Alpha 1 Antitrypsin is an Inhibitor of the SARS-CoV-2–Priming Protease TMPRSS2

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Nurit P Azouz
Andrea M. Klingler
Victoria Callahan
Ivan V. Akhrymuk
Katarina Elez
Lluís Raich
Brandon M. Henry
Justin L. Benoit
Stefanie W. Benoit
Frank Noé
Kylene Kehn-Hall
Marc E. Rothenberg

Abstract

Background: Host proteases have been suggested to be crucial for dissemination of MERS, SARS-CoV, and SARS-CoV-2 coronaviruses, but the relative contribution of membrane versus intracellular proteases remains controversial. Transmembrane serine protease 2 (TMPRSS2) is regarded as one of the main proteases implicated in the coronavirus S protein priming, an important step for binding of the S protein to the angiotensin-converting enzyme 2 (ACE2) receptor before cell entry. 


Methods: We developed a cell-based assay to identify TMPRSS2 inhibitors. Inhibitory activity was established in SARS-CoV-2 viral load systems.


Results: We identified the human extracellular serine protease inhibitor (serpin) alpha 1 antitrypsin (A1AT) as a novel TMPRSS2 inhibitor. Structural modeling revealed that A1AT docked to an extracellular domain of TMPRSS2 in a conformation that is suitable for catalysis, resembling similar serine protease inhibitor complexes. Inhibitory activity of A1AT was established in a SARS-CoV-2 viral load system. Notably, plasma A1AT levels were associated with COVID-19 disease severity. 


Conclusions: Our data support the key role of extracellular serine proteases in SARS CoV-2 infections and indicate that treatment with serpins, particularly the FDA-approved drug A1AT, may be effective in limiting SARS-CoV-2 dissemination by affecting the surface of the host cells.

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References

Hoffmann M, Kleine-Weber H, Schroeder S, Kruger N, Herrler T, Erichsen S, Schiergens TS, Herrler G, Wu NH, Nitsche A, Muller MA, Drosten C, Pohlmann S. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell. 2020;181(2):271-80 e8. doi: 10.1016/j.cell.2020.02.052. PubMed PMID: 32142651; PMCID: PMC7102627.

Qing E, Hantak MP, Galpalli GG, Gallagher T. Evaluating MERS-CoV entry pathways. Methods Mol Biol. 2020;2099:9-20. doi: 10.1007/978-1-0716-0211-9_2. PubMed PMID: 31883084; PMCID: PMC7121971.

Heurich A, Hofmann-Winkler H, Gierer S, Liepold T, Jahn O, Pohlmann S. TMPRSS2 and ADAM17 cleave ACE2 differentially and only proteolysis by TMPRSS2 augments entry driven by the severe acute respiratory syndrome coronavirus spike protein. J Virol. 2014;88(2):1293-307. doi: 10.1128/JVI.02202-13. PubMed PMID: 24227843; PMCID: PMC3911672.

Iwata-Yoshikawa N, Okamura T, Shimizu Y, Hasegawa H, Takeda M, Nagata N. TMPRSS2 Contributes to virus spread and immunopathology in the airways of murine models after coronavirus infection. J Virol. 2019;93(6). doi: 10.1128/JVI.01815-18. PubMed PMID: 30626688; PMCID: PMC6401451.

Matsuyama S, Nao N, Shirato K, Kawase M, Saito S, Takayama I, Nagata N, Sekizuka T, Katoh H, Kato F, Sakata M, Tahara M, Kutsuna S, Ohmagari N, Kuroda M, Suzuki T, Kageyama T, Takeda M. Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells. Proc Natl Acad Sci U S A. 2020;117(13):7001-3. doi: 10.1073/pnas.2002589117. PubMed PMID: 32165541; PMCID: PMC7132130.

Reihill JA, Walker B, Hamilton RA, Ferguson TE, Elborn JS, Stutts MJ, Harvey BJ, Saint-Criq V, Hendrick SM, Martin SL. Inhibition of protease-epithelial sodium channel signaling improves mucociliary function in cystic fibrosis airways. Am J Respir Crit Care Med. 2016;194(6):701-10. doi: 10.1164/rccm.201511-2216OC. PubMed PMID: 27014936.

Strnad P, McElvaney NG, Lomas DA. Alpha1-antitrypsin deficiency. N Engl J Med. 2020;382(15):1443-55. doi: 10.1056/NEJMra1910234. PubMed PMID: 32268028.

