New Results

Efficacy of Host Cell Serine Protease Inhibitor MM3122 against SARS-CoV-2 for Treatment and Prevention of COVID-19

Adrianus C.M. Boon, Traci L. Bricker, Ethan J. Fritch, Sarah R. Leist, Kendra Gully, Ralph S. Baric, Rachel L. Graham, Brigid V. Troan, Matthew Mahoney, James W. Janetka
doi: https://doi.org/10.1101/2024.02.09.579701

ABSTRACT

We have developed a novel class of peptidomimetic inhibitors targeting several host cell human serine proteases including transmembrane protease serine 2 (TMPRSS2), matriptase and hepsin. TMPRSS2 is a membrane associated protease which is highly expressed in the upper and lower respiratory tract and is utilized by SARS-CoV-2 and other viruses to proteolytically process their glycoproteins, enabling host cell receptor binding, entry, replication, and dissemination of new virion particles. We have previously shown that compound MM3122 exhibited sub nanomolar potency against all three proteases and displayed potent antiviral effects against SARS-CoV-2 in a cell-viability assay. Herein, we demonstrate that MM3122 potently inhibits viral replication in human lung epithelial cells and is also effective against the EG.5.1 variant of SARS-CoV-2. Further, we have evaluated MM3122 in a mouse model of COVID-19 and have demonstrated that MM3122 administered intraperitoneally (IP) before (prophylactic) or after (therapeutic) SARS-CoV-2 infection had significant protective effects against weight loss and lung congestion, and reduced pathology. Amelioration of COVID-19 disease was associated with a reduction in pro-inflammatory cytokines and chemokines production after SARS-CoV-2 infection. Prophylactic, but not therapeutic, administration of MM3122 also reduced virus titers in the lungs of SARS-CoV-2 infected mice. Therefore, MM3122 is a promising lead candidate small molecule drug for the treatment and prevention of infections caused by SARS-CoV-2 and other coronaviruses.

IMPORTANCE SARS-CoV-2 and other emerging RNA coronaviruses are a present and future threat in causing widespread endemic and pandemic infection and disease. In this paper, we have shown that the novel host-cell protease inhibitor, MM3122, blocks SARS-CoV-2 viral replication and is efficacious as both a prophylactic and therapeutic drug for the treatment of COVID-19 in mice. Targeting host proteins and pathways in antiviral therapy is an underexplored area of research but this approach promises to avoid drug resistance by the virus, which is common in current antiviral treatments.

INTRODUCTION

The COVID-19 pandemic, caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has illuminated the devastating impact of new emerging viral diseases on both the global economy and health of populations1. It has affected all aspects of human life and highlighted the threat of future outbreaks with other respiratory viruses for which currently available drugs will be ineffective. Incredibly, vaccines as well as several new drug candidates targeting this virus were developed at unprecedented speed because of early countermeasure work that focused on paradigm pathogens within the coronavirus family2–4. This was made possible by the combined efforts of scientists worldwide who elucidated details about the makeup and pathogenesis of SARS-CoV-2 infection. This work also resulted in the identification of several potential therapeutic targets such as the viral entry receptor, angiotensin converting enzyme 2 (ACE2), and host cell proteases, such as transmembrane protease serine 2 (TMPRSS2) and cathepsin L1 (CTSL1), required for entry, replication, and release of the virus5–8.

TMPRSS29–11 has a trypsin-like serine protease domain and belongs to the family of Type II Transmembrane Serine Protease (TTSP) proteolytic enzymes with reported physiological roles in cancer and many other diseases12–15. TMPRSS2 has previously been shown to be important in other coronavirus infections caused by SARS-CoV-116, 17, HKU-118, MERS-CoV19, and others20,21. Furthermore, both TMPRSS2 and the other TTSPs, matriptase (ST14)22, 23 and HAT (human airway trypsin-like protease, TMPRSS11D)24, 25, have been demonstrated to proteolytically process the hemagglutinin (HA) protein on the surface of some influenza A viruses and SARS-CoV-117, allowing viral cell adhesion and entry in these infections24, 26–31. Additionally, TMPRSS2 was found to support replication of other respiratory viruses including human para-influenza virus type 1, 2 and mouse Sendai virus32. This makes TMPRSS2 an excellent target for the development of a broadly acting antiviral inhibitor33 against diverse respiratory viruses24, 34–39.

We recently reported on the discovery and development of a new class of peptidomimetic TMPRSS2 inhibitors39. These inhibitors were rationally designed based on the peptide substrate sequence specificity of TMPRSS215 and molecular docking studies using the X-ray structure of TMPRSS2 bound to another inhibitor nafamostat40, 41. These inhibitors, including MM3122 and MM3144, are significantly more potent than another reported TMPRSS2 inhibitor Camostat41, suggesting improved efficacy in vivo. Unlike MM3122, which has the desired selectivity for TMPRSS2 over the coagulation serine proteases thrombin and Factor Xa, MM3144 does not. This compound was subsequently reported as a matriptase and TMPRSS2 inhibitor by another group as N-038642. We tested the in vitro and in vivo efficacy of the most promising compound at the time, MM3122, in Calu-3 human lung epithelial cells and in a mouse model of SARS-CoV-2. Overall, we showed potent antiviral efficacy of MM3122 against XBB.1.5 and EG.5.1 variant of SARS-CoV-2 in vitro and amelioration of COVID-19 disease in vivo.

RESULTS

MM3122 inhibits authentic SARS-CoV-2 replication in human lung epithelial cells

The ability of MM3122, a TMPRSS2 inhibitor, to inhibit wild-type (wt) SARS-CoV-2 infection and replication was assessed on Calu-3 cells, a human lung epithelial cell line. At a 0.03 µM concentration of MM3122, virus replication was completely inhibited, and no infectious virus was detected in the supernatant of the treated and SARS-CoV-2 infected cells (Fig 1). The inhibitory concentration (IC50) of MM3122 was ∼0.01-0.02 µM against the authentic wt SARS-CoV-2 virus. This is greater than 50 times more potent than Remdesivir which had an IC50 of ∼1 µM, an RNA-dependent RNA polymerase inhibitor developed for other viral infections that is one of the FDA-approved drugs approved to treat SARS-CoV-2 infected patients43. MM3122 was also tested for its activity against the EG.5.1 variant of SARS-CoV-2. Similar to wt SARS-CoV-2, we observed robust inhibition of the virus with an IC50 of ∼0.05-0.1 µM. Taken together, these studies demonstrate the high potential for small molecule TMPRSS2 inhibitors to inhibit replication of SARS-CoV-2 and several of its many variants.

Figure 1:
Figure 1: The TMPRSS2 inhibitor MM3122 inhibits replication of authentic SARS-CoV-2 and variants.

Calu-3 cells, plated in 24-well plates, were infected with 4,000 PFU of A) WA1/2020 and B) EG.5.1 strains of SARS-CoV-2 for one hour at 37°C. After two washes, the cells were incubated for 48 hours in media with different concentrations of MM3122 (red symbol), Remdesivir (grey symbols), or DMSO (black symbols) as the positive and negative controls respectively. Infectious virus titer in the supernatant of the wells 48 hours after infection with SARS-CoV-2. The results are the average of 3 independently repeated assays. The dotted line is the limit of detection.

