WO2024141048A1 - Methods for treating coronavirus infections - Google Patents
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- WO2024141048A1 WO2024141048A1 PCT/CN2023/143398 CN2023143398W WO2024141048A1 WO 2024141048 A1 WO2024141048 A1 WO 2024141048A1 CN 2023143398 W CN2023143398 W CN 2023143398W WO 2024141048 A1 WO2024141048 A1 WO 2024141048A1
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Abstract
A method of treating a coronavirus infection in a subject in need thereof, the method including administering a therapeutically effective amount of an endothelin receptor antagonist to the subject, with the proviso that the endothelin receptor antagonist is not co-administered with dapagliflozin or niclosamide.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of priority of U.S. Provisional Patent Application No. 63/436,305 filed December 30, 2022, the contents of it being hereby incorporated by reference in its entirety for all purposes.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING
The Sequence Listing text file, identified as Sequence_Listing_P25710PCT00. xml, Size: 14.9 KB, created on December 13, 2023, filed herewith, is hereby incorporated by reference in its entirety.
The present disclosure relates to methods and compounds useful for treating coronavirus infections.
Compared to influenza, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) , the causative agent of COVID-19, causes severe vascular damage1. SARS-CoV-2 primarily attacks epithelial cells in the airways via angiotensin-converting enzyme 2 (ACE2) as an entry point2; the abundant expression of ACE2 on vascular endothelium makes it the other major target for virus infection3. A growing body of research points to a key role of vascular damage and thrombosis in COVID-related pulmonary and extrapulmonary multi-organ injury and failure4. It may also contribute to the debilitating and long-lasting post-COVID sequelae such as ‘brain fog’ 5. Sustained elevation of endothelial damage and dysfunction biomarkers4-7, including endothelin-1 (ET-1) –the most potent vasoconstrictor8, 9, was detected in the plasma of hospitalised COVID patients. All the evidence prompts an urgent need for understanding such vasoactive factors in the pathogenesis of COVID-19.
ET-1 has been implicated in various infectious diseases10, e.g., viral pneumonia11. Blockade of endothelin receptors could inhibit human cytomegalovirus and coxsackievirus infection in epithelial and endothelial cells12, 13. Endothelin receptor antagonist was also able to mitigate influenza A/H5N1 virus-induced pulmonary injury in the infected birds14. However, the exact role of ET-1 and its receptors in SARS-CoV-2 infection remains unknown.
There is thus a need to develop a better understanding of ET-1 and its receptors in SARS-CoV-2 infection, which can be used improve methods for treating coronavirus infections.
Herein, it is determined that host-derived ET-1 aggravates SARS-CoV-2 viral entry in humans and hamsters. First, we identified the upregulation of ET-1 and its receptors in human and hamster lung tissues after SARS-CoV-2 infection as well as the other vascular damage and dysfunction manifestations. Second, we purposely deployed an antiviral medication, nirmatrelvir, to inhibit viral replication in infected animals, showing a reduction of ET-1 and its receptors in lung tissues. Intriguingly, blockade of endothelin signalling via an FDA-approved endothelin receptor antagonist, macitentan, could also significantly reduce the viral load of infected lungs and mitigate vascular damage. Third, we confirmed that lung epithelial cells, the primary site of viral insult, increased the production of ET-1 with SARS-CoV-2 challenge and the presence of ET-1 promotes viral entry into lung epithelial cells. Either genetic knockdown or pharmaceutical inhibition of ET receptors prevents viral entry in vitro. Taken together, host-derived vasoconstrictor ET-1 emerges as a novel facilitator for viral entry in SARS-CoV-2 infection.
In a first aspect, provided herein is a method of treating a coronavirus infection in a subject in need thereof, the method comprising administering a therapeutically effective amount of an endothelin receptor antagonist to the subject, with the proviso that the endothelin receptor antagonist is not co-administered with dapagliflozin or niclosamide.
In certain embodiments, the subject does not suffer from pulmonary hypertension.
In certain embodiments, the coronavirus infection is the result of a coronavirus selected from the group consisting of severe acute respiratory syndrome coronavirus (SARS-CoV) , severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) , Middle East respiratory syndrome-related coronavirus (MERS-CoV) , human coronavirus 229E (229E-CoV-2) , human coronavirus NL63 (NL63-CoV) , human coronavirus OC43 (OC43-CoV) , human coronavirus HKU1 (HCoV-HKU1) , and variants thereof.
In certain embodiments, the coronavirus infection is the result of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and variants thereof.
In certain embodiments, the endothelin receptor antagonist is a selective ETA receptor antagonist, a selective ETB receptor antagonist, or a dual ETA receptor and ETB receptor antagonist.
In certain embodiments, the endothelin receptor antagonist is a dual ETA receptor and ETB receptor antagonist.
In certain embodiments, the endothelin receptor antagonist is selected from the group consisting of sitaxsentan, ambrisentan, atrasentan, BQ-123, sparsentan, zibotentan,
aprocitentan, edonentan, bosentan, macitentan, tezosentan, BQ-788, A192621, and mixtures thereof.
In certain embodiments, the endothelin receptor antagonist is bosentan, macitentan, ambrisentan, or a mixture thereof.
In certain embodiments, the endothelin receptor antagonist is macitentan.
In certain embodiments, the coronavirus infection is the result of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the endothelin receptor antagonist is bosentan, macitentan, ambrisentan, or a mixture thereof.
In certain embodiments, the coronavirus infection is the result of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the endothelin receptor antagonist is macitentan.
In certain embodiments, the SARS-CoV-2 is an omicron variant.
In certain embodiments, the subject is a human.
The above and other objects and features of the present disclosure will become apparent from the following description of the disclosure, when taken in conjunction with the accompanying drawings.
Figure 1 depicts experimental results showing the vascular damage after SARS-CoV-2 infection in humans and hamsters. (A) Experimental design for ex vivo human lung tissue underwent mock (n = 1) and SARS-CoV-2 (n = 1) infection. (B) Representative multi-channel immunofluorescent staining for vWF and 4HNE in human lung tissue after mock or SARS-CoV-2 infection. DAPI as counterstain. Scale bar, 50 μm; 10 μm (magnified) . (C) Representative Masson’s trichrome staining of human lung tissue. Scale bar, 200 μm. (D) Experimental design to study the in vivo pathological changes in golden Syrian hamsters after mock (n = 5) or SARS-CoV-2 infection (n = 4) . (E) Representative H&E images showing the changes in vasculature, bronchiole as well as alveoli in lungs in infected golden Syrian hamsters. Intra-endothelium infiltration (rhombus) , epithelium desquamation (arrowhead) , bronchiolar epithelium cell death (arrow) , massive alveolar space infiltration (asterisk) and alveolar wall thickening (circle) were observed. Scale bar, 50 μm. These representative images were selected from a pool of images captured in 5 samples from each group. Semi-quantitative histopathological score indicating the severity of lung damage in each group, different areas were evaluated: overall, vasculature, bronchioles and alveoli. Scale bar, 50 μm. The scoring method is detailed in the Methods section. Data are mean ± S. E. M., Kruskal–Wallis test. Representative multi-channel immunofluorescent staining for vWF and 4HNE in hamster
lungs. DAPI as counterstain. Scale bar, 25 μm; 10 μm (magnified) . Representative Masson’s trichrome staining of hamster lung tissue. Scale bar, 65 μm.
