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A Novel Glucocorticoid and Androgen Receptor Modulator Reduces Viral bioRxiv preprint doi: https://doi.org/10.1101/2021.02.20.432110; this version posted February 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 A novel glucocorticoid and androgen receptor modulator reduces viral 2 entry and innate immune inflammatory responses in the Syrian Hamster 3 model of SARS-CoV-2 4 5 Savannah M. Rocha1,2, Anna C. Fagre1, Amanda S. Latham1,2, Katriana A. Popichak1, Casey P. 6 McDermott2, Clinton C. Dawson1, Jason E. Cummings1, Juliette Lewis1, Philip Reigan3, Tawfik A. 7 Aboellail1, Rebekah C. Kading1, Tony Schountz1, Neil D. Theise4, Richard A. Slayden1* and Ronald B. 8 Tjalkens2* 9 10 1 Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine and 11 Biomedical Sciences, Colorado State University, Fort Collins, CO, USA 12 2 Department of Environmental and Radiological Health Sciences, College of Veterinary Medicine and 13 Biomedical Sciences, Colorado State University, Fort Collins, CO, USA 14 3 Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado Denver Anschutz 15 Medical Campus, Denver, CO, USA 16 4 Department of Pathology, New York University-Grossman School of Medicine, New York, NY 17 18 *Corresponding Author: 19 20 Ronald B. Tjalkens Richard A. Slayden 21 Department of Environmental and Department of Microbiology, 22 Radiological Health Sciences Immunology and Pathology 23 College of Veterinary Medicine and College of Veterinary Medicine and Biomedical Sciences Biomedical Sciences Colorado State University Colorado State University Fort Collins, CO 80523-1680 Fort Collins, CO 80523-1682 Tel: +1 970 491 2825 Tel: +1 970 491 2902 Fax: +1 970 491 7569 Fax: +1 970 491 1815 [email protected] [email protected] 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.20.432110; this version posted February 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 24 25 Running Title: Anti-SARS-CoV-2 activity of a corticosteroid receptor inhibitor 26 27 28 29 Keywords: COVID19, SARS-CoV-2, glucocorticoid receptor, androgen receptor, innate immunity, 30 inflammation, therapeutics 31 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.20.432110; this version posted February 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 32 Abstract 33 Since its initial discovery in late 2019, severe acute respiratory syndrome coronavirus 2 (SARS- 34 CoV-2), the cause of COVID19, has spread worldwide and despite significant research efforts, treatment 35 options remain limited. Replication of SARS-CoV-2 in lung is associated with marked infiltration of 36 macrophages and activation of innate immune inflammatory responses triggered, in part, by heightened 37 production of interleukin-6 (IL-6) that recruits lymphocytes to the site of infection that amplify tissue injury. 38 Antagonists of the glucocorticoid and androgen receptors have shown promise in experimental models of 39 COVID19 and in clinical studies, because cell surface proteins required for viral entry, angiotensin 40 converting enzyme 2 (ACE2) and the transmembrane serine protease 2 (TMPRSS2), are transcriptionally 41 regulated by these receptors. We therefore postulated that the glucocorticoid (GR) and androgen receptor 42 (AR) antagonist, PT150, would reduce infectivity of SARS-CoV-2 and prevent inflammatory lung injury 43 in the Syrian golden hamster model of COVID19. Animals were infected intranasally with 2.5 × 104 44 TCID50/ml equivalents of SARS-CoV-2 (strain 2019-nCoV/USA-WA1/ 2020) and PT150 was 45 administered by oral gavage at 30 and 100 mg/Kg/day for a total of 7 days. Animals were then examined 46 at days 3, 5 and 7 post-infection (DPI) for lung histopathology, viral load and production of proteins 47 regulating the initiation and progression of SARS-CoV-2 infection. Results of these studies indicated that 48 oral administration of PT150 decreased replication of SARS-CoV-2 in lung, as well as expression of ACE2 49 and TMPRSS2 protein. Hypercellularity and inflammatory cell infiltration driven by macrophage responses 50 were dramatically decreased in PT150-treated animals, as was tissue damage and expression of IL-6. 51 Molecular modeling suggested that PT150 binds to the co-activator interface of the ligand binding domain 52 of both AR and GR and thereby acts as an allosteric modulator and transcriptional repressor of these 53 receptors. Phylogenetic analysis of AR and GR across multiple species permissive to SARS-CoV-2 54 infection revealed a high degree of sequence identity maintained across species, including human, 55 suggesting that the mechanism of action and therapeutic efficacy observed in Syrian hamsters would likely 56 be predictive of positive outcomes in patients. PT150 is therefore a strong candidate for further clinical 57 development for the treatment of COVID19 across variants of SARS-CoV-2. 