key: cord-0797138-jry4y3p9
authors: Hoffmann, Markus; Hofmann-Winkler, Heike; Smith, Joan C.; Krüger, Nadine; Sørensen, Lambert K.; Søgaard, Ole S.; Hasselstrøm, Jørgen Bo; Winkler, Michael; Hempel, Tim; Raich, Lluís; Olsson, Simon; Yamazoe, Takashi; Yamatsuta, Katsura; Mizuno, Hirotaka; Ludwig, Stephan; Noé, Frank; Sheltzer, Jason M.; Kjolby, Mads; Pöhlmann, Stefan
title: Camostat mesylate inhibits SARS-CoV-2 activation by TMPRSS2-related proteases and its metabolite GBPA exerts antiviral activity
date: 2020-08-05
journal: bioRxiv
DOI: 10.1101/2020.08.05.237651
sha: 9b8fe78775eb8b9697cebf75c26d67ad738b832d
doc_id: 797138
cord_uid: jry4y3p9

Antiviral therapy is urgently needed to combat the coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The protease inhibitor camostat mesylate inhibits SARS-CoV-2 infection of lung cells by blocking the virus-activating host cell protease TMPRSS2. Camostat mesylate has been approved for treatment of pancreatitis in Japan and is currently being repurposed for COVID-19 treatment. However, potential mechanisms of viral resistance as well as camostat mesylate metabolization and antiviral activity of metabolites are unclear. Here, we show that SARS-CoV-2 can employ TMPRSS2-related host cell proteases for activation and that several of them are expressed in viral target cells. However, entry mediated by these proteases was blocked by camostat mesylate. The camostat metabolite GBPA inhibited the activity of recombinant TMPRSS2 with reduced efficiency as compared to camostat mesylate and was rapidly generated in the presence of serum. Importantly, the infection experiments in which camostat mesylate was identified as a SARS-CoV-2 inhibitor involved preincubation of target cells with camostat mesylate in the presence of serum for 2 h and thus allowed conversion of camostat mesylate into GBPA. Indeed, when the antiviral activities of GBPA and camostat mesylate were compared in this setting, no major differences were identified. Our results indicate that use of TMPRSS2-related proteases for entry into target cells will not render SARS-CoV-2 camostat mesylate resistant. Moreover, the present and previous findings suggest that the peak concentrations of GBPA established after the clinically approved camostat mesylate dose (600 mg/day) will result in antiviral activity.

The outbreak of the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-72 CoV-2) in the city of Wuhan, China, in the winter of 2019 and its subsequent pandemic spread 73 has resulted in more than 14 million cases of coronavirus disease 2019 and more than 600.00 74 deaths (1). Antivirals designed to combat SARS-CoV-2 are not available and repurposing of 75 existing drugs developed against other diseases is considered the fastest option to close this gap 76 (2). Remdesivir, a drug generated to inhibit Ebola virus infection, has recently been shown to 77 reduce the duration of hospitalization for . However, the drug failed to reduce 78 fatality significantly (3) and beneficial effects were not observed in a previous clinical trial (4), 79 indicating that additional therapeutic options are needed. 80 We previously showed that the SARS-CoV-2 spike protein (S) uses the host cell factors 81 angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2) for 82 entry into target cells (5). TMPRSS2 is a cellular type II transmembrane serine protease (TTSP) 83 expressed in human respiratory epithelium that cleaves and thereby activates the viral S protein. 84 Activation is essential for viral infectivity and we found that the protease inhibitor camostat 85 mesylate, which is known to block TMPRSS2 activity (6), inhibits SARS-CoV-2 infection of 86 lung cells (5). Camostat mesylate has been approved for treatment of pancreatitis in Japan (7-9) 87 and it is currently being investigated as a treatment of COVID-19 in several clinical trials in 88 Denmark, Israel and USA (NCT04321096, NCT04353284, NCT04355052, NCT04374019). 89 The activity of TMPRSS2 is essential for SARS-CoV and MERS-CoV lung infection and 90 disease development (10, 11) . Whether TMPRSS2-independent pathways for S protein activation 91 exist and contribute to viral spread outside the lung is not fully understood. The S proteins of 92 SARS-CoV-2 and several other coronaviruses can be activated by the pH-dependent endosomal 93 cysteine protease cathepsin L in certain cell lines (5, (12) (13) (14) (15) . However, this auxiliary S protein 94 activation pathway is not operative in the lung, likely due to low cathepsin L expression (16).