Figure 2 depicts experimental data showing SARS-CoV-2 caused upregulation of endothelin signalling with vascular dysfunction. (A) Serum ET-1 level in non-infected healthy subjects (n = 4) and COVID patients (n = 4) . Data are mean ± S. E. M. Mann-Whitney test was performed to compare between healthy and infected group. (B) Representative multi-channel fluorescent images comparing the expression pattern of ET-1, ETAR, ETBR, and ACE2 in vessels of the mock-treated and SARS-CoV-2-challenged human lung tissue in an ex vivo infection model. Nuclei were counterstained using SN520. Scale bar, 50 μm. These representative images were selected from a pool of images captured in 1 sample from each group. (C) Serum ET-1 level of hamsters in mock infection (n = 5) and SARS-CoV-2 infection (n = 4) group. One data point from the mock group fell out of 1.5 IQR, thus omitted from the analysis. Two-tailed unpaired t-test. (D) Representative images of multi-channel fluorescent staining showing the temporal expression pattern of ET-1, ETAR, ETBR and ACE2 in vessels in the hamster lungs underwent mock or SARS-CoV-2 infection. Nuclei were counterstained using SN520. Scale bar, 50 μm. These representative images were selected from a pool of images captured in 5 samples from each group. (E) H&E staining of whole lung FFPE section. Rectangles represent the selected vessel region (region of interest) to be cut. The enlarged regions of interest with H&E staining were shown on the side, while the same region on the consecutive slide after laser dissection was shown in parallel (post-laser dissection) . (F) Functional enriched items of differential proteins between mock and infection group from DAVID with -log10 (p-value) . (G) A panel of selected endothelial function and extracellular matrix deposition markers were picked and relative quantitation between mock and infection group was shown. Blank bars represent weak signals that failed to be quantified in MaxDIA.
Figure 3 depicts experimental results showing macitentan reduced viral copies in infected hamster lungs and mitigated vascular pathology. (A) Schematic diagram of infected golden Syrian hamsters treated with macitentan (n = 5) and nirmatrelvir (n = 4) intraperitoneally. Treatment started one day post-infection. Plaque assay of lung lysate from hamsters was performed on VeroE6/TMPRSS2 cells. (B) Relative expression of RdRp reflecting viral copies in lungs after macitentan and nirmatrelvir treatment. (C) Viral genome copies were determined relative to β-actin. Data are mean ± S. E. M. One-way ANOVA followed by Tukey’s post hoc test. (D-F) Relative mRNA expression of (I) EDN1, (J) EDNRA and (K) EDNRB in lung lysate from different groups. (G) Representative H&E images of hamster lung histopathological changes 4 dpi with and without macitentan or nirmatrelvir
treatment. The coloured rectangles of the respective areas were magnified on the right. Intra-endothelium infiltration (rhombus) , epithelium desquamation (arrowhead) , bronchiolar epithelium cell death (arrow) , massive alveolar space infiltration (asterisk) and alveolar wall thickening (circle) . Scale bar, 50 μm. (H) Semi-quantitative histopathological score indicating the severity of lung damage in each group, different areas were evaluated: overall, vasculature, bronchioles and alveoli. The scoring method is detailed in the Methods section. Data are mean ± S.E.M. Kruskal Wallis test with Dunn’s multiple comparison test. One sample from nirmatrelvir group was excluded due to the data lying outside 1.5 IQR. (I) Representative multi-channel immunofluorescent staining for vWF and 4HNE in hamster lungs with and without drug treatment. DAPI as counterstain. Scale bar, 25 μm; 10 μm (magnified) . (J) Representative Masson’s trichrome staining of hamster lung tissue with and without drug treatments. Scale bar, 65 μm. (K) Representative images of immunostaining for endothelin-1 (ET-1) in bronchiole in infected hamsters’ lungs. Cell nuclei were counterstained with haematoxylin. Scale bars, 50 μm. These representative images were selected from a pool of 15 images captured in 5 hamsters per group.
Figure 4 depicts experimental results showing ET-1 is a co-factor to mediate SARS-CoV-2 entry (A) Normalised mRNA expression of EDN1 comparing mock and SA-infected epithelial cells in RNA sequencing analysis. Mann-Whitney test. (B) SARS-CoV-2 viral entry assay. A549-hACE2 cells were pre-treated with 100 nM ET-1 for 30 minutes before infection with SARS-CoV-2 D614G spike protein variants pseudotyped particles. Luciferase signals were measured 24h post-infection. Mean ± S. E. M., n = 3 independent experiment. Two-tailed Student’s t-test. (C) Relative mRNA expression of ACE2 with increasing ET-1 concentration in A549-hACE2 cells. One-way ANOVA with Dunnett's multiple comparisons test. (D) Western blot showing no change in ACE2 protein level with increasing concentration of ET-1. (E) Effect of pharmaceutical blockade of endothelin receptors on viral entry. A549-hACE2 was pre-treated with selective or dual endothelin receptor antagonists for 30 minutes before pseudotyped particle infection. BQ123 (ETAR antagonist; 100 μM) ; BQ788 (ETBR antagonist; 100 μM) ; Macitentan (Dual endothelin receptor antagonist; 10 μM) . Luciferase signals were measured 24h post-infection. Mean ± S. E. M., n = 3. One-way ANOVA followed by Tukey’s post hoc test. (F) Effect of endothelin receptors knockdown on viral entry. siRNA was used to knock down either one or both endothelin receptors. Pseudovirus was added 48h after transfection of siRNA. Luciferase signals were measured 24h post-infection. Mean ± S. E. M, n = 4. One-way ANOVA followed by Tukey’s post hoc test. (G) Macitanten inhibited SARS-CoV-2 pseudotyped wildtype virus entry in a dose-dependent manner in A549-TMPRSS2-
ACE2 cells. Data are mean ± S. D., n = 3. (H) Antiviral activity of Macitentan against SARS-CoV-2 Omicron BA. 1 (MOI of 0.01, 48h after infection) in Vero E6-TMPRSS2 cells. One-way ANOVA followed by Sidak’s post-test. (I-J) Western blot showing no change in ACE2 with (I) pharmaceutical agonist and antagonists and (J) genetic knockdown of endothelin receptors using siRNA. ET-1 (100nM) ; BQ3020 (ETBR antagonist, 100nM) ; BQ123 (ETAR antagonist; 100 μM) ; BQ788 (ETBR antagonist; 100 μM) and Macitentan (Dual endothelin receptor antagonist; 10 μM) . (K) Western blot showing ACE2 and ET-1 expression with increasing concentration of spike protein. (L-M) Quantification of (J) ACE2 and (K) ET-1 in western blot. The values were expressed as relative unit of the values for the group with no spike protein. Actin as reference protein. (N) Time-lapse images showing translocation of ETBR in A549-hACE2 after challenge of SARS-CoV-2 S1 receptor binding domain (RBD) . EDNRB was labelled by RFP through transient transfection of plasmid encoding EDNRB-RFP. The upper row shows the merged image of both the brightfield and the fluorescent channel. (O) Graphical summary showing the role of ET-1 in SARS-CoV-2 infection in alveolar epithelial cells.
