key: cord-0948352-hn82d2k7
authors: Rocha, Savannah M.; Fagre, Anna C.; Latham, Amanda S.; Popichak, Katriana A.; McDermott, Casey P.; Dawson, Clinton C.; Cummings, Jason E.; Lewis, Juliette; Reigan, Philip; Aboellail, Tawfik A.; Kading, Rebekah C.; Schountz, Tony; Theise, Neil D.; Slayden, Richard A.; Tjalkens, Ronald B.
title: A novel glucocorticoid and androgen receptor modulator reduces viral entry and innate immune inflammatory responses in the Syrian Hamster model of SARS-CoV-2
date: 2021-02-22
journal: bioRxiv
DOI: 10.1101/2021.02.20.432110
sha: b5565b380dc9b69d3257d36192d26fe0b604817e
doc_id: 948352
cord_uid: hn82d2k7

Since its initial discovery in late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of COVID19, has spread worldwide and despite significant research efforts, treatment options remain limited. Replication of SARS-CoV-2 in lung is associated with marked infiltration of macrophages and activation of innate immune inflammatory responses triggered, in part, by heightened production of interleukin-6 (IL-6) that recruits lymphocytes to the site of infection that amplify tissue injury. Antagonists of the glucocorticoid and androgen receptors have shown promise in experimental models of COVID19 and in clinical studies, because cell surface proteins required for viral entry, angiotensin converting enzyme 2 (ACE2) and the transmembrane serine protease 2 (TMPRSS2), are transcriptionally regulated by these receptors. We therefore postulated that the glucocorticoid (GR) and androgen receptor (AR) antagonist, PT150, would reduce infectivity of SARS-CoV-2 and prevent inflammatory lung injury in the Syrian golden hamster model of COVID19. Animals were infected intranasally with 2.5 × 104 TCID50/ml equivalents of SARS-CoV-2 (strain 2019-nCoV/USA-WA1/ 2020) and PT150 was administered by oral gavage at 30 and 100 mg/Kg/day for a total of 7 days. Animals were then examined at days 3, 5 and 7 post-infection (DPI) for lung histopathology, viral load and production of proteins regulating the initiation and progression of SARS-CoV-2 infection. Results of these studies indicated that oral administration of PT150 decreased replication of SARS-CoV-2 in lung, as well as expression of ACE2 and TMPRSS2 protein. Hypercellularity and inflammatory cell infiltration driven by macrophage responses were dramatically decreased in PT150-treated animals, as was tissue damage and expression of IL-6. Molecular modeling suggested that PT150 binds to the co-activator interface of the ligand binding domain of both AR and GR and thereby acts as an allosteric modulator and transcriptional repressor of these receptors. Phylogenetic analysis of AR and GR across multiple species permissive to SARS-CoV-2 infection revealed a high degree of sequence identity maintained across species, including human, suggesting that the mechanism of action and therapeutic efficacy observed in Syrian hamsters would likely be predictive of positive outcomes in patients. PT150 is therefore a strong candidate for further clinical development for the treatment of COVID19 across variants of SARS-CoV-2.

Emerging in late December 2019 in Wuhan, China, several unidentified cases of severe pneumonia 59 were reported of which were epidemiologically linked to a seafood and wet animal wholesale market.

Through deep sequencing of lower respiratory samples from these patients, a novel betacoronavirus was 61 identified [1, 2] and has since been distinguished as the initial point source of the biological threat that has 62 now developed into a global pandemic. As of late 2020, severe acute respiratory syndrome coronavirus 2 63 (SARS-CoV-2) has infected over 40 million people and has been responsible for over 1.14 million deaths 

Coronaviruses (Order: Nidovirales; Family: Coronaviridae) are enveloped, non-segmented, 69 positive-sense single-stranded RNA viruses that contain very large genomes up to 33.5 kilobases (kb). The 70 four subtypes (genera) of these viruses -alpha, beta, gamma, and delta-coronaviruses -share a highly 71 conserved genome organization comprising a large replicase gene followed by structural and accessory 72 genes. The organization of the coronavirus genome is organized from the 5'-leader-UTR, replicase, 73 S(spike), E(envelope), M(membrane), N(nucleocapsid) to the 3' UTR poly (A) tail [5] . Notably, production 74 of the spike protein has been linked to severity of disease. The spike protein is a proteolytically processed 75 glycoprotein that extends from the viral membrane and modulates virus-cell membrane fusion. In order to 76 acquire functionality, the protein must undergo multiple stepwise endoproteolytic cleavages [6] . The spike 77 glycoprotein of SARS-CoV-2 has two domains, the S1 domain comprising residues 12 -667 and the S2 78 domain, comprising residues 668 -1273. The S1 subunit contains the receptor binding domain (RBD), 79 which interacts with the ACE2 receptor, whereas the S2 subunit remains associated with the viral envelope 80 [7] . Viral infection requires proteolytic cleavage at Arg685-Ser686 at the S1 site by the transmembrane 81 protease, serine 2 (TMPRSS2), followed by cleavage at the S2 site at Arg815-Ser816 [6] . Proteolytic 82 5 cleavage of the spike protein then enables membrane fusion and entry into the host cell in complex with the 83 ACE2 receptor [8] .