Whether this pathway contributes to the recently reported extrapulmonary spread of SARS-CoV-96 2 is unknown (17) . Similarly, it is unclear whether TTSPs other than TMPRSS2 can promote 97 extrapulmonary SARS-CoV-2 spread. Finally, camostat mesylate is rapidly hydrolyzed into the 98 active metabolite 4-(4-guanidinobenzoyloxy)phenylacetic acid (GBPA) in patients (18-20) but it 99 is unknown to what extend GBPA inhibits TMPRSS2 activity. 100 Here, we identify TTSPs other than TMPRSS2 that can be used by SARS-CoV-2 for S 101 protein activation and demonstrate that they are inhibited by camostat mesylate. Moreover, we 102 provide evidence that camostat mesylate is rapidly converted into GBPA in cell culture and that 103 GBPA inhibits SARS-CoV-2 entry with almost identical efficiency as compared to camostat 104 mesylate when cells are preincubated with these compounds. 121 The TTSP family comprises several enzymes which have previously been shown to activate 122 surface glycoproteins of coronaviruses and other viruses, at least upon directed expression (21-123 23). Therefore, we asked whether the S protein of SARS-CoV-2 (SARS-2-S) can employ TTSPs 124 other than TMPRSS2 for its activation. For this, we expressed different TTSPs along with the 125 SARS-CoV-2 receptor, ACE2, in the otherwise poorly susceptible BHK-21 cells, treated the cells 126 with ammonium chloride, which blocks the cathepsin L-dependent, auxiliary activation pathway, 127 and transduced the cells with previously described vesicular stomatitis virus (VSV)-based 128 pseudotypes bearing SARS-2-S (5). Ammonium chloride treatment strongly reduced SARS-2-S-129 driven transduction and this effect was rescued upon expression of TMPRSS2 (Fig. 1) , as 130 expected. Notably, this effect was also efficiently rescued by expression of TMPRSS13 and, to a 131 lesser degree, TMPRSS11D, TMPRSS11E and TMPRSS11F (Fig. 1) . Thus, SARS-2-S can use 132 diverse TTSPs for S protein activation upon overexpression, with S protein activation by 133 TMPRSS13 being particularly robust. In order to obtain insights into whether SARS-2-S activating TTSPs could contribute to viral 137 spread in the infected host, we asked whether these enzymes are expressed in viral target cells.

For this, we analyzed single-cell RNA-Seq datasets collected from human lungs (24) and airways 139 (25). As previously reported (26-31), ACE2 was expressed in the lung epithelial compartment, 140 particularly including alveolar type 2 cells, secretory (goblet/club) cells, and ciliated cells (Fig. 141 2A and Fig. S1 ). TMPRSS2 and TMPRSS13 were similarly expressed across epithelial cells, 142 although TMPRSS13 expression was generally less robust. In contrast, expression of 143 7 TMPRSS11-family members was only rarely detected ( Fig. 2A) . We found that 53% of ACE2 + 144 cells in the lung co-express TMPRSS2, while 21% of ACE2 + cells do not express TMPRSS2 but 145 do express another TTSP capable of activating SARS-CoV-2 (Fig. S1 ). Within the airways, we 146 observed ACE2 expression in secretory cells, ciliated cells, and suprabasal cells in both the nasal 147 turbinate and the trachea (Fig. 2B) . Interestingly, the expression pattern of the TTSPs in the 148 airways was largely distinct: TMPRSS2 was primarily expressed in ciliated and secretory cells,

TMPRSS11D was primarily expressed in basal cells, TMPRSS11E was primarily expressed in 150 ionocytes, and TMPRSS13 was primarily expressed in nasal secretory cells (Fig. 2B ). Within this 151 dataset, 21% of ACE2 + cells co-expressed TMPRSS2, while 24% of ACE2 + cells co-expressed a 152 different TTSP (Fig. S1 ). In total, these results suggest that TMPRSS2 is the dominant SARS-

CoV-2-activating protease in the lung, in keeping with findings made for SARS-CoV and MERS-

CoV, while the virus may use other activating proteases for spread in the airways.