Figure 5 depicts experimental results showing the presence of viral components in lungs after SARS-CoV-2 infection Representative immunohistochemical staining for SARS-CoV-2 nucleocapsid protein (NP) in (A) human lung tissue after ex vivo infection and (B) hamster lungs after in vivo infection.
Figure 6 depicts experimental results showing targeted mass spectrometry assay shows upregulation of plasma ET-1 in COVID patients (A) Pattern of typical inflammatory profiles in serum between healthy subjects and SARS-CoV-2 patients (H1-4 = Healthy subjects, P1-4 = COVID-19 patients) was evaluated by targeted mass spectrometry (MS) assay. Samples fell out of the detection limit represented by a cross. (B-C) Serum (B) IL-1b and (C) TNF level in non-infected healthy subjects (n = 4) and COVID patients (n = 4) . Data are mean ± S. E. M. Mann-Whitney test was performed to compare between healthy and infected group.
Figure 7 depicts experimental results showing the expression pattern of ET-1, its receptors and ACE2 in bronchial epithelial cells upon SARS-CoV-2 infection in golden Syrian hamsters Representative images of multi-channel fluorescent staining showing the expression pattern of ET-1, ETAR, ETBR, and ACE2 in bronchiole in the lungs of hamsters underwent mock or SARS-CoV-2 infection. Nuclei were counterstained using SN520. Scale bar, 50 μm. These representative images were selected from a pool of images captured in 4 samples from each group.
Figure 8 depicts experimental results showing RNA sequencing shows upregulation of ET-1 in lung epithelial cells with SARS-CoV-2 infection. (A) Differentially expressed genes related to endothelin signalling and cytokines between mock and SARS-CoV-2 (SA) infected Calu-3 epithelial cells using RNA sequencing. (B-C) Normalised mRNA expression of IL-1βand TNF in mock vs SA-infected epithelial cells. Mann-Whitney test.
Figure 9 depicts experimental results showing single-cell sequencing analysis of epithelial cells in COVID-19 patients. (A) The UMAP plot displays 16 clusters of epithelial cells from lungs of the control and COVID-19 groups. Expression levels of EDN1 across epithelial cells of control groups. (The EDN1 expression of each cell was measured as the ln (EDN1 counts *10000 /total counts in the cell) . (B) The UMAP plot displaying the main 5 cell types identified in epithelial cells of each group was in the right panel. (C) Violin plots displaying the expression of EDN1 in both AT1 cells and AT2 cells of two groups. The y-axis shows the normalized EDN1 counts. Mann-Whitney test was performed to compare between control and COVID-19 group.
Figure 10 depicts experimental results showing co-localisation of SARS-CoV-2 spike protein and endothelin receptors in endothelial cells. Confocal images of Flag-tagged SARS-CoV-2 S protein in human umbilical cord endothelial cells (HUVEC) 24h post-transfection with wildtype SARS-CoV-2 full-length spike plasmid for mammalian cell expression. Endothelin receptors, ETAR and ETBR, were stained as indicated in the figures. Nuclei are stained with DAPI. Merged and magnified images are shown. Arrows in the magnified images highlight S protein/ET receptor colocalization. Scale bar, 50 μm (magnified, 25 μm) .
Definitions
Throughout the present disclosure, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising" , will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. It is also noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as “comprises” , “comprised” , “comprising” and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean “includes” , “included” , “including” , and the like; and that terms such as “consisting essentially of” and “consists essentially of” have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the present invention.
Furthermore, throughout the present disclosure and claims, unless the context requires otherwise, the word “include” or variations such as “includes” or “including” , will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.
The use of the singular herein includes the plural (and vice versa) unless specifically stated otherwise. In addition, where the use of the term "about" is before a quantitative value, the present teachings also include the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term "about" refers to a ±10%, ±7%, ±5%, ±3%, ±1%, or ±0%variation from the nominal value unless otherwise indicated or inferred.
The term "therapeutically effective amount" as used herein, means that amount of the compound or therapeutic agent that elicits a biological and/or medicinal response in a cell culture, tissue system, subject, animal, or human that is being sought by a researcher, veterinarian, clinician, or physician, which includes alleviation of the symptoms of the disease, condition, or disorder being treated.
As used herein, the terms “treat” , "treating" , "treatment" , and the like refer to reducing or ameliorating a disorder/disease and/or symptoms associated therewith. It will be appreciated, although not precluded, treating a disorder or condition does not require that the disorder, condition, or symptoms associated therewith be completely eliminated. In certain embodiments, treatment includes prevention of a disorder or condition, and/or symptoms associated therewith. The term “prevention” or “prevent” as used herein refers to any action that inhibits or at least delays the development of a disorder, condition, or symptoms associated therewith. Prevention can include primary, secondary and tertiary prevention levels, wherein: a) primary prevention avoids the development of a disease; b) secondary prevention activities are aimed at early disease treatment, thereby increasing opportunities for interventions to prevent progression of the disease and emergence of symptoms; and c) tertiary prevention reduces the negative impact of an already established disease by restoring function and reducing disease-related complications.
As used herein, the term “subject” refers to any animal (e.g., a mammal) , including, but not limited to, humans, non-human primates, canines, felines, and rodents.