MM3122 is a multi-targeted serine protease inhibitor with activity against some cathepsins

To determine the target specificity of MM3122 for TMPRSS2, we tested for its inhibitory activity against 53 serine and cysteine proteases (Table 1). In our previous paper39, we profiled MM3122 for its inhibition of hepatocyte growth factor activator (HGFA), matriptase, hepsin, thrombin, and Factor Xa. For the 47 other proteases we contracted Reaction Biology Co. (Malvern, PA) to determine the IC50 values of MM3122 against a large panel of serine and cysteine proteases of high importance. A previous group had reported the selectivity profile for Camostat and Nafamostat against these same proteases44 which is also shown in Table 1. In addition to TMPRSS2, MM3122 has potent activity (0.01 to 10 nM) against only 7 other proteases, matriptase, hepsin, matriptase-2, plasma kallikrein, trypsin, tryptase b2 and tryptase g1. It has moderate inhibitory activity (10 nM to 1 µM) against HGFA, Factor Xa, kallikrein 1 (KLK1), KLK5, KLK14, plasmin, and proteinase K and surprisingly against the cysteine protease cathepsin S with an IC50 of 590 nM. Furthermore, MM3122 also inhibited the other cysteine proteases cathepsin C, cathepsin L and papain, with IC50s of 1.4 µM, 12.8 µM, and 1.1 µM. Comparing the selectivity profile of MM3122 to that of Camostat and Nafamostat reveals that the cysteine protease activity is absent in the latter. Otherwise, the profiles are generally similar with some exceptions, notably decreased activity of MM3122 against thrombin, plasmin, Factor VIIA and XIA, KLK12, KLK13 and KLK14, urokinase, matriptase-2, trypsin and the 2 tryptases. We also found that MM3122 does not inhibit furin or the SARS-CoV-2 proteases, Mpro and PLpro.

View this table:
Table 1: Protease selectivity profile of MM3122, Camostat and Nafamostat tested against a panel of 53 serine and cysteine proteases.

MM3122 ameliorates SARS-CoV-2 induced disease in mice

To assess the antiviral activity of MM3122 in vivo, four groups of mice were treated with 50 mg/kg or 100 mg/kg MM3122 30 minutes prior to (prophylactic) and 24 hours after (therapeutic) intranasal infection with the MA10 strain of SARS-CoV-2. A mock infected and vehicle treated group were included as controls. Intranasal inoculation of vehicle treated mice with SARS-CoV-2 resulted in significant weight loss compared to mock infected animals (Fig 2A). Importantly, none of the MM3122 groups lost any significant amount of body weight. Five days after infection, lung congestion was assessed using an independently developed scoring system as described in the Methods. Vehicle-treated mice that were infected with SARS-CoV-2 MA10 demonstrated evidence of congestion with scores ranging from 1-3. These scores were reduced to 0 for infected mice that received 50 and 100 mg/kg MM3122 prophylactically (P < 0.01), and 0.5 for mice that received 50 and 100 mg/kg MM3122 therapeutically. Mice that received MM3122 prior to infection also had reduced virus titers (5000-10,000-fold) compared to vehicle treated and infected animals (Fig 2B). However, mice that received MM3122 24 h after infection had similar amounts of virus in the lungs compared to the vehicle treated animals. Finally, we performed pathological analysis of the lungs of MM3122 and control treated mice. Global pneumonia, used to assess the % of lung affected, was between 3 (>50%) and 4 (>80%) for the SARS-CoV-2 infected and vehicle treated mice. This score was reduced to 1.2 (P < 0.05) and 1.6 in the mice that received 50 mg/kg and 100 mg/kg of MM3122 prior to virus infection. No change in the global pneumonia score was observed for the mice that received 50 (score = 3) and 100 (score = 3.2) mg/kg of MM3122 after virus infection. Similarly, the bronchointerstitial pneumonia score was reduced in the animals that received MM3122 prior to infection (1.2 and 1.5 for 50 mg/kg (P < 0.05) and 100 mg/kg respectively) but not after SARS-CoV-2 infection. Finally, vasculitis and endotheliitis replicated control baseline levels (average score = 0.3) in the prophylactically treated group (score = 0.4 and 0.3 for 50 mg/kg and 100 mg/kg respectively). A reduction in score was also observed for the mice that received MM3122 after infection (score = 1.2 and 1.8 for 50 mg/kg and 100 mg/kg respectively), but this was not statistically significant compared to the infected and vehicle control treated animals (score = 2.8).

Figure 2:
Figure 2: MM3122 protects mice against SARS-CoV-2 disease in mice.

Eleven-to twelve-month-old female mice received MM3122 30 minutes before and 24 hours after intranasal inoculation with 1,000 PFU of MA10. (A) Weight loss was measured daily for five days. (B) Infectious virus titer in left lung lobe 5 days after infection. (C) Lung congestion score 5 days after infection. (D) Representative images and magnifications of H&E sections of lungs from uninfected and vehicle control treated mice (left panel), SARS-CoV-2 infected, and vehicle control treated mice (middle panels), and MM3122 treated and SARS-CoV-2 infected mice (right panels). (E) Global pneumonia, vasculitis and endotheliitis, and bronchointerstitial pathology scores of these same animals. All data was analyzed by a non-parametric one-way ANOVA (Kruskal-Wallis) with multiple comparisons corrects against the SARS-CoV-2 + vehicle group. The dotted line is the limit of detection. Each data point is an individual mouse, and the data are from a single experiment with 3-4 mice per group. ** = P < 0.01, ns = not significant.

MM3122 reduces inflammatory cytokine and chemokine production after SARS-CoV-2 infection

Weight loss and severe disease after SARS-CoV-2 infection is associated with exacerbated inflammatory responses resulting in lung congestion and immunopathology. To assess inflammation, cytokine and chemokine concentrations were quantified in lung tissue homogenates using a mouse cytokine 23-plex assay. Compared to uninfected and vehicle control animals, the levels of IL-6, KC, G-CSF, CCL2, CCL3, CCL4, IL1 alpha, IL-12p40 and CCL5 were increased three-fold or more five days after infection with the MA10 strain of SARS-CoV-2 (Fig 3). Prophylactic and therapeutic treatment with MM3122 significantly (P < 0.001) reduced the amount of IL-6, KC, G-CSF, CCL2 and CCL3 in the lungs of these mice. Smaller reductions in cytokine and chemokine productions were observed for CCL4, IL-1 alpha, IL-12p40, and CCL5. Combined, these data show that the TMPRSS2 inhibitor MM3122 reduces virus titers and inflammation after SARS-CoV-2 infection.

Figure 3:
Figure 3: Prophylactic and therapeutic administration of MM3122 reduces inflammation in mice.

Eleven-to twelve-month-old female mice received MM3122 30 minutes before and 24 hours after intranasal inoculation with 1,000 PFU of SARS-CoV-2 MA10. Cytokine and chemokine concentrations in left lung lobe homogenates collected from these same animals. Data was analyzed by a parametric ordinary one-way ANOVA with multiple comparisons corrects against the SARS-CoV-2 + vehicle group. Each data point is an individual mouse, and the data are from a single experiment with 3-4 mice per group. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, ns = not significant.