Downregulating expression of ACE2 and TMPRSS2 has therefore emerged as an important 85 therapeutic strategy for the treatment of COVID19 in order to increase host defense by preventing entry of 86 SARS-CoV-2 into cells, thereby limiting viral replication. Clinical evidence supports this hypothesis, where 87 a prospective study reported a decrease in the rate of intensive care unit admissions in men who had been 88 prescribed anti-androgens for six months prior to hospitalization [9] . Both ACE2 and TMPRSS2 are highly 

Both ACE2 and TMPRSS2 are also significantly regulated by inflammation. ACE2, as well as the 93 inflammatory cytokine interleukin-6 (IL-6), are non-canonical interferon-responsive genes (ISGs) that are 94 highly expressed following infection with SARS-CoV-2 [10] . Inflammation also directly upregulates 95 expression of TMPRSS2 [11] . Steroids have therefore been extensively used to treat COVID19 patients, 96 albeit with mixed results. A recent study in The Lancet indicated that clinical evidence does not support 97 corticosteroid treatment for SARS-CoV-2-related lung injury. Patients who were given corticosteroids were 98 more likely to require mechanical ventilation, vasopressors, and renal replacement therapy [12] .

Corticosteroids such as dexamethasone have been shown to increase expression of ACE2 [13, 14] , which 100 would enhance viral entry and replication and could therefore worsen infection when given too early.

6 stages of RNA synthesis and may confer species specificity for virulence [5, 15] . In part because a number 109 of human RNA viruses have glucocorticoid receptor cis-acting elements in their 5-UTR's, antagonists of 110 the glucocorticoid receptor are one potential class of drugs for therapeutic modulation of SARS-CoV-2 111 infection. A report comparing the host inflammatory response to SARS-CoV (SARS1) and HCoV-EMC 112 (MERS) revealed a unique set of 207 genes dysregulated during the course of infection. Based on these 113 data, the authors predicted that selected kinase inhibitors and glucocorticoid receptor modulators could 114 function as potential antiviral compounds [16] . These studies suggested two important points about 115 modulating infection with human coronaviruses: 1) targeting cellular responses has been shown to inhibit 116 viral replication and 2) immunomodulatory drugs that reduce the excessive host inflammatory response to 117 respiratory viruses have therapeutic benefit, as seen with influenza virus infections [16] .

However, classical antagonists of glucocorticoid function that compete for interaction at the steroid 119 binding pocket of the receptor ligand binding domain (LBD) could be problematic, due to excessive 120 blockade of cortisol function. Thus, allosteric modulators of both AR and GR that could dampen 121 transcriptional activation through these receptors would be preferable. Such interactions tend to favor 122 stabilization of transcriptional co-repressor proteins on chromatin, such as CoREST, HDAC2/3/4 and 123 NCoR2, that prevent binding of co-activator proteins in response to activation of cis-acting transcription 124 factors [17, 18] . To address the potential for a transcriptional modulator to protect against SARS-CoV-2 125 infection through downregulation of AR/GR-dependent expression of ACE2 and TMPRS22, we examined 126 the therapeutic efficacy of (11β,17β)-11-(1,3-benzodioxol-5-yl)-17-hydroxy-17-(1-propynyl)-estra-4,9-127 dien-3-one (designated as "PT150"), a synthetic AR and GR modulator shown to antagonize human GR 128 [19, 20] were mounted onto poly-ionic slides. Sections were then deparaffinized and immunostained using the Leica 176 RX m automated robotic staining system. Antigen retrieval was performed by using Bond Epitope Retrieval