As used herein, the term “variant” when used in connection with a virus (such as, SARS-CoV-2) means a virus that is a progeny of a reference (or “parent” ) virus that possesses one or more changes in its genome (e.g., a RNA genome) , or a virus that is genetically
engineered to have one or more changes in its genome, relative to a reference (or “parent” ) virus, which may or may not result in changes to the proteins encoded by the RNA sequence (e.g., one or more proteins of a variant virus may include substitutions, deletions, or insertions compared to a parent strain) . For example, known variants of SARS-CoV-2 include, but are not limited to, B. 1.1.7 (alpha variant) , B. 1.351 (beta variant) , P. 1 (gamma variant) , B. 1.617.2 (delta variant) , and B. 1.1.529 (omicron variant) . A variant of a virus may comprise a genome sequence that shares about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or up to about 100%sequence identity or homology with the reference (or “parent” ) genome sequence.
As used herein the term “substantially silences EDNRB and/or EDNRA” means that expression or protein activity or level of the target gene or protein encoded by the target gene in the presence of the introduced inhibitory nucleic acid molecule targeting a EDNRB and/or EDNRA nucleic acid sequence is reduced by about 10%to 100%, 10%to 90%, 20%to 90%, 30%to 90%, 40%to 90%, 50%to 90%, 50%to 80%, 50%to 70%, 55%to 70%, 55%to 69.2%, 70%to 90%, or 80%to 90% as compared to the expression or protein activity or level of the target gene or protein encoded by the target gene seen when the inhibitory nucleic acid molecule targeting a endothelin-1 nucleic acid sequence is not present. Generally, when a gene is substantially silenced, it will have at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100%reduction in expression or protein activity or level of the target gene or protein encoded by the target gene as compared to when the inhibitory nucleic acid molecule is not present.
Provided herein is a method of treating a coronavirus infection in a subject in need thereof, the method comprising administering a therapeutically effective amount of an endothelin receptor antagonist to the subject.
In certain embodiments, the endothelin receptor antagonist is not co-administered with a human renal sodium-dependent glucose transporter 2 (SGLT2) inhibitor, such as dapagliflozin. In certain embodiments, the endothelin receptor antagonist is not co-administered with an anthelmintic, such as niclosamide.
In certain embodiments, the subject does not suffer from pulmonary arterial hypertension (PAH) , cancer, cardiovascular disease, hypertension, pulmonary hypertension, diabetic nephropathy, renal disease, occlusive vascular disease, congestive heart failure, a brain
haemorrhage, cerebral aneurysm, a glomerular disorder, nephropathy, congestive heart failure, or a connective tissue disorder.
The endothelin receptor antagonist can be an antibody, an antibody fragment (such as Fab, Fab’, F (ab’) 2, Fv, or single chain (ScFv) ) , a peptide, an inhibitory nucleic acid molecule, or a small molecule (synthetic or naturally isolated or derived) , wherein the inhibitory nucleic acid molecule substantially silences EDNRB and/or EDNRA.
In certain embodiments, the endothelin receptor antagonist is a selective ETA receptor antagonist, such as sitaxsentan, ambrisentan, atrasentan, zibotentan, darusentan, avosentan, and clazosentan, a selective ETB receptor antagonist, or a dual ETA receptor and ETB receptor antagonist, such as bosentan, tezosentan, and macitentan.
Exemplary the endothelin receptor antagonist include, but are not limited to, sitaxsentan, ambrisentan, atrasentan, BQ-123 (cyclo (-D-Trp-D-Asp-Pro-D-Val-Leu-) ) , sparsentan, zibotentan, aprocitentan, edonentan, bosentan, macitentan, tezosentan, BQ-788 (sodium N- { [ (2R, 6S) -2, 6-dimethyl-1-piperidinyl] carbonyl} -4-methyl-L-leucyl-N- [ (1R) -1-carboxylatopentyl] -1- (methoxycarbonyl) -D-tryptophanamide) , A192621 ( (2R, 3R, 4S) -4- (1, 3-benzodioxol-5-yl) -1- [2- [ (2, 6-diethylphenyl) amino] -2-oxoethyl] -2- (4-propoxyphenyl) pyrrolidine-3-carboxylic acid) , and mixtures thereof.
In certain embodiments, the endothelin receptor antagonist is macitentan
The coronavirus infection can be caused by an existing coronavirus, such as severe acute respiratory syndrome coronavirus (SARS-CoV) , severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) , Middle East respiratory syndrome-related coronavirus (MERS-CoV) , human coronavirus 229E (229E-CoV-2) , human coronavirus NL63 (NL63-CoV) , human coronavirus OC43 (OC43-CoV) , and human coronavirus HKU1 (HCoV-HKU1) or novel variants thereof.
In certain embodiments, SARS-CoV is wild type SARS-CoV , B. 1.1.7 (alpha variant) , B.1.351 (beta variant) , P. 1 (gamma variant) , B. 1.617.2 (delta variant) , B. 1.1.529 (omicron variant) , or a novel variant thereof.
The subject can be asymptomatic or symptomatic. In instances in which the subject tis symptomatic, the subject can exhibit one or more symptoms selected from the group consisting of fever, cough, fatigue, shortness of breath, muscle pain, joint pain, sore throat, headache, and chills (e.g., fever, cough, and shortness of breath) . In certain embodiments, the one or more symptoms can appear from 2-14 days after the subject's exposure to coronavirus.
The subject can optionally be diagnosed with the coronavirus infection prior to the step of administering the therapeutically effective amount of the endothelin-1 antagonist. The
coronavirus can be diagnosed using any method known in the art including, but not limited to, polymerase chain reaction (PCR) , such as reverse transcription PCR (RT-PCR) , real-time PCR (e.g., quantitative PCR (qPCR) ) , and real-time RT-PCR (rRT-PCR) , or an antigen test, such as a lateral flow test.
The methods described herein can also be employed prior to exposure to the coronavirus or immediately after exposure or presumed exposure to the coronavirus.
Also provided herein is method of prophylactic treatment of a subject prior to exposure or potential exposure with a coronavirus, the method comprising administering a therapeutically effective amount of an endothelin receptor antagonist to the subject. Prophylactic treatment can be administered to a subject who is at high risk of exposure to infected subject, for example, immunocompromised subjects, medical personnel, subjects with known exposure, and essential workers.
Results
Vascular damage after SARS-CoV-2 infection in humans and hamsters
We performed SARS-CoV-2 infection of human lung biopsies ex vivo (Fig. 1A) . Detection of SARS-CoV-2 nucleocapsid protein confirmed the success of viral infection in lung tissues (Fig. 5A) . Von Willebrand factor (vWF) that mediates platelet adhesion and triggers thrombus formation was increased in pulmonary micro-vessels post-viral infection. In addition to intravascular changes, we observed accumulation of oxidative stress as indicated by increased expression of 4-hydroxynonenal (4HNE) (Fig. 1B) , and collagen deposition in the extravascular regions shown by Masson’s trichrome staining in lung tissue after SARS-CoV-2 infection (Fig. 1C) .