A recent study provided evidence for extrapulmonary replication of SARS-CoV-2 in liver, 156 colon, heart, kidney and blood in some patients (17). Therefore, we asked whether ACE2, 157 TMPRSS2 and related SARS-2-S-activating proteases are expressed in these organs, using 158 published resources (32, 33) . Liver, colon, heart and kidney expressed robust levels of ACE2 159 (Fig. 2C) . Similarly, TMPRSS2 expression in colon, liver and kidney was readily detectable, 160 although expression levels were lower than those measured for lung (Fig. 2C ). In contrast, little 161 to no expression of TMPRSS11D, TMPRSS11E, TMPRSS11F, TMPRSS13 was detected in 162 liver, colon, heart and kidney. Finally, TMPRSS13 was expressed in lung and blood cells and Newly identified SARS-2-S activators are camostat mesylate sensitive 170 We next asked whether S protein activation by TTSP other than TMPRSS2 can be inhibited by 171 camostat mesylate. To address this question, we performed the rescue assay as described above Multiple studies show that camostat mesylate is rapidly converted into its active metabolite, 4-(4-191 guanidinobenzoyloxy)phenylacetic acid (GBPA) in animals and humans, followed by further 192 conversion of GBPA into the inactive metabolite 4-guanidinobenzoic acid (GBA) (18-20, 34) 193 ( Fig. 6A) . However, the capacity of GBPA to inhibit the enzymatic activity of TMPRSS2 has not 194 been examined. To address this question, we compared inhibition of recombinant TMPRSS2 by 195 camostat mesylate, GBPA and GBA. For this, we used FOY-251, a methanesulfonate of GBPA. 196 We found that FOY-251 exerted a 10-fold reduced capacity to inhibit TMPRSS2 as compared to 197 camostat mesylate, although both compounds completely suppressed TMPRSS2 activity at 1 µM 198 or higher (Fig. 4) . In contrast, GBA was less active (Fig. 4) . Thus, FOY-251 blocks TMPRSS2 199 activity but with reduced efficiency as compared to camostat mesylate. to form a long-lived covalent complex that is the main source of inhibition (36). However, the 206 population of the short-lived precomplex directly relates to the inhibitory activity (35). By 207 computing the TMPRSS2-GBPA binding kinetics (35), we find that (i) the noncovalent 208 TMPRSS2-GBPA complex is metastable, rendering it suitable to form a covalent inhibitory 209 complex, and (ii) its population is 40% lower compared to camostat at equal drug concentrations, 210 consistent with the finding that FOY-251 is a viable but less potent inhibitor (Fig. 4) .

Structurally, we find that GBPA binds in the same manner as camostat (Fig. 5, (35) ). The main 212 stabilizing interaction is its Guanidinium group binding into TMPRSS2's S1 pocket which is in viral spread in type II pneumocytes has been reported (46) but viral spread in mice is 286 TMPRSS2 independent (44, 47) . 287 The present study shows that also SARS-CoV-2 can use TTSPs other than TMPRSS2 for 288 S protein activation. Whether the TTSPs found here to activate SARS-2-S upon directed 289 expression play a role in viral spread in the host remains to be investigated. Expression analyses 290 suggest that they may. TMPRSS13 activated SARS-2-S with similar efficiency as TMPRSS2 and 291 TMPRSS13 mRNA was found to be coexpressed with ACE2 in type II pneumocytes, goblet and In addition, fresh culture medium (without cells) was also incubated with CellTiter-Glo substrate 454 in order to define the assay background. Following incubation, the samples were transferred into 455 white opaque-walled 96-well plates and luminescence was recorded (200 msec/sample) using a 456 Hidex Sense plate luminometer (Hidex).

The SARS-CoV-2 isolate hCoV-19/Germany/FI1103201/2020 (GISAID accession EPI- All statistical analyses were performed using GraphPad Prism (version 8.4.2, GraphPad Software, 492 Inc.). Statistical significance of differences between two datasets was analyzed by paired, two- containing culture medium was analyzed by paired, two-tailed student's t-test (p ≤ 0.01, **). 