DISCUSSION

The COVID-19 pandemic began 4 years ago and is still a major economic catastrophe and medical problem worldwide costing over $14 trillion in economic losses in the US45 and resulting in excess mortality rates estimated at exceed 24 million people worldwide through early 202346. While highly efficacious vaccines have saved millions of lives globally47, they are not 100% effective, even less so for variants, and a large percentage of the population refuses to be vaccinated so there is a dire need for new drugs to prevent and treat this life-threatening disease. There are some small molecule FDA-approved drugs to treat COVID-191 including the viral polymerase inhibitors remdesivir and molnupiravir, as well as SARS-CoV-2 Mpro protease inhibitor nirmatrelvir, sold under the brand name Paxlovid. With the exception immunomodulatory treatments for COVID-19 such as the JAK1/JAK2 inhibitor baricitinib48–50, which target the symptoms of infection and not viral pathogenesis directly, there are no approved antiviral drugs, which target the host and confer protection against multiple different viruses from diverse viral families. Concomitantly, others, and we have reported on the first potential drugs, targeting the TMPRSS2 and matriptase, to treat SARS-CoV-2 and COVID-1939, 42. TMPRSS2, and other host cell transmembrane proteases51 including matriptase are essential for viral entry of many respiratory RNA viruses into the lung tissue. In this communication, we have demonstrated that one lead candidate drug that we have developed, MM3122, exhibited significant protective effects against weight loss, lung congestion (gross lung discoloration), and inflammation administered prophylactically at both low and high doses, in aged mice infected with mouse-adapted SARS-CoV-2. These effects were less pronounced in the therapeutic groups. Interestingly, these protections did not fully correspond with protection from viral replication, as titers were lower or absent in the prophylactic group but were not significantly different from the infected vehicle control in the therapeutic group. These protective effects of COVID-19 in the lung can potentially be explained by inhibition of the proteolytic activation of other substrates by TMPRSS2, matriptase, and hepsin or the other proteases, which MM3122 targets such as tryptase (Table 1). In summary, these promising results suggest MM3122 is a potential clinical candidate for the treatment and prevention of diseases including COVID-19, caused from infection by SARS-CoV-2 and other coronaviruses.

Limitations of the study

We note several limitations of our study. (a) The in vivo efficacy of MM3122 was not tested against more recent variants of SARS-CoV-2 in mice or in Syrian hamsters. For the latter, we will need to perform PK studies, which is beyond the scope of this study. Also, the more recent variants of SARS-CoV-2 are attenuated compared to MA10 strain of SARS-CoV-2, creating challenges in observing effects on weight loss and disease of MM3122. Also, given that MM3122 has similar activity against EG.5.1 in vitro, we do not expect differences in vivo. (b) We did not evaluate the efficacy of MM3122 in male mice. Males are considered more susceptible to SARS-CoV-2 infection and therefore we expect more disease in the untreated animals with continued amelioration of disease in the MM3122 treated animals. (c) To increase efficacy, we are currently working on developing improved compounds with longer half-life and higher compound exposure compared to MM3122.39 One potential way to achieve increased efficacy with MM3122 would be to develop formulations for inhalation or nebulization as alternative administration routes, which would deliver compound directly to the respiratory tract at the primary site of viral entry and the infection. In summary, these studies demonstrate that MM3122 is effective in inhibiting SARS-CoV-2 replication in vitro and that administration of MM3122 in vivo reduces COVID-19 disease in mice.

MATERIALS AND METHODS

Cells and Viruses

Vero cells expressing human angiotensin converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2) (Vero-hACE2-hTMPRSS252, 53, gift from Adrian Creanga and Barney Graham, NIH) were cultured at 37°C in Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES (pH 7.3), 100 U/mL of Penicillin, 100 µg/mL of Streptomycin, and 10 µg/mL of puromycin. Vero cells expressing TMPRSS2 (Vero-hTMPRSS2)53 were cultured at 37°C in DMEM supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES (pH 7.3), 100 U/mL of Penicillin, 100µg/mL of Streptomycin, and 5 µg/mL of blasticidin. Calu-3 cells were cultured in DMEM media supplemented with 1.0 mM sodium pyruvate, non-essential amino-acids (NEAA), 100 U/mL of penicillin, 100 µg/mL streptomycin, 2.0 mM L-glutamine, 10 mM HEPES, and 10% Fetal Bovine Serum (FBS).

The Lineage A variant of SARS-CoV-2 (WA1/2020), or the XBB.1.5 and E.G.5.1 (from Mehul Suthar) variants of SARS-CoV-2 were propagated on Vero-hTMPRSS2 cells. The virus stocks were subjected to next-generation sequencing, and the S protein sequences were identical to the original isolates. The infectious virus titer was determined by plaque and focus-forming assay on Vero-hACE2-hTMPRSS2 or Vero-hTMPRSS2 cells.

Baric laboratory-generated stock of SARS-CoV-2 MA10, a mouse-adapted virulent mutant generated from a recombinantly derived synthesized sequence of the Washington strain that causes severe acute and chronic disease in mice54, 55. Virus was maintained at low passage (P2-P3) to prevent the accumulation of additional potentially confounding mutations.

Drug Preparation and Administration

MM312239 was freshly prepared at 8 mg/mL in 5% DMSO in PBS. MM3122 was administered intraperitoneal (IP) at 50 and 100 mg/kg bodyweight in 100µL volumes to mice starting at 30 minutes before infection (prophylactic treatment) or 24 hours after infection (day 1, therapeutic treatment). Subsequent doses were administered at approximately the same times each day post-infection.

SARS-CoV-2 challenge studies

All studies with mice were conducted under the University of North Carolina IACUC approval (20-114). Aged (11-to 12-month-old) female BALB/c mice obtained from Envigo (retired breeders) were acclimated for 7 days in the Biosafety laboratory level 3 prior to any experimentation. Food and water were provided ad libitum, and the animal room maintained a 12-hour light/dark cycle. Prior to inoculation with SARS-CoV-2, animals were anesthetized intraperitoneal with a combination of 50 mg/kg Ketamine and 15 mg/kg Xylazine in 50 µL, and infected intranasally with 1,000 PFU of sequence-and titer-verified SARS-CoV-2 MA10 in 50 µL PBS. Mice were monitored daily for weight loss and disease. At 5 days post-infection, mice were euthanized following sedation by isoflurane and thoracotomy, and lungs were collected for assessments of virus titer, inflammatory cytokines and chemokine levels, histological analysis, and lung congestion score. Lung congestion score was measured using an independently defined scale of 0-4 (0: no congestion; 1: one lobe involved; 2: two lobes involved; 3: three lobes involved; 4: all four lobes involved; all scores have 0.5-point intervals). Lung sections used in all experiments for various assessments were as follows: lower right lobe, histology; upper right lobe, RNA; left lobe and central lobe, virus titer. Prior to virus titration and cytokine analysis, the lung lobes were homogenized with glass beads in 1.0 mL PBS, clarified by centrifugation and stored at -80°C. Infectious virus titers were quantified by plaque assay on Vero E6 cells and calculated at PFU/mL of homogenized tissue.