We recapitulated vascular damage in a well-established golden Syrian hamster model of COVID-1915. Hamsters were randomly divided into 2 groups receiving either PBS as mock infection or SARS-CoV-2. Sera and lungs were collected 4 days post-infection (dpi) (Fig. 1D) . Nucleocapsid proteins were detected in the lung tissue of hamsters after infection (Fig. 5B) . Histopathological analysis by haematoxylin and eosin (H&E) staining showed massive area of inflammation, thrombosis and alveolar space infiltration in the acute phase of infection (Fig. 1E) . Semiquantitative analysis suggested the most significant changes were observed in lung microvasculature compared to other parts (Fig. 1F) . Similar to our observation in human tissues, increased expression of vWF and 4HNE was detected in the infected hamster lungs (Fig. 1G) , as well as extensive collagen deposition in the extravascular regions (Fig. 1H) .
SARS-CoV-2 caused upregulation of endothelin signalling with vascular dysfunction
Using a targeted mass spectrometry assay, we found that serum ET-1, but not interleukin-1 beta (IL-1β) and tumour necrosis factor (TNF) , of COVID patients was upregulated when compared to healthy subjects (Fig. 2A and Fig. 6) , which is in accordance with the published clinical data16. We further confirmed the upregulation of endothelin signalling after SARS-CoV-2 infection with an ex vivo infection model of human lung biopsies, showing higher expression of ET-1 and its receptors (Fig. 2B) .
To confirm the endothelin expression pattern in vivo, we measure systemic and local ET-1 in the lungs of the infected hamsters. Similar to COVID patients, infected hamsters showed a higher level of ET-1 in serum (Fig. 2B) . Immunofluorescence staining indicated an elevation of ET-1 and endothelin type B receptor (ETBR) on both vessels (Fig. 2D) and bronchioles (Fig. 7) in infected lung tissues. We then performed spatial proteomics analysis on hamster lung samples, selecting three individual micro-vessels on a section from mock and SARS-CoV-2 infection groups (Fig. 2E) . The region of interest was cut by laser dissection and we identified 5302 proteins in total. Among all, 501 proteins showed a change above 2-fold between mock and SARS-CoV-2 infection groups. Functional annotation by DAVID was performed to show the differentially expressed proteins between two groups were highly correlated with endothelial function and blood coagulation (Fig. 2F) . To confirm SARS-CoV-2-induced intra-and extra-vascular changes, a few typical markers for endothelial functions and extracellular matrix deposition were chosen for quantification based on data-independent acquisition (DIA) analysis (Fig. 2G) .
Macitentan reduced viral copies in infected lungs and mitigated vascular pathology
Given the observed association between ET-1 and pulmonary vascular damage upon SARS-CoV-2 infection, we investigated the treatment efficacy of blocking endothelin receptors by macitentan, a US Food and Drug Administration (FDA) -approved drug for pulmonary hypertension. Hamsters infected with SARS-CoV-2 were administered macitentan intraperitoneally at a dose of 0.3 mg per kg body weight 1 day after virus exposure. Nirmatrelvir, a commonly used antiviral drug, was administered as a positive control for the same duration. Treatment was performed daily and the animals were sacrificed 4 days post-infection (Fig. 3A) . Although macitentan was not as effective as nirmatrelvir in reducing infectious viral particles (Fig. 3B) , both drugs significantly lowered viral copies in lungs after 4 days of infection (Fig. 3C) . Surprisingly, nirmatrelvir significantly lowered the gene expression of EDN1 and EDNRB, showing a positive association with virus count in the lungs
(Fig. 3D-F) . Both macitentan and nirmatrelvir attenuated vascular damage and inflammatory cell infiltration in the alveolar space after viral infection (Fig, 3G and H) . Immunofluorescence staining for vWF and 4HNE showed that macitentan and nirmatrelvir could improve vascular function as well as lowering oxidative stress around the vessels (Fig. 3I) . The drugs also averted excessive accumulation of collagen around the blood vessels in the lungs, preventing the infected animals from developing pulmonary fibrosis (Fig. 3J) . Moreover, we also observed a lowered expression of ET-1 in bronchioles after drug treatment (Fig. 3K) . The result suggests that ET-1 and its receptors could possibly be a therapeutic target in treating SARS-CoV-2 infection.
ET-1 is a co-factor to mediate SARS-CoV-2 entry
ET-1 is found to be predominantly expressed in endothelial cells. The results presented herein also demonstrate an increase in the expression of ET-1 in lung epithelial cells after infection. To confirm lung epithelial cells as a source of ET-1 upon infection, RNA sequencing of a commonly used lung epithelial cell line, Calu-3, infected by SARS-CoV-2 was carried out and a list of inflammatory cytokines was investigated (Fig. 8) . Again, ET-1, but not IL-1β and TNF, was significantly upregulated after SARS-CoV-2 challenge (Fig. 4A and Fig. 8) .
To investigate the types of epithelial cells that govern the production of ET-1, single-cell sequencing analysis was performed from public databases of the lungs from normal control and infected patients. 15 clusters of epithelial cells were recovered and further classified into five major categories (Fig. 9A-B) . EDN1 was mostly expressed in alveolar type 1 epithelial cells (AT1) and alveolar type 2 epithelial cells (AT2) (Fig. 9B) . Significantly higher levels of EDN1 were detected in both AT1 and AT2 cells in COVID-19 patients (Fig. 9C) .
To elucidate the role of ET-1 in SARS-CoV-2 infection, it was investigated how ET-1 alters the infectivity of the virus in lung alveolar epithelial cells in vitro. Lentiviral particles pseudotyped with SARS-CoV-2 D614G spike protein were employed to infect human lung alveolar epithelial cells overexpressed with ACE2 (A549-hACE2) . It was found that the addition of ET-1 significantly increased viral entry into A549-hACE2 (Fig. 4B) . Notably, ET-1 reduced the mRNA and did not cause significant changes in protein level of ACE2, a major host cell receptor for SARS-CoV-2 entry, in a dose-dependent manner (Fig. 4C and D) . Either pharmaceutical blockade or genetic knockdown of endothelin receptors reduced the infectivity of SARS-CoV-2 S-pseudotyped virions in A549-hACE2 cells (Fig. 4E and F) . Here, an FDA-approved endothelin receptor antagonist, macitentan, was used to study its therapeutic potential in SARS-CoV-2 infection. Macitentan inhibited SARS-CoV-2 pseudotyped virus entry in a
dose-dependent manner with IC50=0.29 μM (Fig. 4G) . It could not suppress authentic SARS-CoV-2 Omicron BA. 1 replication in VeroE6-TMPRSS2 cells until at a high dose of 10 μM (Fig. 4H) . Moreover, neither endothelin receptor agonists nor antagonists or knockdown alter the protein level of ACE2 (Fig. 4I and J) . Stimulation of lung epithelial cells by spike protein resulted in a decrease in ACE2 and an increase in ET-1 expression (Fig. 4K-M) . This might imply that endothelin receptors as alternate host receptors independent of ACE2 for SARS-CoV-2.