Baer A, Kehn-Hall K. Viral concentration determination through plaque assays: Using traditional and novel overlay systems. J Vis Exp. 2014(93):e52065. doi: 10.3791/52065. PubMed PMID: 25407402; PMCID: PMC4255882.

Dominguez C, Boelens R, Bonvin AM. HADDOCK: A protein-protein docking approach based on biochemical or biophysical information. J Am Chem Soc. 2003;125(7):1731-7. doi: 10.1021/ja026939x. PubMed PMID: 12580598.

Dementiev A, Simonovic M, Volz K, Gettins PG. Canonical inhibitor-like interactions explain reactivity of alpha1-proteinase inhibitor pittsburgh and antithrombin with proteinases. J Biol Chem. 2003;278(39):37881-7. doi: 10.1074/jbc.M305195200. PubMed PMID: 12860985.

Rensi S, Keys A, Y-C, Derry A, McInnes G, Liu T, Altman R. Homology modeling of TMPRSS2 yields candidate drugs that may inhibit entry of SARS-CoV-2 into human cells. ChemRxiv. 2020. doi: 10.26434/chemrxiv.12009582.v1. PMID: 32511288.

Xue LC, Dobbs D, Honavar V. HomPPI: A class of sequence homology based protein-protein interface prediction methods. BMC Bioinformatics. 2011;12:244. doi: 10.1186/1471-2105-12-244. PubMed PMID: 21682895; PMCID: PMC3213298.

Tim Hempel LR, Simon Olsson, Nurit P. Azouz, Andrea M. Klingler, Marc E. Rothenberg, Frank Noé. Molecular mechanism of SARS-CoV-2 cell entry inhibition via TMPRSS2 by camostat and nafamostat mesylate. bioRxiv2020.

Guttman O, Baranovski BM, Schuster R, Kaner Z, Freixo-Lima GS, Bahar N, Kalay N, Mizrahi MI, Brami I, Ochayon DE, Lewis EC. Acute-phase protein alpha1-anti-trypsin: Diverting injurious innate and adaptive immune responses from non-authentic threats. Clin Exp Immunol. 2015;179(2):161-72. doi: 10.1111/cei.12476. PubMed PMID: 25351931; PMCID: PMC4298394.

McElvaney OJ, McEvoy NL, McElvaney OF, Carroll TP, Murphy MP, Dunlea DM, Ni Choileain O, Clarke J, O’Connor E, Hogan G, Ryan D, Sulaiman I, Gunaratnam C, Branagan P, O’Brien ME, Morgan RK, Costello RW, Hurley K, Walsh S, de Barra E, McNally C, McConkey S, Boland F, Galvin S, Kiernan F, O’Rourke J, Dwyer R, Power M, Geoghegan P, Larkin C, O’Leary RA, Freeman J, Gaffney A, Marsh B, Curley GF, McElvaney NG. Characterization of the inflammatory response to severe COVID-19 illness. Am J Respir Crit Care Med. 2020;202(6):812-21. doi: 10.1164/rccm.202005-1583OC. PubMed PMID: 32584597; PMCID: PMC7491404.

Chu H, Chan JF, Yuen TT, Shuai H, Yuan S, Wang Y, Hu B, Yip CC, Tsang JO, Huang X, Chai Y, Yang D, Hou Y, Chik KK, Zhang X, Fung AY, Tsoi HW, Cai JP, Chan WM, Ip JD, Chu AW, Zhou J, Lung DC, Kok KH, To KK, Tsang OT, Chan KH, Yuen KY. Comparative tropism, replication kinetics, and cell damage profiling of SARS-CoV-2 and SARS-CoV with implications for clinical manifestations, transmissibility, and laboratory studies of COVID-19: An observational study. Lancet Microbe. 2020;1(1):e14-e23. doi: 10.1016/S2666-5247(20)30004-5. PubMed PMID: 32835326; PMCID: PMC7173822.

Oguntuyo KY, Stevens CS, Siddiquey MN, Schilke RM, Woolard MD, Zhang H, Acklin JA, Ikegame S, Hung CT, Lim JK, Cross RW, Geisbert TW, Ivanov SS, Kamil JP, Lee B. In plain sight: The role of alpha-1-antitrypsin in COVID-19 pathogenesis and therapeutics. bioRxiv. 2020. doi: 10.1101/2020.08.14.248880. PubMed PMID: 32817940; PMCID: PMC7430570.