Cytokine Analysis

Homogenized lung samples were subjected to cytokine and chemokine analysis using the Bio-Rad Mouse Cytokine 23-plex assay (Cat # M60009RDPD) per the manufacturer’s protocol. Cytokine assays were performed in non-inactivated samples at BSL3. The data were analyzed on a Luminex MAGPIX machine, and cytokine concentrations in the lung homogenates were extrapolated using the provided standards.

Histological analysis

Lung tissues from the lower right lobe were fixed for a minimum of 7 days in 10% formalin, paraffin embedded, sectioned, and stained with hematoxylin and eosin (H&E). H&E sections were submitted for graded blindly for vasculitis/endotheliitis, Bronchointerstitial pneumonia, and global pneumonia severity score by a board-certified veterinary pathologist. Details on the histology scoring system are provided in the Supplementary Material.

MM3122 in vitro inhibition assays

Calu-3 cells (5 x 105 cells/well) were seeded in 24-well culture plates in infection medium (DMEM + 1.0 mM Sodium pyruvate, NEAA, 100 U/mL of penicillin, 100 µg/mL streptomycin, 2.0 mM L-glutamine, 10 mM HEPES, and 2% FBS) and incubated overnight at 37°C and 5% CO2. After 24 h, media was removed and fresh 250 µL media was added to each well containing MM3122 or Remdesivir starting at 60 µM concentration and diluted 3-fold to 20, 6, 2, 0.6 and 0.2 µM. Media alone, and DMSO were included as negative controls. Next, the cells were transferred to the BSL3 laboratory and 250 µL of media containing 4,000 PFU of SARS-CoV-2 was added for 1 h at 37°C and 5% CO2. Note, that the final concentration of MM3122 and Remdesivir is 30, 10, 3, 1, 0.3, and 0.1 µM. After 1 h, the virus inoculum was removed, the cells were washed twice with infection media and fresh infection media containing MM3122, Remdesivir or DMSO was added to each well. At 48 h post-infection, culture supernatant is collected and used to quantify virus titers by plaque assay as described below.

Virus titration assays

Plaque assays were performed on Vero-hACE2-hTRMPSS2 cells in 24-well plates. Lung tissue homogenates or nasal washes were diluted serially by 10-fold, starting at 1:10, in cell infection medium (DMEM + 100 U/mL of penicillin, 100 µg/mL streptomycin, and 2% FBS). Two hundred and fifty microliters of the diluted virus were added to a single well per dilution per sample. After 1 h at 37°C, the inoculum was aspirated, the cells were washed with PBS, and a 1% methylcellulose overlay in MEM supplemented with 2% FBS was added. Seventy-two to ninety-six hours after virus inoculation, dependent on the virus strain, the cells were fixed with 4% formalin and the monolayer was stained with crystal violet (0.5% w/v in 25% methanol in water) for 30 min at 20°C. The number of plaques were counted and used to calculate the plaque forming units/mL (PFU/mL).

AUTHOR CONTRIBUTIONS

T.L.B. performed the in vitro inhibition studies on Calu-3 cells. E.J.F., S.R.L., K.G., R.S.B, R.L.G. performed all the in vivo mouse studies with MM3122 and SARS-CoV-2. B.V.T performed the histology experiments. M.M. synthesized the MM3122 compound. T.L.B., A.C.M.B., R.L.G., R.S.B., J.W.J. analyzed the data. A.C.M.B. performed the statistical analysis. A.C.M.B. had unrestricted access to all the data. A.C.M.B, J.W.J. provided key reagents, supervised experiments, and acquired funding. A.C.M.B. and J.W.J. wrote the manuscript and all authors reviewed and edited the final version. All authors agreed to submit the manuscript, read, and approved the final draft, and take full responsibility of its content.

DECLARATION OF INTERESTS

The Boon laboratory has received unrelated funding support in sponsored research agreements from AI Therapeutics, GreenLight Biosciences Inc., Moderna Inc., and Nano targeting & Therapy Biopharma Inc. The Boon laboratory has received funding support from AbbVie Inc., for the commercial development of SARS-CoV-2 mAb. R.S.B. is a member of the advisory board of VaxArt and Invivyd and has collaborations with Takeda, Janssen Pharmaceuticals, Pfizer, Moderna, Ridgeback Biosciences, and Gilead that are unrelated to this work. J.W.J. has a patent application covering the MM3122 compound. R.S.B. and S.R.L. hold a patent on the MA10 strain of SARS-CoV-2.

SUPPLEMENTARY INFORMATION

Scoring criteria for Global pneumonia severity.

Scoring criteria for Vasculitis / Endotheliitis.

Scoring criteria for Bronchointerstitial pneumonia.

ACKNOWLEDGEMENTS

This study was supported by the NIH (NIAID Center of Excellence for Influenza Research and Response (CEIRR)) contract 75N93021C00016 and R01 AI169022 to A.C.M.B and by Washington University School of Medicine, Siteman Cancer Center grant SCC P30CA091842 and Barnes Jewish Hospital Foundation award BJHF 4984 to J.W.J. We also acknowledge preclinical services provided by the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under contract HHSN272201700036I/75N93020F00001 to the University of North Carolina-Chapel Hill.

