2):108254. doi: 10.1016/j.celrep.2020.108254. Epub 2020 Sep 23. PMID: 33007239; PMCID: PMC7510585.↩︎

  14. Anas Shamsi, Taj Mohammad, Saleha Anwar, Mohamed F. AlAjmi, Afzal Hussain, Md. Tabish Rehman, et al. Glecaprevir and Maraviroc are high-affinity inhibitors of SARS-CoV-2 main protease: possible implication in COVID-19 therapy. Biosci Rep. 2020 26 June 2020;40(6): BSR20201256. doi: https://doi.org/10.1042/BSR20201256↩︎

  15. Anas Shamsi, Taj Mohammad, Saleha Anwar, Mohamed F. AlAjmi, Afzal Hussain, Md. Tabish Rehman, et al. Glecaprevir and Maraviroc are high-affinity inhibitors of SARS-CoV-2 main protease: possible implication in COVID-19 therapy. Biosci Rep. 2020 26 June 2020;40(6): BSR20201256. doi: https://doi.org/10.1042/BSR20201256↩︎

  16. Anas Shamsi, Taj Mohammad, Saleha Anwar, Mohamed F. AlAjmi, Afzal Hussain, Md. Tabish Rehman, et al. Glecaprevir and Maraviroc are high-affinity inhibitors of SARS-CoV-2 main protease: possible implication in COVID-19 therapy. Biosci Rep. 2020 26 June 2020;40(6): BSR20201256. doi: https://doi.org/10.1042/BSR20201256↩︎

  17. Baggen, J., Vanstreels, E., Jansen, S. et al. Cellular host factors for SARS-CoV-2 infection. Nat Microbiol, 2021;6(1219–123). doi: https://doi.org/10.1038/s41564-021-00958-0↩︎

  18. Baggen, J., Vanstreels, E., Jansen, S. et al. Cellular host factors for SARS-CoV-2 infection. Nat Microbiol, 2021;6(1219–123). doi: https://doi.org/10.1038/s41564-021-00958-0↩︎

  19. Baggen, J., Vanstreels, E., Jansen, S. et al. Cellular host factors for SARS-CoV-2 infection. Nat Microbiol, 2021;6(1219–123). doi: https://doi.org/10.1038/s41564-021-00958-0↩︎

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