Geleris J, Sun Y, Platt J, Zucker J, Baldwin M, Hripcsak G, Labella A, Manson DK, Kubin C, Barr RG, Sobieszczyk ME, Schluger NW. Observational study of hydroxychloroquine in hospitalized patients with COVID-19. N Engl J Med. 2020;382(25):2411-8. doi: 10.1056/NEJMoa2012410. PubMed PMID: 32379955; PMCID: PMC7224609.

Kalligeros M, Shehadeh F, Atalla E, Mylona EK, Aung S, Pandita A, Larkin J, Sanchez M, Touzard-Romo F, Brotherton A, Shah R, Cunha CB, Mylonakis E. Hydroxychloroquine use in hospitalized patients with COVID-19: An observational matched cohort study. J Glob Antimicrob Resist. 2020. doi: 10.1016/j.jgar.2020.07.018. PubMed PMID: 32763357; PMCID: PMC7403006.

Tianling Ou HM, Lizhou Zhang, Amrita Ojha, Hyeryun Choe, Michael Farzan. Hydroxychloroquine-mediated inhibition of SARS-CoV-2 entry is attenuated by TMPRSS2. BioRxiv2020.

Donaldson SH, Hirsh A, Li DC, Holloway G, Chao J, Boucher RC, Gabriel SE. Regulation of the epithelial sodium channel by serine proteases in human airways. J Biol Chem. 2002;277(10):8338-45. doi: 10.1074/jbc.M105044200. PubMed PMID: 11756432.

Kim TS, Heinlein C, Hackman RC, Nelson PS. Phenotypic analysis of mice lacking the Tmprss2-encoded protease. Mol Cell Biol. 2006;26(3):965-75. doi: 10.1128/MCB.26.3.965-975.2006. PubMed PMID: 16428450; PMCID: PMC1347042.

Camostat Mesylate in COVID-19 Outpatients. ClinicalTrials.gov identifier: NCT04353284. Posted April 20, 2020. Updated November 24, 2020. https://clinicaltrials.gov/ct2/show/NCT04353284?term=camostat&cond=Covid-19&draw=2&rank=1.

The Utility of Camostat Mesylate in Patients With COVID-19 Associated Coagulopathy (CAC) and Cardiovascular Compli. ClinicalTrials.gov identifier: NCT04435015. Posted June 17, 2020. Updated October 28, 2020. https://clinicaltrials.gov/ct2/show/NCT04435015?term=camostat&cond=Covid-19&draw=2&rank=2.

Clinical Efficacy of Nafamostat Mesylate for COVID-19 Pneumonia. ClinicalTrials.gov identifier: NCT04418128. Posted June 5, 2020. Updated June 9, 2020. https://clinicaltrials.gov/ct2/show/NCT04418128?term=nafamostat&cond=Covid-19&draw=2.

Study to Evaluate the Safety and Efficacy of Liquid Alpha1-Proteinase Inhibitor (Human) in Hospitalized Participants With Coronavirus Disease (COVID-19) 2020. ClinicalTrials.gov identifier: NCT04547140. Posted September 4, 2020. Updated March 1, 2021. https://clinicaltrials.gov/ct2/show/NCT04547140?term=alpha+1&cond=Covid19&draw=2&rank=1.

Study to Evaluate the Safety and Efficacy of Prolastin in Hospitalized Subjects With COVID-19 2020. ClinicalTrials.gov identifier: NCT04495101. Posted July 31, 2020. Updated February 21, 2021. https://clinicaltrials.gov/ct2/show/NCT04495101?term=alpha+1&cond=Covid19&draw=2&rank=6.

Song J, Li Y, Huang X, Chen Z, Li Y, Liu C, Chen Z, Duan X. Systematic analysis of ACE2 and TMPRSS2 expression in salivary glands reveals underlying transmission mechanism caused by SARS-CoV-2. J Med Virol. 2020;92(11):2556-66. doi: 10.1002/jmv.26045. PubMed PMID: 32441816; PMCID: PMC7280739.

Yap JKY, Moriyama M, Iwasaki A. Inflammasomes and pyroptosis as therapeutic targets for COVID-19. J Immunol. 2020;205(2):307-12. doi: 10.4049/jimmunol.2000513. PubMed PMID: 32493814; PMCID: PMC7343621.