REFERENCES

  1. 1.↵
    Shoichet BK, Craik CS. Preparing for the next pandemic. Science. 2023;382(6671):649–50. Epub 20231109. doi: 10.1126/science.adk5868. PubMed PMID: 37943911.
    OpenUrlCrossRefPubMed
  2. 2.↵
    Sheahan TP, Sims AC, Zhou S, Graham RL, Pruijssers AJ, Agostini ML, Leist SR, Schäfer A, Dinnon KH, 3rd, Stevens LJ, Chappell JD, Lu X, Hughes TM, George AS, Hill CS, Montgomery SA, Brown AJ, Bluemling GR, Natchus MG, Saindane M, Kolykhalov AA, Painter G, Harcourt J, Tamin A, Thornburg NJ, Swanstrom R, Denison MR, Baric RS. An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice. Sci Transl Med. 2020;12(541). Epub 20200406. doi: 10.1126/scitranslmed.abb5883. PubMed PMID: 32253226; PMCID: PMC7164393.
    OpenUrlAbstract/FREE Full Text
  3. 3.
    Sheahan TP, Sims AC, Graham RL, Menachery VD, Gralinski LE, Case JB, Leist SR, Pyrc K, Feng JY, Trantcheva I, Bannister R, Park Y, Babusis D, Clarke MO, Mackman RL, Spahn JE, Palmiotti CA, Siegel D, Ray AS, Cihlar T, Jordan R, Denison MR, Baric RS. Broad-spectrum antiviral GS-5734 inhibits both epidemic and zoonotic coronaviruses. Sci Transl Med. 2017;9(396). doi: 10.1126/scitranslmed.aal3653. PubMed PMID: 28659436; PMCID: PMC5567817.
    OpenUrlAbstract/FREE Full Text
  4. 4.↵
    Corbett KS, Edwards DK, Leist SR, Abiona OM, Boyoglu-Barnum S, Gillespie RA, Himansu S, Schäfer A, Ziwawo CT, DiPiazza AT, Dinnon KH, Elbashir SM, Shaw CA, Woods A, Fritch EJ, Martinez DR, Bock KW, Minai M, Nagata BM, Hutchinson GB, Wu K, Henry C, Bahl K, Garcia-Dominguez D, Ma L, Renzi I, Kong WP, Schmidt SD, Wang L, Zhang Y, Phung E, Chang LA, Loomis RJ, Altaras NE, Narayanan E, Metkar M, Presnyak V, Liu C, Louder MK, Shi W, Leung K, Yang ES, West A, Gully KL, Stevens LJ, Wang N, Wrapp D, Doria-Rose NA, Stewart-Jones G, Bennett H, Alvarado GS, Nason MC, Ruckwardt TJ, McLellan JS, Denison MR, Chappell JD, Moore IN, Morabito KM, Mascola JR, Baric RS, Carfi A, Graham BS. SARS-CoV-2 mRNA vaccine design enabled by prototype pathogen preparedness. Nature. 2020;586(7830):567-71. Epub 20200805. doi: 10.1038/s41586-020-2622-0. PubMed PMID: 32756549; PMCID: PMC7581537.
    OpenUrlCrossRefPubMed
  5. 5.↵
    Rahbar Saadat Y, Hosseiniyan Khatibi SM, Zununi Vahed S, Ardalan M. Host Serine Proteases: A Potential Targeted Therapy for COVID-19 and Influenza. Front Mol Biosci. 2021;8:725528. Epub 20210830. doi: 10.3389/fmolb.2021.725528. PubMed PMID: 34527703; PMCID: PMC8435734.
    OpenUrlCrossRefPubMed
  6. 6.
    Zhou P, Yang XL, Wang XG, Hu B, Zhang L, Zhang W, Si HR, Zhu Y, Li B, Huang CL, Chen HD, Chen J, Luo Y, Guo H, Jiang RD, Liu MQ, Chen Y, Shen XR, Wang X, Zheng XS, Zhao K, Chen QJ, Deng F, Liu LL, Yan B, Zhan FX, Wang YY, Xiao GF, Shi ZL. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020;579(7798):270-3. Epub 20200203. doi: 10.1038/s41586-020-2012-7. PubMed PMID: 32015507; PMCID: PMC7095418.
    OpenUrlCrossRefPubMed
  7. 7.
    Hoffmann M, Kleine-Weber H, Schroeder S, Krüger N, Herrler T, Erichsen S, Schiergens TS, Herrler G, Wu NH, Nitsche A, Müller MA, Drosten C, Pöhlmann 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. Epub 20200305. doi: 10.1016/j.cell.2020.02.052. PubMed PMID: 32142651; PMCID: PMC7102627.
    OpenUrlCrossRefPubMed
  8. 8.↵
    Gomes CP, Fernandes DE, Casimiro F, da Mata GF, Passos MT, Varela P, Mastroianni-Kirsztajn G, Pesquero JB. Cathepsin L in COVID-19: From Pharmacological Evidences to Genetics. Front Cell Infect Microbiol. 2020;10:589505. Epub 20201208. doi: 10.3389/fcimb.2020.589505. PubMed PMID: 33364201; PMCID: PMC7753008.
    OpenUrlCrossRefPubMed
  9. 9.↵
    Vaarala MH, Porvari KS, Kellokumpu S, Kyllonen AP, Vihko PT. Expression of transmembrane serine protease TMPRSS2 in mouse and human tissues. The Journal of pathology. 2001;193(1):134–40. Epub 2001/02/13. doi: 10.1002/1096-9896(2000)9999:9999<::AID-PATH743>3.0.CO;2-T. PubMed PMID: 11169526.
    OpenUrlCrossRefPubMedWeb of Science
  10. 10.
    Vaarala MH, Porvari K, Kyllonen A, Lukkarinen O, Vihko P. The TMPRSS2 gene encoding transmembrane serine protease is overexpressed in a majority of prostate cancer patients: detection of mutated TMPRSS2 form in a case of aggressive disease. Int J Cancer. 2001;94(5):705–10. Epub 2001/12/18. doi: 10.1002/ijc.1526. PubMed PMID: 11745466.
    OpenUrlCrossRefPubMedWeb of Science
  11. 11.↵
    Afar DE, Vivanco I, Hubert RS, Kuo J, Chen E, Saffran DC, Raitano AB, Jakobovits A. Catalytic cleavage of the androgen-regulated TMPRSS2 protease results in its secretion by prostate and prostate cancer epithelia. Cancer Res. 2001;61(4):1686–92. Epub 2001/03/14. PubMed PMID: 11245484.
    OpenUrlAbstract/FREE Full Text
  12. 12.↵
    Damalanka VC, Janetka JW. Recent progress on inhibitors of the type II transmembrane serine proteases, hepsin, matriptase and matriptase-2. Future Med Chem. 2019;11(7):743–69. Epub 20190404. doi: 10.4155/fmc-2018-0446. PubMed PMID: 30945556.
    OpenUrlCrossRefPubMed
  13. 13.
    Tanabe LM, List K. The role of type II transmembrane serine protease-mediated signaling in cancer. FEBS J. 2017;284(10):1421–36. Epub 2016/11/22. doi: 10.1111/febs.13971. PubMed PMID: 27870503; PMCID: PMC5432387.
    OpenUrlCrossRefPubMed
  14. 14.
    Kuhn N, Bergmann S, Kasnitz N, Lambertz RL, Keppner A, van den Brand JM, Pohlmann S, Weiss S, Hummler E, Hatesuer B, Schughart K. The proteolytic activation of A (H3N2) Influenza virus hemagglutinin is facilitated by different type II transmembrane serine proteases. Journal of Virology. 2016. Epub 2016/02/19. doi: 10.1128/JVI.02693-15. PubMed PMID: 26889029.