Solutions 1 and 2 for 20 minutes each in conjunction with base plate heat application. Sections were then 178 permeabilized (0.1% Triton X in 1X TBS) and blocked with 1% donkey serum. Primary antibodies were 179 diluted to their optimized dilutions in tris-buffered saline and incubated on the tissue for 1 hour/antibody:

Rabbit SARS nucleocapsid protein (SARS-CoV-2; Rockland; 1:500), goat ionized calcium binding adaptor 181 molecule 1 (IBA1; Abcam; 1:50), goat angiotensin converting enzyme 2 (ACE2;R&D Systems; 1:500), 182 rabbit transmembrane serine protease 2 (TMPRSS2; Abcam; 1:500), mouse interleukin 6 (IL-6; 183 ThermoFisher; 1:500). Sections were then stained for DAPI (Sigma) and were mounted on glass coverslips 184 using ProLong Gold Anti-Fade medium and stored at 4°C until imaging. 

Clinical observations and levels of SARS-CoV-2 in the lungs of Syrian hamsters treated with PT150. Oral 240 gavage with PT150 or vehicle began on the same day as infection with 2.5 x 10 4 TCID50 SARS-CoV-2 241 (USA-WA1-2020 strain) by intranasal inoculation. Body weights were monitored daily for each animal, 242 with a noted decline in average body weight in each experimental group that reached a maximum loss by 243 day 5 with a total overall loss of eight-percent body weight (Fig 1A,B) . This finding is consistent with other 244 longitudinal studies in Syrian golden hamsters infected with SARS-CoV-2 that demonstrate the maximal 245 clinical severity of disease at day 5 post-infection [28, 29] . Infected hamsters treated with vehicle-only 246 showed the greatest decline in body weight relative to controls, that was prevented by treatment with PT150 247 at 30 and 100 mg/Kg/day (Fig 1A) . Two-way ANOVA analysis indicated a difference with treatment 

In addition, the number of apoptotic endothelial cells in pulmonary bronchi was greatly reduced by 284 treatment with 100 mg/Kg/day PT150 (Fig 2G-I 

Analysis of immune cell infiltration and broncho-interstitial pneumonia in lung tissue of animals exposed 292 to SARS-CoV-2. Lungs were examined for the extent of immune cell infiltration at 3, 5 and 7 days post-293 infection (DPI) by quantitative digital image analysis (Fig 3) . Whole mount sections of paraffin-embedded 294 lung tissue were stained with H&E and bright field grayscale images were collected using a microscope 295 equipped with a scanning motorized stage. Pseudo-colored H&E images are depicted in blue, overlaid with 296 ROIs detected by intensity thresholding in red. Hematoxylin-positive immune cell soma were rendered as 297 focal points within the regions of interest to calculate the percent hypercellularity of tissue following 298 infection with SARS-CoV-2. By 3 DPI, lung tissue showed significant infiltration of immune cells in the 299 SARS-CoV-2 + vehicle group in addition to widespread hemorrhaging (Fig 3A) . Immune cell infiltration 300 was decreased in dose-dependent fashion by treatment with PT150 at 30 and 100 mg/Kg/day (Fig 3D-F 303   3J ). The percent of total lung area displaying immune cell hypercellularity was quantified by ROI 304 thresholding and normalizing to the total lung section area (Fig 3K) . This effectively showed the post- Fig 4) . Comparing sequences of the androgen receptor (Fig 4A) (Fig 4B) ranged from 89.96 (golden hamster) and 90.89 (greater horseshoe bat) to 318 95.00 (sunda pangolin). Sequence similarity between these genes amongst difference species is relevant for 319 both potential zoonotic propagation of SARS-CoV-2 as well as to the testing of potential therapeutic 320 compounds acting through the androgen and glucocorticoid receptors. facilitate entry of SARS-CoV-2 into cells. We therefore examined production of these proteins in lung 336 tissue from infected hamsters with and without PT150 treatment. ACE2 intensity measurements were 337 quantified by immunofluorescence imaging (Fig 5) , where pseudostratified columnar epithelium lining 338 bronchioles showed marked decreases in ACE2 production relative to vehicle control (Fig 5J) with the 339 administration of 100mg/kg/day of PT150 at the 3-day timepoint as well as the 7-day timepoint (Fig 5G-I,   340 16 K). Decreased ACE2 production was also observed in the 30mg/kg/day PT150 lung sections at 7DPI (Fig   341   5F ). Production of ACE2 in vehicle-treated SARS-CoV-2 lung sections were similar to control until the 7-342 day timepoint, at which point production reached a maximum (Fig 5A-C) . Expression of TMPRSS2 within 343 bronchiolar cells was increased in the untreated lung sections infected with SARS-CoV-2 (Fig 6A-C, K) ,