Chen J, Wu H, Yu Y, Tang N. Pulmonary alveolar regeneration in adult COVID-19 patients. Cell Res. 2020;30(8):708-10. doi: 10.1038/s41422-020-0369-7. PubMed PMID: 32632255; PMCID: PMC7338112.

Harbig A, Mernberger M, Bittel L, Pleschka S, Schughart K, Steinmetzer T, Stiewe T, Nist A, Bottcher-Friebertshauser E. Transcriptome profiling and protease inhibition experiments identify proteases that activate H3N2 influenza A and influenza B viruses in murine airways. J Biol Chem. 2020;295(33):11388-407. doi: 10.1074/jbc.RA120.012635. PubMed PMID: 32303635; PMCID: PMC7450141.

Gierer S, Bertram S, Kaup F, Wrensch F, Heurich A, Kramer-Kuhl A, Welsch K, Winkler M, Meyer B, Drosten C, Dittmer U, von Hahn T, Simmons G, Hofmann H, Pohlmann S. The spike protein of the emerging betacoronavirus EMC uses a novel coronavirus receptor for entry, can be activated by TMPRSS2, and is targeted by neutralizing antibodies. J Virol. 2013;87(10):5502-11. doi: 10.1128/JVI.00128-13. PubMed PMID: 23468491; PMCID: PMC3648152.

Shirato K, Kawase M, Matsuyama S. Middle East respiratory syndrome coronavirus infection mediated by the transmembrane serine protease TMPRSS2. J Virol. 2013;87(23):12552-61. doi: 10.1128/JVI.01890-13. PubMed PMID: 24027332; PMCID: PMC3838146.

Hatesuer B, Bertram S, Mehnert N, Bahgat MM, Nelson PS, Pohlmann S, Schughart K. Tmprss2 is essential for influenza H1N1 virus pathogenesis in mice. PLoS Pathog. 2013;9(12):e1003774. doi: 10.1371/journal.ppat.1003774. PubMed PMID: 24348248; PMCID: PMC3857797.

Sakai K, Ami Y, Tahara M, Kubota T, Anraku M, Abe M, Nakajima N, Sekizuka T, Shirato K, Suzaki Y, Ainai A, Nakatsu Y, Kanou K, Nakamura K, Suzuki T, Komase K, Nobusawa E, Maenaka K, Kuroda M, Hasegawa H, Kawaoka Y, Tashiro M, Takeda M. The host protease TMPRSS2 plays a major role in in vivo replication of emerging H7N9 and seasonal influenza viruses. J Virol. 2014;88(10):5608-16. doi: 10.1128/JVI.03677-13. PubMed PMID: 24600012; PMCID: PMC4019123.

Engh R, Lobermann H, Schneider M, Wiegand G, Huber R, Laurell CB. The S variant of human alpha 1-antitrypsin, structure and implications for function and metabolism. Protein Eng. 1989;2(6):407-15. doi: 10.1093/protein/2.6.407. PubMed PMID: 2785270.

Bao J, Pan G, Poncz M, Wei J, Ran M, Zhou Z. Serpin functions in host-pathogen interactions. PeerJ. 2018;6:e4557. doi: 10.7717/peerj.4557. PubMed PMID: 29632742; PMCID: PMC5889911.

Dementiev A, Dobo J, Gettins PG. Active site distortion is sufficient for proteinase inhibition by serpins: Structure of the covalent complex of alpha1-proteinase inhibitor with porcine pancreatic elastase. J Biol Chem. 2006;281(6):3452-7. doi: 10.1074/jbc.M510564200. PubMed PMID: 16321984.

Blaurock N, Schmerler D, Hunniger K, Kurzai O, Ludewig K, Baier M, Brunkhorst FM, Imhof D, Kiehntopf M. C-terminal alpha-1 antitrypsin peptide: A new sepsis biomarker with immunomodulatory function. Mediators Inflamm. 2016;2016:6129437. doi: 10.1155/2016/6129437. PubMed PMID: 27382189; PMCID: PMC4921625.

Risor MW, Juhl DW, Bjerring M, Mathiesen J, Enghild JJ, Nielsen NC, Otzen DE. Critical influence of cosolutes and surfaces on the assembly of serpin-derived amyloid fibrils. Biophys J. 2017;113(3):580-96. doi: 10.1016/j.bpj.2017.06.030. PubMed PMID: 28793213; PMCID: PMC5549687.