    OpenUrlAbstract/FREE Full Text
  15. 15.↵
    Lucas JM, Heinlein C, Kim T, Hernandez SA, Malik MS, True LD, Morrissey C, Corey E, Montgomery B, Mostaghel E, Clegg N, Coleman I, Brown CM, Schneider EL, Craik C, Simon JA, Bedalov A, Nelson PS. The androgen-regulated protease TMPRSS2 activates a proteolytic cascade involving components of the tumor microenvironment and promotes prostate cancer metastasis. Cancer Discov. 2014;4(11):1310–25. Epub 2014/08/15. doi: 10.1158/2159-8290.CD-13-1010. PubMed PMID: 25122198; PMCID: PMC4409786.
    OpenUrlAbstract/FREE Full Text
  16. 16.↵
    Glowacka I, Bertram S, Muller MA, Allen P, Soilleux E, Pfefferle S, Steffen I, Tsegaye TS, He Y, Gnirss K, Niemeyer D, Schneider H, Drosten C, Pohlmann S. Evidence that TMPRSS2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response. J Virol. 2011;85(9):4122–34. Epub 2011/02/18. doi: 10.1128/JVI.02232-10. PubMed PMID: 21325420; PMCID: PMC3126222.
    OpenUrlAbstract/FREE Full Text
  17. 17.↵
    Bertram S, Glowacka I, Müller MA, Lavender H, Gnirss K, Nehlmeier I, Niemeyer D, He Y, Simmons G, Drosten C, Soilleux EJ, Jahn O, Steffen I, Pöhlmann S. Cleavage and activation of the severe acute respiratory syndrome coronavirus spike protein by human airway trypsin-like protease. J Virol. 2011;85(24):13363–72. Epub 20111012. doi: 10.1128/jvi.05300-11. PubMed PMID: 21994442; PMCID: PMC3233180.
    OpenUrlAbstract/FREE Full Text
  18. 18.↵
    Saunders N, Fernandez I, Planchais C, Michel V, Rajah MM, Baquero Salazar E, Postal J, Porrot F, Guivel-Benhassine F, Blanc C, Chauveau-Le Friec G, Martin A, Grzelak L, Oktavia RM, Meola A, Ahouzi O, Hoover-Watson H, Prot M, Delaune D, Cornelissen M, Deijs M, Meriaux V, Mouquet H, Simon-Lorière E, van der Hoek L, Lafaye P, Rey F, Buchrieser J, Schwartz O. TMPRSS2 is a functional receptor for human coronavirus HKU1. Nature. 2023;624(7990):207-14. Epub 20231025. doi: 10.1038/s41586-023-06761-7. PubMed PMID: 37879362.
    OpenUrlCrossRefPubMed
  19. 19.↵
    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. Epub 2013/09/13. doi: 10.1128/JVI.01890-13. PubMed PMID: 24027332; PMCID: PMC3838146.
    OpenUrlAbstract/FREE Full Text
  20. 20.↵
    Bertram S, Dijkman R, Habjan M, Heurich A, Gierer S, Glowacka I, Welsch K, Winkler M, Schneider H, Hofmann-Winkler H, Thiel V, Pohlmann S. TMPRSS2 activates the human coronavirus 229E for cathepsin-independent host cell entry and is expressed in viral target cells in the respiratory epithelium. J Virol. 2013;87(11):6150–60. Epub 2013/03/29. doi: 10.1128/JVI.03372-12. PubMed PMID: 23536651; PMCID: PMC3648130.
    OpenUrlAbstract/FREE Full Text
  21. 21.↵
    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. Epub 2013/03/08. doi: 10.1128/JVI.00128-13. PubMed PMID: 23468491; PMCID: PMC3648152.
    OpenUrlAbstract/FREE Full Text
  22. 22.↵
    Beaulieu A, Gravel É, Cloutier A, Marois I, Colombo É, Désilets A, Verreault C, Leduc R, Marsault É, Richter MV. Matriptase Proteolytically Activates Influenza Virus and Promotes Multicycle Replication in the Human Airway Epithelium. Journal of Virology. 2013;87(8):4237–51. doi: doi:10.1128/jvi.03005-12.
    OpenUrlAbstract/FREE Full Text
  23. 23.↵
    Whittaker GR, Straus MR. Human matriptase/ST 14 proteolytically cleaves H7N9 hemagglutinin and facilitates the activation of influenza A/Shanghai/2/2013 virus in cell culture. Influenza Other Respir Viruses. 2020;14(2):189–95. Epub 2019/12/11. doi: 10.1111/irv.12707. PubMed PMID: 31820577; PMCID: PMC7040964.
    OpenUrlCrossRefPubMed
  24. 24.↵
    Baron J, Tarnow C, Mayoli-Nussle D, Schilling E, Meyer D, Hammami M, Schwalm F, Steinmetzer T, Guan Y, Garten W, Klenk HD, Bottcher-Friebertshauser E. Matriptase, HAT, and TMPRSS2 activate the hemagglutinin of H9N2 influenza A viruses. J Virol. 2013;87(3):1811–20. Epub 2012/11/30. doi: 10.1128/JVI.02320-12. PubMed PMID: 23192872; PMCID: PMC3554176.
    OpenUrlAbstract/FREE Full Text
  25. 25.↵
    Bottcher E, Matrosovich T, Beyerle M, Klenk HD, Garten W, Matrosovich M. Proteolytic activation of influenza viruses by serine proteases TMPRSS2 and HAT from human airway epithelium. J Virol. 2006;80(19):9896–8. Epub 2006/09/16. doi: 10.1128/JVI.01118-06. PubMed PMID: 16973594; PMCID: PMC1617224.
    OpenUrlAbstract/FREE Full Text
  26. 26.↵
    Bottcher-Friebertshauser E, Freuer C, Sielaff F, Schmidt S, Eickmann M, Uhlendorff J, Steinmetzer T, Klenk HD, Garten W. Cleavage of influenza virus hemagglutinin by airway proteases TMPRSS2 and HAT differs in subcellular localization and susceptibility to protease inhibitors. Journal of Virology. 2010;84(11):5605–14. Epub 2010/03/20. doi: 10.1128/JVI.00140-10. PubMed PMID: 20237084; PMCID:
    OpenUrlAbstract/FREE Full Text
  27. 27.
    pmcid:2876594.
  28. 27.
    Paszti-Gere E, Czimmermann E, Ujhelyi G, Balla P, Maiwald A, Steinmetzer T. In vitro characterization of TMPRSS2 inhibition in IPEC-J2 cells. J Enzyme Inhib Med Chem. 2016;31(sup2):123–9. Epub 2016/06/10. doi: 10.1080/14756366.2016.1193732. PubMed PMID: 27277342.
    OpenUrlCrossRefPubMed
  29. 28.
    Bertram S, Glowacka I, Blazejewska P, Soilleux E, Allen P, Danisch S, Steffen I, Choi SY, Park Y, Schneider H, Schughart K, Pohlmann S. TMPRSS2 and TMPRSS4 facilitate trypsin-independent spread of influenza virus in Caco-2 cells. J Virol. 2010;84(19):10016–25. Epub 2010/07/16. doi: 10.1128/JVI.00239-10. PubMed PMID: 20631123; PMCID: PMC2937781.
    OpenUrlAbstract/FREE Full Text
  30. 29.