indicating induction associated with enhanced levels of viral entry, replication and dissemination. In 345 contrast, the 100mg/kg/day PT150 treated animals showed significant decreases in TMPRSS2 protein 346 levels at all timepoints (Fig 6G-I ) similar to levels observed in the control group (Fig 6J-K) . There was 347 also reduction in TMPRSS2 proteins levels within the 30 mg/kg/day treatment groups at 5DPI and 7DPI 348 (Fig 6D-F, K) , similar to levels in control animals at 7DPI.

Syrian hamsters infected with SARS-CoV-2 in parallel with decreased viral load. The peak of infectivity 352 and viral replication within the Syrian hamster model at sampled time points was observed at 3DPI. Using 353 quantitative immunofluorescence scanning microscopy, we evaluated the extent of lung area containing 354 both SARS-CoV-2 viral protein and infiltrating IBA1+ macrophages (Fig 7) . In the SARS-CoV-2 + vehicle 355 group, staining for viral nucleocapsid protein indicated the peak of viral protein production at 3DPI, which 356 declined at both 5 and 7DPI (Fig 7A-E) . Increased infiltration of macrophages was present at 3DPI, peaked 357 at 5DPI and was then followed by a decline at 7DPI (Fig 7A-E) . Control animals (Fig 7S,T) showed no 358 staining for SARS-CoV-2 and only background levels of IBA1. Viral replication was decreased by 359 administration of 30mg/kg/day of PT150 (Fig 7G-L) , and was further decreased by the administration of 360 100mg/kg/day of PT150 (Fig 7M-R) . In parallel to the decrease in viral nucleocapsid protein, there was a 361 decrease in the percent of total lung area occupied by infiltrating macrophages at 30 and 100 mg/Kg/day 362 PT150 (Fig 7G-L,M-R) . Quantification of immunofluorescence staining, as measured by the percent of 363 total sampled lung area positive for SARS-CoV-2, revealed marked decreases in both percent lung area 364 expressing viral replication, as well as the number of IBA1+ cells/mm 2 (Fig 7U,V) . This demonstrates a dose-response relationship in therapeutic efficacy of PT150 for reducing the viral burden of SARS-CoV-2 366 in lung, as well as a corresponding decrease in the extent of infiltrating macrophages.

In Syrian golden hamsters infected with SARS-CoV-2, there was a significant increase in 368 macrophage-derived IL-6 within the bronchiolar epithelial layer (Fig 8A-C) . Immunofluorescence images 369 of infected hamster lung tissue at 3DPI revealed cells within the bronchiolar epithelial layer co-producing 370 high levels of IL-6 (green) with SARS-CoV-2 nucleocapsid protein (red). Nuclei were counterstained with 371 DAPI (blue) and IBA1+ macrophages are shown in cyan. In infected animals, triple label 372 immunofluorescence images show cells staining intensely for IL-6 and co-localizing with expression of 373 SARS-CoV-2 nucleocapsids protein, adjacent to IBA1+ macrophages (Fig 8A and inset) . Expression of 374 IL-6 persisted at 5 and 7DPI, even after SARS-CoV-2 nucleocapsid protein was no longer evident ( Fig   375   8B ,C). Treatment with PT150 and 30 mg/Kg/day (Fig 8D-F) and 100 mg/Kg/day (Fig 8G-I 

CoV-2 and show a high degree of sequence homology in both AR and GR (Fig 4) , it is likely that SARS-

CoV-2 infected patients will response similarly to PT150 treatment.

Clinical symptoms of SARS-CoV-2 infection were observed in infected vehicle-only treated 401 animals as early 3DPI. Behavioral and physical changes were more prominent at the 5-day timepoint and 402 included labored breathing, ruffled fur, akinesia and maximal weight loss (5-10%). Weight loss observed 403 5DPI similar to other studies in Syrian hamsters [43] [44] [45] [46] . Animals that received PT150 at clinically relevant 404 low (30mg/kg/day) and high (100mg/Kg/day) doses did not display as severe clinical manifestations and 405 weight loss was minimal at the 5-day timepoint, when compared to untreated control animals (Fig. 1) .