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Acknowledgments: We are grateful for in-depth discussions with Katarina Elez

FU Berlin) and the members of the JEDI COVID-19 grand 680 challenge. Funding: Research in the Sheltzer Lab was supported by NIH grants 1DP5OD021385 681 and R01CA237652-01, a Damon Runyon-Rachleff Innovation award, an American Cancer 682

Society Research Scholar Grant, and a grant from the New York Community Trust. The Noé lab 683 was supported by Deutsche Forschungsgemeinschaft DFG (SFB/TRR 186

The Pöhlmann lab 686 was supported by BMBF (RAPID Consortium, 01KI1723D)

Competing interests: J.C.S. is a co-founder of Meliora Therapeutics and is an employee of 693

This work was performed outside of her affiliation with Google and used no 694 proprietary knowledge or materials from Google

is a member of the Advisory Board of Tyra Biosciences, and is a co-founder of 696

As part of its mission the Deutsches Primatenzentrum

Data availability 699 statement: All data associated with this 700 29 study are shown in the paper or the Supplementary Materials. All of the data used in this 701 manuscript to determine protease expression are described in Table S1 of (29) and the code used 702 for performing these analyses is available at github

Different TTSPs can activate SARS-2-S in transfected cells. BHK-21 cells transiently 727 expressing ACE2 and one of the indicated type-II transmembrane serine protease (or empty 728 vector) were pre-incubated with either 50 mM ammonium chloride or DMSO (control, indicated 729 by dashed line) for 2 h

SARS-2-S-driven cell entry of viral pseudotypes was analyzed by 731 measuring the activity of virus-encoded luciferase activity in cell lysates. Data were further 732 normalized and entry efficiency in the absence of ammonium chloride was set as 100 %. Shown 733 are the average (mean) data obtained from three biological replicates, each performed in 734 quadruplicates. Error bars indicate the standard error of the mean (SEM)

Cells expressing the coronavirus receptor ACE2 are 740 highlighted in the right panel. These panels are reproduced with permission from Smith et al. 741 (29)

Cells expressing various S-activating proteases in the 744 human airway are highlighted. (C) Log2-normalized expression data of the

Activation of SARS-2-S by TMPRSS2-related proteases can be suppressed by 748 camostat mesylate. The experiment was performed as described for figure 1 with the 749 modifications

At 16 h post inoculation with viral particles bearing SARS-2-S, pseudotype entry was 753 analyzed by measuring the activity of virus-encoded luciferase activity in cell lysates. Data were 754 further normalized and entry efficiency into control-treated cells was set as 100 %. Shown are the 755 average (mean) data obtained from three biological replicates

Statistical significance of differences in entry efficiency in 757 ammonium chloride-, camostat mesylate-or ammonium chloride + camostat mesylate-treated 758 cells versus control-treated cells was analyzed by two-way ANOVA with Dunnett's posttest

Camostat mesylate and FOY-251 inhibit the activity of recombinant TMPRSS2

TMPRSS2 cleaved Boc-Gln-Ala-Arg-MCA as substrate and produced the potent fluorophore

TMPRSS2 enzyme activity was evaluated by measuring the 764 fluorescence intensity using Envision plate reader and all of the data were normalized against the 765 intensity of the absence of test compounds. The concentration-response data for each test 766 compound was plotted and modeled by a four-parameter logistic fit to determine the 50% 767 inhibitory concentration (IC 50 ) value. Inhibitory activity of camostat mesylate (blue), FOY-768 251(light blue) and GBA (red) against TMPRSS2 recombinant protein were visualized and curve 769 fitting were performed using GraphPad Prism. The average of two independent experiments, each 770 32 performed with quadruplicate (camostat mesylate and FOY-251) or duplicate samples (GBA) is 771 shown

A TMPRSS2 structure model is 774 shown in the left panel, the active site is highlighted in cyan and catalytic triad residues are 775 shown in black. The representative structure of GBPA bound to TMPRSS2 in a reactive complex 776 is shown in the right panel. The GBPA guanidinium head forms a salt bridge with D435 inside 777 the S1 pocket. This transient complex, which is similar for Camostat

Metabolization of camostat mesylate. (B) LC-MS/MS determination of camostat, 782 GBPA and GBA in culture medium containing FCS. Camostat mesylate was added to FCS-783 containing culture medium at a concentration of 15 µM. Samples were taken after incubation for 784 1, 15, 30, 60, 120, 240, 480, and 1,440 min at 37 °C, snap-frozen and stored at -80 °C. Samples 785 were analyzed by LC-MS/MS and quantified regarding their content of intact camostat mesylate 786 and its metabolites GBPA (active) and GBA (inactive)

For normalization, the combined values of camostat mesylate and GBPA were set as 100 % and 793 the relative fractions of the compounds were calculated