    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. Epub 2013/12/19. doi: 10.1371/journal.ppat.1003774. PubMed PMID: 24348248; PMCID: PMC3857797.
    OpenUrlCrossRefPubMed
  31. 30.↵
    Lambertz RLO, Gerhauser I, Nehlmeier I, Gartner S, Winkler M, Leist SR, Kollmus H, Pohlmann S, Schughart K. H2 influenza A virus is not pathogenic in Tmprss2 knock-out mice. Virol J. 2020;17(1):56. Epub 2020/04/24. doi: 10.1186/s12985-020-01323-z. PubMed PMID: 32321537; PMCID: PMC7178614.
    OpenUrlCrossRefPubMed
  32. 31.↵
    Tarnow C, Engels G, Arendt A, Schwalm F, Sediri H, Preuss A, Nelson PS, Garten W, Klenk HD, Gabriel G, Bottcher-Friebertshauser E. TMPRSS2 is a host factor that is essential for pneumotropism and pathogenicity of H7N9 influenza A virus in mice. J Virol. 2014;88(9):4744–51. Epub 2014/02/14. doi: 10.1128/jvi.03799-13. PubMed PMID: 24522916; PMCID: 3993819.
    OpenUrlAbstract/FREE Full Text
  33. 32.↵
    Abe M, Tahara M, Sakai K, Yamaguchi H, Kanou K, Shirato K, Kawase M, Noda M, Kimura H, Matsuyama S, Fukuhara H, Mizuta K, Maenaka K, Ami Y, Esumi M, Kato A, Takeda M. TMPRSS2 is an activating protease for respiratory parainfluenza viruses. J Virol. 2013;87(21):11930–5. Epub 20130821. doi: 10.1128/jvi.01490-13. PubMed PMID: 23966399; PMCID: PMC3807344.
    OpenUrlAbstract/FREE Full Text
  34. 33.↵
    Yang H, Lin X, Yu Q, Awadasseid A, Zhang W. Repurposing and discovery of transmembrane serine protease 2 (TMPRSS2) inhibitors as prophylactic therapies for new coronavirus disease 2019 (COVID-19). Pharmazie. 2023;78(11):217–24. doi: 10.1691/ph.2023.3578. PubMed PMID: 38178286.
    OpenUrlCrossRefPubMed
  35. 34.
    Meyer D, Sielaff F, Hammami M, Bottcher-Friebertshauser E, Garten W, Steinmetzer T. Identification of the first synthetic inhibitors of the type II transmembrane serine protease TMPRSS2 suitable for inhibition of influenza virus activation. Biochem J. 2013;452(2):331–43. Epub 2013/03/27. doi: 10.1042/BJ20130101. PubMed PMID: 23527573.
    OpenUrlAbstract/FREE Full Text
  36. 35.
    Colombo E, Duchene D, Desilets A, Beaulieu A, Gravel E, Najmanovich R, Richter M, Leduc R, Marsault E. New matriptase inhibitors as a potential treatment against influenza. Abstracts of Papers of the American Chemical Society. 2013;245. PubMed PMID: ISI:000324303602432.
    OpenUrlPubMedWeb of Science
  37. 36.
    Colombo E, Desilets A, Hassanzadeh M, Lemieux G, Marois I, Cliche D, Delbrouck JA, Murza A, Jean F, Marsault E, Richter MV, Leduc R, Boudreault PL. Optimization of Ketobenzothiazole-Based Type II Transmembrane Serine Protease Inhibitors to Block H1N1 Influenza Virus Replication. ChemMedChem. 2023:e202300458. Epub 20231021. doi: 10.1002/cmdc.202300458. PubMed PMID: 37864572.
    OpenUrlCrossRefPubMed
  38. 37.
    Pilgram O, Keils A, Benary GE, Müller J, Merkl S, Ngaha S, Huber S, Chevillard F, Harbig A, Magdolen V, Heine A, Böttcher-Friebertshäuser E, Steinmetzer T. Improving the selectivity of 3-amidinophenylalanine-derived matriptase inhibitors. European Journal of Medicinal Chemistry. 2022;238:114437. doi: 10.1016/j.ejmech.2022.114437.
    OpenUrlCrossRef
  39. 38.↵
    Bestle D, Heindl MR, Limburg H, van TVL, Pilgram O, Moulton H, Stein DA, Hardes K, Eickmann M, Dolnik O, Rohde C, Klenk H-D, Garten W, Steinmetzer T, Böttcher-Friebertshäuser E. TMPRSS2 and furin are both essential for proteolytic activation of SARS-CoV-2 in human airway cells. Life Science Alliance. 2020;3(9):e202000786. doi: 10.26508/lsa.202000786.
    OpenUrlAbstract/FREE Full Text
  40. 39.↵
    Mahoney M, Damalanka VC, Tartell MA, Chung DH, Lourenço AL, Pwee D, Mayer Bridwell AE, Hoffmann M, Voss J, Karmakar P, Azouz NP, Klingler AM, Rothlauf PW, Thompson CE, Lee M, Klampfer L, Stallings CL, Rothenberg ME, Pöhlmann S, Whelan SPJ, O’Donoghue AJ, Craik CS, Janetka JW. A novel class of TMPRSS2 inhibitors potently block SARS-CoV-2 and MERS-CoV viral entry and protect human epithelial lung cells. Proc Natl Acad Sci U S A. 2021;118(43). doi: 10.1073/pnas.2108728118. PubMed PMID: 34635581; PMCID: PMC8694051.
    OpenUrlAbstract/FREE Full Text
  41. 40.↵
    Fraser BJ, Beldar S, Seitova A, Hutchinson A, Mannar D, Li Y, Kwon D, Tan R, Wilson RP, Leopold K, Subramaniam S, Halabelian L, Arrowsmith CH, Benard F. Structure and activity of human TMPRSS2 protease implicated in SARS-CoV-2 activation. Nat Chem Biol. 2022;18(9):963–71. Epub 20220608. doi: 10.1038/s41589-022-01059-7. PubMed PMID: 35676539.
    OpenUrlCrossRefPubMed
  42. 41.↵
    Hoffmann M, Hofmann-Winkler H, Smith JC, Kruger N, Arora P, Sorensen LK, Sogaard OS, Hasselstrom JB, Winkler M, Hempel T, Raich L, Olsson S, Danov O, Jonigk D, Yamazoe T, Yamatsuta K, Mizuno H, Ludwig S, Noe F, Kjolby M, Braun A, Sheltzer JM, Pohlmann S. Camostat mesylate inhibits SARS-CoV-2 activation by TMPRSS2-related proteases and its metabolite GBPA exerts antiviral activity. EBioMedicine. 2021;65:103255. Epub 2021/03/08. doi: 10.1016/j.ebiom.2021.103255. PubMed PMID: 33676899; PMCID: PMC7930809.
    OpenUrlCrossRefPubMed
  43. 42.↵
    Shapira T, Monreal IA, Dion SP, Buchholz DW, Imbiakha B, Olmstead AD, Jager M, Désilets A, Gao G, Martins M, Vandal T, Thompson CAH, Chin A, Rees WD, Steiner T, Nabi IR, Marsault E, Sahler J, Diel DG, Van de Walle GR, August A, Whittaker GR, Boudreault P-L, Leduc R, Aguilar HC, Jean F. A TMPRSS2 inhibitor acts as a pan-SARS-CoV-2 prophylactic and therapeutic. Nature. 2022;605(7909):340-8. doi: 10.1038/s41586-022-04661-w.
    OpenUrlCrossRefPubMed
  44. 43.↵