Intriguingly, we observed colocalisation of spike protein with endothelin receptors when SARS-CoV-2 spike protein was transiently transfected into endothelial cells, suggesting a possible physical interaction between them (Fig. 10) . Changes in ETBR expression were more prominent in the lungs after infection and treatment compared to ETAR. We then further investigate if ETBR translocates to the surface like ACE2 upon SARS-CoV-2 infection to aid viral entry17. A time-lapse video recorded immediately after spike protein challenge showed a tendency of surface translocation of ETBR away from the nucleus (Fig. 4N) . Taken together, SARS-CoV-2 infection triggers ET-1 production in lung epithelial cells, which in turn hastens viral entry into lung epithelial cells in a vicious cycle, leading to both intravascular and extravascular damage (Fig. 4O) .
A novel function of ET-1 is disclosed herein, which was originally known as a vasoconstrictor, in the context of SARS-CoV-2 virus-host interaction in the pathogenesis of COVID-19. Plasma ET-1 level is known to increase with the age18, 19 and age-related diseases such as hypertension20, 21 and diabetes22, 23. These findings might explain why the older adults are more susceptible for SARS-CoV-2 infection. Given the intimate relationship with disease severity, ET-1 is a plausible biomarker for prognostication of COVID. We have also shown that ET-1 promotes SARS-CoV-2 viral entry via its two G protein-coupled receptors. Endothelin receptor antagonism attenuates viral entry. Blockade of endothelin signalling emerges as an alternative therapeutic strategy in COVID-19 management.
Distinct activation pattern of ET-1, ETAR and ETBR after viral infection
ET-1 transduces its biological function through two G protein-coupled receptors, i.e., ETAR and ETBR, encoded by EDNRA and EDNRB. ETBR was believed as a clearance receptor for ET-1. In the present study, a more prominent increase in ETBR expression than ETAR after SARS-CoV-2 infection in both humans and hamsters was observed, as well as elevated ET-1. Notably, nirmatrelvir significantly lowered the gene expression level of EDN1 and EDNRB,
but not EDNRA, in the infected lung tissues of hamsters. It indicates the involvement of the ET-1/ETBR axis in SARS-CoV-2 infection.
Similar to SARS-CoV-2, human cytomegalovirus and coxsackievirus infection also activated ET-1 and ETBR, but not ETAR in epithelial and endothelial cells12, 13. It has been shown that human coxsackie-adenovirus receptor was regulated by agonistic and antagonistic binding of ETBR, but not ETAR13. In contrast, influenza A/H5N1 virus increased the mRNA expression level of ET-1 and ETAR in infected bird lung14. Taken together, the activation pattern of endothelin signalling is seemingly virus-specific.
Endothelin receptors as an alternate host receptor for SARS-CoV-2 entry
Mounting evidence has suggested that ACE2 is not the sole receptor of host cells for SARS-CoV-2 entry24-27. The virus also attacks host cells without or with a very low expression level of ACE228, 29. Other receptors with more extensive distribution in different types of cells may explain infection throughout the body. Although ET-1 and its receptors can mostly be found on endothelial cells, they are distributed widely among a variety of cell types from epithelial cells to immune cells30. In our study, without adding ET-1, either genetic knockdown or pharmaceutical blockade of each endothelin receptor could reduce viral entry. Noteworthily, it will not significantly change the expression of ACE2 at protein level. Furthermore, there are two pieces of indirect evidence in the present study indicating endothelin receptors as alternate host receptors to interact with viral spikes. First, upon spike protein challenge, endothelin receptor migrated from the cytoplasm towards the membrane. Second, we were able to co-localise viral spike with endothelin receptors in host cells. These findings warrant further investigation into the exact binding mechanism and sites.
Endothelin receptor antagonists emerge as an alternative anti-viral medication
Current COVID-19 remedy relies heavily on antiviral drugs such as remdesivir, molnupiravir and nirmatrelvir, prescribed to COVID patients within days of symptom onset, to inhibit virus replication. However, clinical use of these antiviral drugs is limited to a narrow time window of treatment immediately after virus exposure, high risk of virus rebound after treatment discontinues31, and significant side effects32, 33 including its lethal mutagenesis property against viral RNA causing genotoxicity34. Monoclonal antibodies targeting the spike protein of SARS-CoV-2 have also been developed to prevent the virus from entering host cells35. However, the efficacy of such neutralizing antibodies is largely affected by the variants of concern and it may trigger some immune-mediated reactions36. With the emergence of new variants of SARS-CoV-2 in a short period of time, the development of neutralizing antibodies might not be a cost-effective approach. Furthermore, the new variants have evolved to become
resistant to the neutralising antibodies and some commonly used antiviral drugs37, 38. Therefore, repurposing FDA-approved medication such as macitentan will be an advantage for emergency use for antiviral treatment with the readily available pharmacokinetics and toxicity data. Combined treatment for both viral entry blockade and viral replication inhibition may generate synergistic effects, by lowering the dosage of anti-viral drugs and thus reducing their toxicity and other side-effects.
The possible link between ET-1 and post-COVID interstitial lung disease
Post-COVID interstitial lung disease is a new fibroinflammatory disease entity39. The relative risk of interstitial lung disease in hospitalized COVID patients is as high as 18.73 within 90 days of infection40, in which plasma ET-1 significantly increased with upregulation of other endothelial damage and dysfunction markers4-9. Mounting evidence suggested that epithelial-mesenchymal transition plays a role in COVID-associated lung fibrosis41, 42. ET-1 is known as a pro-fibrotic factor in a variety of tissues16, 43, 44. ET-1 has been shown to induce epithelial-mesenchymal transition (EMT) of alveolar epithelial cells and collagen production in a TGF-β-dependent manner via endothelin type A receptor45, 46. Here, we observed collagen deposition in infected lung interstitium in both humans and hamsters along with activation of endothelin signalling components after SARS-CoV-2 infection. This evidence implies a possible link between ET-1 and post-COVID interstitial lung disease, worthwhile for further investigations.
In conclusion, this work sheds light on the pivotal role of endothelin signalling in SARS-CoV-2 infection. It opens a door for drug repurposing and mechanism-based development of ET-1-targeted therapy for COVID.