Pathological analysis of infected lung tissue revealed inflammatory cellular infiltration involvement at 407 3DPI, of which peaked at 5DPI and included mixed inflammatory cell populations resulting in 408 bronchiointerstital pneumonia (Fig. 2) . The majority of the parenchymal space at the 5-day timepoint was 409 reduced due to intense inflammatory cell infiltration and showed extravasating cells from pulmonary 410 vessels being recruited to the epithelial layers of the bronchi. The multifocal inflammation began to resolve 411 by 7DPI and was replaced with fibrotic lung tissue and consolidated areas of focal inflammation.

Inflammatory pathology within the treated animals was decreased in severity and showed marked 

Digital image analysis of whole-lung scans in the infected vehicle-only group (Fig 3) showed 416 extensive hypercellularity and bronchiointerstital pneumonia, with markedly increased hypercellularity in 417 19 bronchioles, pulmonary arteries and the lung parenchyma. Lung hypercellularity in infected animals 418 increased throughout the course of the 7-day study, similar to the progressive pneumonia experienced by 419 SARS-CoV-2 patients in response to the overwhelming amount of edema and cellular infiltration into the 420 parenchyma of the lungs. Treatment with PT150 markedly reduced hypercellularity at 5 and 7DPI, with the 421 low-dose PT150 group showing reduction at 7DPI. This indicates that PT150 may be regulating cellular 422 infiltration and recruitment into the lung tissue, resulting in reduced pathology and inflammation.

We performed phylogenetic analysis and molecular docking studies to characterize the putative 424 molecular targets by which PT150 could modulate disease progression (Fig 4) . Sequence concordance 425 values for both AR and GR were very high across species representative of divergent taxonomic groups,

including the greater horseshoe bat, sunda pangolin, Syrian golden hamster and human (amongst other 427 species). This underscores not only the likely similar patterns of regulation of AR/GR-dependent genes 428 across species that are required for entry of SARS-CoV-2, such as ACE2 and TMPRSS2, but also the 429 predictive potential for therapeutic modulation of these receptors using PT150, given the sequence 430 similarity between the hamster and human genes. Similarity in these regulatory proteins also highlights the 431 potential for cross-species transmission of SARS-CoV-2 and related β-coronaviruses and the importance 432 of identifying compounds that can modulate signaling of AR and GR to increase host defense.

Molecular docking studies (Fig 4C,D 

To further investigate the molecular targets of PT150 in vivo, expression of ACE2 was determined 463 in lung in bronchiolar epithelial cells (Fig 5) . ACE2 levles in untreated animals remained constant until 464 preaking at 7DPI. Co-localization of SARS-CoV-2 and ACE2 were observed in bronchiolar epithelial cells 465 at 3DPI and 5DPI, supporting that ACE2 is a vital component in viral binding and entry (Fig 5A-C) . This 466 demonstrates that viral infectivity and replication may transcriptionally regulate ACE2 production in favor 467 of viral propagation and survival. Treatment with 100 mg/Kg PT150 decreased the overall amount of ACE2 468 in addition to reducing co-localization with SARS-CoV-2. This reduction of ACE2 could be due to 469 21 inhibition of androgen receptor binding to the promoter of Ace2, thereby decreasing transcriptional 470 activation, as reported for other anti-androgens [50] . Expression of TMPRSS2 was also investigated due to 471 the requirement of this cell surface serine protease for processing of the viral S1 spike protein that is 472 necessary for viral entry in complex with ACE2 (Fig 6) . 

These data suggest that the anti-viral activity of PT150 is due to direct modulation of host defense that 480 decreases viral entry points.

To determine if PT150 was effective at reducing overall viral replication, infection and immune 482 responses, whole lung sections were analyzed and viral infectivity was determined by assessment of the 483 SARS-CoV-2 nucleocapsid protein (Fig 7) . In untreated animals, there was a significant change from 484 control in the percentage of lung area infected with SARS-CoV-2 at 3DPI that decreased by 5DPI and 7DPI, 485 as seen in previously published studies [44, 52, 53] . Viral loads in animals administered PT150 at 100 486 mg/Kg were not different from control at 3DPI, demonstrating a dramatic reduction in viral attachment, 487 replication and dissemination throughout lung tissue, both in the bronchi and in the parenchyma.

Macrophage infiltration per area of tissue in untreated animals was increased at 3DPI and peaked at 5DPI, and is closely associated with the hyperimmune response observed in patients characterized by severe 496 infiltration of macrophages and lymphocytes into lung tissue. IL-6 production was investigated in the 497 bronchiolar cell layer to determine inflammatory recruitment potential during the course of disease (Fig 8) .