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.20.432110; this version posted February 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 58 Introduction 59 Emerging in late December 2019 in Wuhan, China, several unidentified cases of severe pneumonia 60 were reported of which were epidemiologically linked to a seafood and wet animal wholesale market. 61 Through deep sequencing of lower respiratory samples from these patients, a novel betacoronavirus was 62 identified [1, 2] and has since been distinguished as the initial point source of the biological threat that has 63 now developed into a global pandemic. As of late 2020, severe acute respiratory syndrome coronavirus 2 64 (SARS-CoV-2) has infected over 40 million people and has been responsible for over 1.14 million deaths 65 [3] and continues to surge in many countries. The infection can be spread through respiratory droplet 66 transmission from asymptomatic, pre-symptomatic, and symptomatic carriers [4] making diagnosis and 67 quarantine efforts difficult, ultimately leading to increased propagation and dissemination of infectious 68 virus. 69 Coronaviruses (Order: Nidovirales; Family: Coronaviridae) are enveloped, non-segmented, 70 positive-sense single-stranded RNA viruses that contain very large genomes up to 33.5 kilobases (kb). The 71 four subtypes (genera) of these viruses – alpha, beta, gamma, and delta-coronaviruses – share a highly 72 conserved genome organization comprising a large replicase gene followed by structural and accessory 73 genes. The organization of the coronavirus genome is organized from the 5’-leader-UTR, replicase, 74 S(spike), E(envelope), M(membrane), N(nucleocapsid) to the 3’ UTR poly (A) tail [5]. Notably, production 75 of the spike protein has been linked to severity of disease. The spike protein is a proteolytically processed 76 glycoprotein that extends from the viral membrane and modulates virus-cell membrane fusion. In order to 77 acquire functionality, the protein must undergo multiple stepwise endoproteolytic cleavages [6]. The spike 78 glycoprotein of SARS-CoV-2 has two domains, the S1 domain comprising residues 12 – 667 and the S2 79 domain, comprising residues 668 -1273. The S1 subunit contains the receptor binding domain (RBD), 80 which interacts with the ACE2 receptor, whereas the S2 subunit remains associated with the viral envelope 81 [7]. Viral infection requires proteolytic cleavage at Arg685-Ser686 at the S1 site by the transmembrane 82 protease, serine 2 (TMPRSS2), followed by cleavage at the S2 site at Arg815-Ser816 [6]. Proteolytic 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.20.432110; this version posted February 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 83 cleavage of the spike protein then enables membrane fusion and entry into the host cell in complex with the 84 ACE2 receptor [8]. 85 Downregulating expression of ACE2 and TMPRSS2 has therefore emerged as an important 86 therapeutic strategy for the treatment of COVID19 in order to increase host defense by preventing entry of 87 SARS-CoV-2 into cells, thereby limiting viral replication. Clinical evidence supports this hypothesis, where 88 a prospective study reported a decrease in the rate of intensive care unit admissions in men who had been 89 prescribed anti-androgens for six months prior to hospitalization [9]. Both ACE2 and TMPRSS2 are highly 90 expressed in bronchiolar epithelial cells and transcriptionally regulated at the promoter level through the 91 androgen receptor (AR) [8]. In a model of SARS-CoV-2 infection, inhibitors of AR transcriptional activity 92 markedly reduced viral infectivity and decreased inflammatory lung injury in experimental animals [8]. 93 Both ACE2 and TMPRSS2 are also significantly regulated by inflammation. ACE2, as well as the 94 inflammatory cytokine interleukin-6 (IL-6), are non-canonical interferon-responsive genes (ISGs) that are 95 highly expressed following infection with SARS-CoV-2 [10]. Inflammation also directly upregulates 96 expression of TMPRSS2 [11]. Steroids have therefore been extensively used to treat COVID19 patients, 97 albeit with mixed results. A recent study in The Lancet indicated that clinical evidence does not support 98 corticosteroid treatment for SARS-CoV-2-related lung injury. Patients who were given corticosteroids were 99 more likely to require mechanical ventilation, vasopressors, and renal replacement therapy [12]. 100 Corticosteroids such as dexamethasone have been shown to increase expression of ACE2 [13, 14], which 101 would enhance viral entry and replication and could therefore worsen infection when given too early. 102 Dexamethasone also modestly increased expression of TMPRSS2 in these studies, which could likewise 103 potentially enhance membrane fusion and vesicular uptake of SARS-CoV-2.
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