Methods
Cells, SARS-CoV-2 virus and SARS-CoV-2 spike protein
Different cell lines used in this study were chosen because of their susceptibility to virus infection and sensitivity to replication of coronaviruses. A549-hACE2, A549-TMPRSS2-ACE2 and VeroE6-TMPRSS2 were maintained in DMEM culture medium supplemented with 10%heat-inactivated FBS, 50 U ml-1 penicillin and 50 μg ml-1 streptomycin. SARS-CoV-2 (HKU-001a strain, GenBank accession number: MT230904) was isolated from the nasopharyngeal aspirate specimen of a COVID-19 patient47. The virus was amplified by VeroE6 cells as previously described48. Various concentrations of SARS-CoV-2 (2019-nCoV) spike S1+S2 ECD-His recombinant protein (Sino Biological) was used in in vitro experiments.
Drugs
Endothelin receptor blockers BQ123 (Sigma, 100 μM) , BQ788 (Sigma, 100 μM) and macitentan (Allschwil, 10 μM) were used to block ETAR, ETBR and both receptors respectively. BQ3020 (Sigma, 100nM) is an endothelin receptor type B agonist. Remdesivir (MCE, 10 μM) was used as a positive control for inhibiting virus replication. All other chemicals used in this study were purchased from Sigma-Aldrich unless otherwise specified.
Golden Syrian hamster SARS-CoV-2 infection model
The animal experiments were approved by the HKU Committee on the Use of Live Animals in Teaching and Research (CULATR) . Briefly, each 10-12-week-old female golden Syrian hamster (Mesocricetus auratus) was intranasally challenged with 105 PFU of SARS-CoV-2 in 50 μl PBS under anaesthesia using ketamine (200 mg/kg) -xylazine (10 mg/kg) cocktail in the Biosafety Level 3 animal facility at HKU Department of Microbiology according to our established protocol49, 50. Mock-infected animals were treated the same way with 50 μl PBS. Sera and lungs from the hamsters were collected on 4 dpi for virological and histopathological analyses as previously described51, 52. Viral titres and copies in the lung tissue homogenates were detected by RT–qPCR53. ELISA was performed to determine the level of ET-1 and IL-1β in hamster sera.
Human lung ex vivo SARS-CoV-2 infection model
Human lung tissue for ex vivo infection was obtained from patients undergoing surgical operations at Queen Mary Hospital, Hong Kong as previously described54. Written consent from the donors was obtained and approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (UW13-364) before procedures were performed. The infection of lung pieces was performed as previously published55. Briefly, the diced specimens were infected with SARS-CoV-2 with an inoculum of 1 × 106 PFU ml-1 at 500 μl per well. Samples were collected 24 hours post-infection and used for subsequent analysis.
Macitentan and nirmatrelvir treatment
Our pilot study showed high doses of macitentan (3 mg/kg and 10 mg/kg) caused bone loss in mice56. In this study, 0.3 mg per kg which is equivalent to 0.039 mg per kg in human was used, the conversion was based on body surface area among different species57. Assuming the average weight of a person is 60 kg, 0.039 mg per kg means 2.34 mg per day. Nirmatrelvir (Biochempartner) (150 mg/kg) was used as the positive control. Both drugs were administered intraperitoneally daily for 4 days starting from one-day post-infection to mimic an early
treatment effect. Animals were sacrificed at 4 dpi. A total of n = 5 for macitentan-treated group and n = 4 for nirmatrelvir-treated group.
Targeted Mass Spectrometry (MS) Assay
Pseudo PRM-based targeted quantitation of a selected panel of proteins was performed according to previous reports58, 59. Briefly, serum protein samples from healthy subjects or patients were harvested with boiling in 50 mM TEABC /4%SDS. Standard filter-aided sample preparation was then performed in a 10 KDa filter unit (Millipore) . Reduction and alkylation were done by 50 mM TCEP and 55 mM IAA, followed by trypsin digestion overnight at 37 ℃. Tryptic digested samples were then desalted using C18 ziptip (Thermo) before subject to analysis on a timsTOF Pro Mass Spectrometer (Bruker) . MS acquisition was done under PRM mode targeting peptides for each selected protein at a cycle time of 50 mS. Data obtained were then analyzed by Skyline by comparing to an in-house peptide library. Only peptides with Peak Found Ratio of 100%were considered as identified and their total area of MS1 was calculated and exported by Skyline. The FOC (Fold of Change) against the average peak intensity of each targeted peptide in each sample was then calculated to plot the heatmap.
Spatial proteomics for hamster lung sections
Briefly, consecutive FFPE slides were prepared from the lung of a hamster in mock and SARS-CoV-2 infection group. One slide was used for H&E staining as reference, while three individual vessels were selected on the consecutive slide and cut by laser dissection using Leica LMD 7 laser microdissection microscope. The cut pieces of tissue were collected and dewaxed. The protein extraction was done using SDC-containing extraction buffer and processed by SP3 protocol as previously reported60. Twenty ng of tryptic digested peptides from each cut spot were analysed using a TimsTOF Flex Mass Spectrometry under data-independent acquisition (DIA) acquisition mode using default parameters. A proteome library was first built by using peptides extracted from all vessel regions dissected from one slide from mock and SARS-CoV-2 infection groups. MaxDIA was used for DIA identification and quantitation with default parameters as reported61 against Mesocricetus auratus database downloaded from Uniprot. GO enrichment of significantly changed proteins was conducted by searching against DAVID Bioinformatics Resources (https: //david. ncifcrf. gov/) using species database as background.
Measurement of serum ET-1 level by ELISA
Blood was collected from the hamsters when sacrificed and rested at room temperature for 20 minutes before centrifugation at 1500 x g for 10 minutes at 4℃ to get sera. Endothelin-1 ELISA kit (ab133030, Abcam) were used to quantify ET-1 in sera according to the
manufacturer’s protocol. Heat-inactivated serum samples were added to antibody-coated plates for the assay. The absorbance was measured at 450 nm and the concentration was determined. Results were expressed as mean ± S. E. M. of serum samples.
Pseudovirus preparation and pseudotype-based inhibition assay
Pseudovirus-bearing D614G spike protein was produced using Lenti-X lentiviral packaging plasmid (632673, Takara) according to the manufacturer’s protocol. In brief, the plasmid was first amplified and diluted before mixing with the transfection reagent. The resultant nanoparticles were then added to 293T cells. The supernatant was then harvested and virus titer was determined. A549-hACE2 cells were infected with pseudovirus and collected for analysis after 24 hours of incubation.