Infected animals treated with vehicle showed peak IL-6 production at the 3DPI timepoint, decreasing 499 steadily to the 7-day timepoint. Interestingly, high and low dose PT150 treated groups showed highly 500 reduced levels of IL-6 at all timepoints. This highlights the efficacy of PT150 in reducing innate immune 501 responses concomitant to mitigating the severity of lung pathology in response to SARS-CoV-2.

In conclusion, these data demonstrate disease progression and pathology within the lungs of SARS-

CoV-2-infected animals depends upon production of both ACE2 and TMPRSS2, which facilitate viral entry 504 and replication, leading to recruitment of macrophages that initiate a severe innate immune response leading 505 to broncho-interstitial pneuomonia and consolidation of the lung parenchyma. Expression of IL-6 is 506 necessary for recruitment of immune cells to the site of infection, which leads to the 'cytokine storm' and 507 decreased prognosis for patients [55, 56] . PT150 treatment interrupts this progression of disease, limiting 508 viral entry, thereby reducing viral loads and decreasing the severity of the immune response to SARS-CoV-509 2 infection. This may occur through allosteric inhibition of the androgen and glucocorticoid receptors, 510 which decreases protein levels of ACE2 and TMPRSS2 and mitigates excessive immune responses through 511 inhibition of inflammatory gene expression. Importantly, decreased production of IL-6 production by 512 resident immune cells within the lung tissue is likely to correlate with an improve prognosis for patients.

The novel mechanism of action of PT150 as both an inhibitor of viral entry and an immunomodulator makes 

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Distinct expression of SARS-CoV-2 receptor ACE2 correlates with endotypes of 543 chronic rhinosinusitis with nasal polyps

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SARS-CoV-2 + 100 mg/Kg/day PT150

Quantification of the total 670 area of the lung tissue affected with broncho-interstitial pneumonia was conducted using automated focal 671 point determination within ROIs following manual thresholding. Pseudo colored grayscale images of H&E 672 sections are depicted in blue, overlaid with ROI's detected by intensity thresholding in red

Phylogenetic analysis of the androgen receptor (A) and glucocorticoid receptor 677 (B) indicate a high degree of homology across species, including between human and hamster

CoV-2 via modulation of these receptors. The putative binding site on the androgen (C) and glucocorticoid 680 (D) receptors was analyzed by computer modeling studies using the human receptor

In Syrian golden 683 hamsters infected with SARS-CoV-2, there was a modest decrease in expression of the ACE2R in 684 bronchiolar epithelial cells at day 7 post-infection in animals given PT150 at 100 mg/Kg/day. Experimental 685 groups were (A-C) SARS-CoV-2 + vehicle

CoV-2 + 100 mg/Kg/day PT150, (J) Control + vehicle. K, Quantification of ACE2R expression in lung

Figure 6. PT150 administration decreases overall TMPRSS2 expression in bronchiolar epithelial 690 cells. In Syrian golden hamsters infected with SARS-CoV-2, there was a significant decrease in expression 691 of the TMPRSS2 in bronchiolar epithelial cells at all timepoints in animals given PT150 at

Experimental groups were (A-C) SARS-CoV-2 + vehicle

G-I) SARS-CoV-2 + 100 mg/Kg/day PT150, (J) Control + vehicle. K, Quantification 694 of TMPRSS2 protein expression in lung tissue

SARS-CoV-2 viral protein expression and macrophage infiltration is reduced by oral 697 administration of PT150. Viral load was determined by immunofluorescence staining of the SARS-CoV-698 2 nucleocapsid protein. Total viral replication and immune cell infiltration is seen within lung sections at 699 the 3DPI, 5DPI, and 7DPI timepoints

V) was performed using adaptive intensity 702 thresholding and cellular co-localization of protein expression. N=6 animals per group with a sampling of 703 5 lobes of tissue per animal. Differences were determined by one-way ANOVA

There was a significant decrease in expression of TMPRSS2 708 in bronchiolar epithelial cells of Syrian golden hamsters infected with SARS-CoV-2 at all timepoints in 709 animals given PT150 at 30 and 100 mg/Kg/day. Experimental groups were (A-C) SARS-CoV-2 + vehicle, 710 (D-F), SARS-CoV-2 + 30 mg/Kg/day PT150, (G-I) SARS-CoV-2 + 100 mg/Kg/day PT150, (J) Control + 711 vehicle. K, Quantification of IL-6 protein expression in lung tissue