Potere N, Del Buono MG, Niccoli G, Crea F, Toldo S, Abbate A. Developing LRP1 agonists into a therapeutic strategy in acute myocardial infarction. Int J Mol Sci. 2019;20(3). doi: 10.3390/ijms20030544. PubMed PMID: 30696029; PMCID: PMC6387161.

Chandrika Bhattacharyya CD, Arnab Ghosh, Animesh K. Singh, Souvik Mukherjee, Partha P. Majumder, Analabha Basu, Nidhan K. Biswas. Global spread of SARS-CoV-2 subtype with spike protein mutation D614G is shaped by human genomic variations that regulate expression of TMPRSS2 and MX1 genes. BioRxiv. 2020. doi: 10.1101/2020.05.04.075911

Serban KA, Petrusca DN, Mikosz A, Poirier C, Lockett AD, Saint L, Justice MJ, Twigg HL, 3rd, Campos MA, Petrache I. Alpha-1 antitrypsin supplementation improves alveolar macrophages efferocytosis and phagocytosis following cigarette smoke exposure. PLoS One. 2017;12(4):e0176073. doi: 10.1371/journal.pone.0176073. PubMed PMID: 28448535; PMCID: PMC5407578.

Qin C, Zhou L, Hu Z, Zhang S, Yang S, Tao Y, Xie C, Ma K, Shang K, Wang W, Tian DS. Dysregulation of immune response in patients with coronavirus 2019 (COVID-19) in Wuhan, China. Clin Infect Dis. 2020;71(15):762-8. doi: 10.1093/cid/ciaa248. PubMed PMID: 32161940; PMCID: PMC7108125.

Wang D, Hu B, Hu C, Zhu F, Liu X, Zhang J, Wang B, Xiang H, Cheng Z, Xiong Y, Zhao Y, Li Y, Wang X, Peng Z. Clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in Wuhan, China. JAMA. 2020;323(11):1061-9. doi: 10.1001/jama.2020.1585. PubMed PMID: 32031570; PMCID: PMC7042881.

Henry BM, de Oliveira MHS, Benoit S, Plebani M, Lippi G. Hematologic, biochemical and immune biomarker abnormalities associated with severe illness and mortality in coronavirus disease 2019 (COVID-19): A meta-analysis. Clin Chem Lab Med. 2020;58(7):1021-8. doi: 10.1515/cclm-2020-0369. PubMed PMID: 32286245.

Ren Y, He QY, Fan J, Jones B, Zhou Y, Xie Y, Cheung CY, Wu A, Chiu JF, Peiris JS, Tam PK. The use of proteomics in the ciscovery of serum biomarkers from patients with severe acute respiratory syndrome. Proteomics. 2004;4(11):3477-84. doi: 10.1002/pmic.200400897. PubMed PMID: 15378763; PMCID: PMC7167722.

Chen X, Zhao B, Qu Y, Chen Y, Xiong J, Feng Y, Men D, Huang Q, Liu Y, Yang B, Ding J, Li F. Detectable serum SARS-CoV-2 viral load (RNAaemia) is closely correlated with drastically elevated interleukin 6 (IL-6) level in critically ill COVID-19 patients. Clin Infect Dis. 2020. doi: 10.1093/cid/ciaa449. PubMed PMID: 32301997; PMCID: PMC7184354.

Ulhaq ZS, Soraya GV. Interleukin-6 as a potential biomarker of COVID-19 progression. Med Mal Infect. 2020;50(4):382-3. doi: 10.1016/j.medmal.2020.04.002. PubMed PMID: 32259560; PMCID: PMC7129451.

Zhou F, Yu T, Du R, Fan G, Liu Y, Liu Z, Xiang J, Wang Y, Song B, Gu X, Guan L, Wei Y, Li H, Wu X, Xu J, Tu S, Zhang Y, Chen H, Cao B. Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: A retrospective cohort study. Lancet. 2020;395(10229):1054-62. doi: 10.1016/S0140-6736(20)30566-3. PubMed PMID: 32171076; PMCID: PMC7270627.

Janciauskiene S, Welte T. Well-known and less well-known functions of alpha-1 antitrypsin. Its role in chronic obstructive pulmonary disease and other disease developments. Ann Am Thorac Soc. 2016;13 Suppl 4:S280-8. doi: 10.1513/AnnalsATS.201507-468KV. PubMed PMID: 27564662.