    Lin HXJ, Cho S, Meyyur Aravamudan V, Sanda HY, Palraj R, Molton JS, Venkatachalam I. Remdesivir in Coronavirus Disease 2019 (COVID-19) treatment: a review of evidence. Infection. 2021;49(3):401–10. Epub 20210102. doi: 10.1007/s15010-020-01557-7. PubMed PMID: 33389708; PMCID: PMC7778417.
    OpenUrlCrossRefPubMed
  45. 44.↵
    Shrimp JH, Kales SC, Sanderson PE, Simeonov A, Shen M, Hall MD. An Enzymatic TMPRSS2 Assay for Assessment of Clinical Candidates and Discovery of Inhibitors as Potential Treatment of COVID-19. ACS Pharmacology & Translational Science. 2020;3(5):997–1007. doi: 10.1021/acsptsci.0c00106.
    OpenUrlCrossRef
  46. 45.↵
    Walmsley T, Rose A, John R, Wei D, Hlávka JP, Machado J, Byrd K. Macroeconomic consequences of the COVID-19 pandemic. Economic Modelling. 2023;120:106147. doi: 10.1016/j.econmod.2022.106147.
    OpenUrlCrossRef
  47. 46.↵
    Msemburi W, Karlinsky A, Knutson V, Aleshin-Guendel S, Chatterji S, Wakefield J. The WHO estimates of excess mortality associated with the COVID-19 pandemic. Nature. 2023;613(7942):130-7. doi: 10.1038/s41586-022-05522-2.
    OpenUrlCrossRefPubMed
  48. 47.↵
    Agrawal V, Sood N, Whaley CM. The Impact of the Global COVID-19 Vaccination Campaign on All-Cause Mortality. National Bureau of Economic Research Working Paper Series. 2023;No. 31812. doi: 10.3386/w31812.
    OpenUrlCrossRef
  49. 48.
    Hall FC, Cheriyan J, Cope AP, Galloway J, Wilkinson I, Bond S, Norton S, Banham-Hall E, Bayes H, Kostapanos M, Nodale M, Petchey WG, Sheeran T, Underwood J, Jayne DR, Group T-RI. Efficacy and safety of baricitinib or ravulizumab in adult patients with severe COVID-19 (TACTIC-R): a randomised, parallel-arm, open-label, phase 4 trial. Lancet Respir Med. 2023;11(12):1064–74. Epub 20231114. doi: 10.1016/S2213-2600(23)00376-4. PubMed PMID: 37977159; PMCID: PMC10682367.
    OpenUrlCrossRefPubMed
  50. 49.↵
    Dastan F, Jamaati H, Barati S, Varmazyar S, Yousefian S, Niknami E, Tabarsi P. The effects of combination-therapy of tocilizumab and baricitinib on the management of severe COVID-19 cases: a randomized open-label clinical trial. Front Pharmacol. 2023;14:1265541. Epub 20231019. doi: 10.3389/fphar.2023.1265541. PubMed PMID: 37927607; PMCID: PMC10620525.
    OpenUrlCrossRefPubMed
  51. 50.↵
    Song W, Sun S, Feng Y, Liu L, Gao T, Xian S, Chen J. Efficacy and safety of baricitinib in patients with severe COVID-19: A systematic review and meta-analysis. Medicine (Baltimore). 2023;102(48):e36313. doi: 10.1097/MD.0000000000036313. PubMed PMID: 38050265; PMCID: PMC10695502.
    OpenUrlCrossRefPubMed
  52. 51.↵
    Fuentes-Prior P. Priming of SARS-CoV-2 S protein by several membrane-bound serine proteinases could explain enhanced viral infectivity and systemic COVID-19 infection. J Biol Chem. 2021;296:100135. Epub 20201206. doi: 10.1074/jbc.REV120.015980. PubMed PMID: 33268377; PMCID: PMC7834812.
    OpenUrlAbstract/FREE Full Text
  53. 52.↵
    Chen RE, Zhang X, Case JB, Winkler ES, Liu Y, VanBlargan LA, Liu J, Errico JM, Xie X, Suryadevara N, Gilchuk P, Zost SJ, Tahan S, Droit L, Turner JS, Kim W, Schmitz AJ, Thapa M, Wang D, Boon ACM, Presti RM, O’Halloran JA, Kim AHJ, Deepak P, Pinto D, Fremont DH, Crowe JE, Jr., Corti D, Virgin HW, Ellebedy AH, Shi PY, Diamond MS. Resistance of SARS-CoV-2 variants to neutralization by monoclonal and serum-derived polyclonal antibodies. Nat Med. 2021. Epub 2021/03/06. doi: 10.1038/s41591-021-01294-w. PubMed PMID: 33664494.
    OpenUrlCrossRefPubMed
  54. 53.↵
    Zang R, Gomez Castro MF, McCune BT, Zeng Q, Rothlauf PW, Sonnek NM, Liu Z, Brulois KF, Wang X, Greenberg HB, Diamond MS, Ciorba MA, Whelan SPJ, Ding S. TMPRSS2 and TMPRSS4 promote SARS-CoV-2 infection of human small intestinal enterocytes. Sci Immunol. 2020;5(47). Epub 2020/05/15. doi: 10.1126/sciimmunol.abc3582. PubMed PMID: 32404436.
    OpenUrlAbstract/FREE Full Text
  55. 54.↵
    Leist SR, Dinnon KH, 3rd, Schäfer A, Tse LV, Okuda K, Hou YJ, West A, Edwards CE, Sanders W, Fritch EJ, Gully KL, Scobey T, Brown AJ, Sheahan TP, Moorman NJ, Boucher RC, Gralinski LE, Montgomery SA, Baric RS. A Mouse-Adapted SARS-CoV-2 Induces Acute Lung Injury and Mortality in Standard Laboratory Mice. Cell. 2020;183(4):1070–85.e12. Epub 20200923. doi: 10.1016/j.cell.2020.09.050. PubMed PMID: 33031744; PMCID: PMC7510428.
    OpenUrlCrossRefPubMed
  56. 55.
    Dinnon KH, 3rd, Leist SR, Okuda K, Dang H, Fritch EJ, Gully KL, De la Cruz G, Evangelista MD, Asakura T, Gilmore RC, Hawkins P, Nakano S, West A, Schäfer A, Gralinski LE, Everman JL, Sajuthi SP, Zweigart MR, Dong S, McBride J, Cooley MR, Hines JB, Love MK, Groshong SD, VanSchoiack A, Phelan SJ, Liang Y, Hether T, Leon M, Zumwalt RE, Barton LM, Duval EJ, Mukhopadhyay S, Stroberg E, Borczuk A, Thorne LB, Sakthivel MK, Lee YZ, Hagood JS, Mock JR, Seibold MA, O’Neal WK, Montgomery SA, Boucher RC, Baric RS. SARS-CoV-2 infection produces chronic pulmonary epithelial and immune cell dysfunction with fibrosis in mice. Sci Transl Med. 2022;14(664):eabo5070. Epub 20220928. doi: 10.1126/scitranslmed.abo5070. PubMed PMID: 35857635; PMCID: PMC9273046.
    OpenUrlCrossRefPubMed
Back to top
Posted February 12, 2024.
Download PDF
Email
Efficacy of Host Cell Serine Protease Inhibitor MM3122 against SARS-CoV-2 for Treatment and Prevention of COVID-19
Adrianus C.M. Boon, Traci L. Bricker, Ethan J. Fritch, Sarah R. Leist, Kendra Gully, Ralph S. Baric, Rachel L. Graham, Brigid V. Troan, Matthew Mahoney, James W. Janetka
bioRxiv 2024.02.09.579701; doi: https://doi.org/10.1101/2024.02.09.579701
Twitter logo Facebook logo LinkedIn logo Mendeley logo
Citation Tools

Subject Area