To investigate the effect of genetic knockdown of endothelin receptors on viral entry, siRNA targeting EDNRA and EDNRB (SilencerTM Select Pre-Designed s4465 and s4468, Invitrogen) were used. A549-hACE2 cells were transfected with siRNA using Lipofectamine RNAiMAX Transfection Reagent (#13778075, Invitrogen) . After 48h of transfection, SARS-CoV-2 spike pseudotyped virus was added to the cells. To investigate the effect of different endothelin receptor blockers on viral entry, A549-ACE2 were pretreated with receptor antagonists (100 μM BQ123, selective ETAR blocker; 100 μM BQ788, selective ETBR blocker, 10 μM macitentan, dual endothelin receptor blocker) for 30 minutes before inoculation with SARS-CoV-2 spike pseudotyped virus. In both genetic knockdown and pharmaceutical inhibition experiment, the inoculum was switched to fresh medium after 6-h inoculation. The activity of firefly luciferase was measured using Luciferase Assay System (Promega) for quantification after 24h of transduction. The results of these experiment are shown in Fig. 4.
Histological analysis
Lung samples were harvested at 4 dpi, fixed and pathogens inactivated. The specimens then underwent progressive dehydration with ethanol, followed by infiltration with xylene and embedded in paraffin. Five-micron sections were prepared and stained with haematoxylin and eosin (H&E) (ab245880, Abcam) for histopathological evaluation and Masson’s trichrome (ab150686, Abcam) for assessing the parenchymal fibrotic changes in lung sections. The staining was performed following the manufacturers’ instructions. Images were captured using OLYMPUS BX51WI.
Histopathological scores were evaluated as previously described62. Briefly, the assessment areas were divided into 4 categories. General evaluation was firstly done to estimate the inflammation area of the total cutting area, followed by a detailed assessment of bronchioles,
alveoli and vasculature, mainly to check the presence of infiltration and inflammation. A score from 0 to 4 was given to each category. The overall score for each sample was calculated by Overall score = score of the general assessment × the scores accumulated from individual category assessment (vasculature, bronchiole and alveoli) .
For immunohistochemistry, antigen retrieval and quenching of endogenous peroxidase activity were performed. The lung sections were incubated with primary antibodies overnight at 4℃ after blocking. Antibodies used: Anti-SARS-CoV-2 spike protein RBD (MA5-36247) , Anti-SARS-CoV-2 nucleocapsid protein (MA17404, Invitrogen) , Anti-endothelin-1 (ab117757, Abcam) , Anti-ETAR (ab117521, Abcam) and Anti-ETBR (ab117529, Abcam) , Anti-ACE2 (ab108252, Abcam) , Anti-vWF (ab6994, Abcam) and anti-4HNE (ab46545, Abcam) . Visualisation of positive signals was done either by 3, 3'-diaminobenzidine (DAB) staining (Vector Lab, US) or multi-channel IHC detection kit (Neon, Histova, China) .
For multi-channel staining, each primary antibody was incubated at 4℃ overnight with tyramide signal amplification (TSA) performed the following day. Visualisation of positive signals was done with horseradish peroxidase (HRP) secondary antibody and different fluorophores: NEON420, NEON520, NEON570, NEON650 and SN520. OLYMPUS BX51WI, Leica TCS SPE confocal microscope and Nikon ECLIPSE Ti2 microscope were used to capture the images. All image analyses were performed using Image-Pro Plus 6.0.
Detection of ACE2 and ET-1 by western blot
The amount of ACE2 and ET-1 in cells was detected by western blot. Briefly, cells were lysed by RIPA buffer with protease and phosphatase inhibitor cocktail (Thermo Scientific) . The proteins were centrifuged at 16,000 x g for 20 minutes at 4℃. Supernatant is collected and boiled. The proteins were then separated in a 10%SDS-PAGE gel and transferred to nitrocellulose filter membranes. Primary antibodies were applied after blocking with 5%milk. Visualisation of bands was performed the next day using horseradish peroxidase (HRP) conjugated secondary antibodies. Antibodies used: Anti-endothelin-1 (ab117757, 1/3000) , Anti-ACE2 (ab108252, 1/5000) and Anti-Actin (ab179467, 1/5000) . ImageJ v. 1.51 was used to perform quantitative analysis of the bands.
Viral load reduction assay
The viral load reduction assay was performed in a 96-well plate format. Briefly, pre-seeded VeroE6-TMPRSS2 were infected with SARS-CoV-2 and/or its variants with a multiplicity of infection (MOI) of 0.01 for 1 hour in a 37℃, 5%CO2 incubator. The infectious
culture was then removed and replaced with fresh medium with 10-fold diluted concentrations of the drug compound. The supernatant was collected at 48 hours post-infection for viral load quantification by RT-qPCR method.
RNA extraction and quantitative RT-PCR
The viral RNA was extracted via RNeasy mini kits (74104, Qiagen) following the manufacturer’s handbook. The viral load was quantified by One-step TB Green RT-PCR kit (RR086A, TaKaRa) with LightCycler 480 Real-time PCR system (Roche) . The amplified signal was acquired every cycle in the PCR reaction after the annealing step. The primers used were listed in the following table.
Plaque assay
The plaque assay was performed on the VeroE6/TMPRSS2 cell line. In brief, the lung homogenate was serial diluted in free DMEM and infected with cells for 1 hour in 37℃incubator. The inoculum was removed from the cells. The cells were overlaid with 1%low melting agarose and incubated for another two days. The cells were fixed in 4%PFA solution overnight, followed by staining by crystal violet.
Public scRNA/snRNA-seq data analysis
ScRNA/snRNA-seq datasets from the lungs of healthy individuals and COVID-19 patients were downloaded from Gene Expression Omnibus (GEO accession: GSE171524, GSE171668, GSE149878, GSE161382, GSE163919 and GSE136831) . We imported these datasets into the Seurat (v 4.3.0) R package63 and merged them into a Seurat object. All functions used in downstream analysis were run with default parameters, unless otherwise specified. Low quality cells were excluded if the number of detected genes was smaller than 200 or greater than 5000. Cells containing more than 10%mitochondrial genes were also
removed. Next, we used NormalizeData () to normalize the gene expression matrix and applied FindVariableFeatures () to detect highly variable genes. After using ScaleData () to scale the data, highly variable genes were utilized to perform RunPCA () for principal component analysis. The harmony R package (v 0.1.1) 64 was used to remove batch effect by different patients. PCs 1-20 were used for FindNeighbors () and FindClusters () to determine the clusters (resolution=0.6) . The clustering result was visualized with the UMAP scatter plot using the PCs 1-20.
We identified the EPCAM+ epithelial cells and further performed clustering for epithelial cells with PCs 1-15 and a resolution parameter of 0.3. We categorized these cells into 5 types based on known markers: AGER, CLIC5 (AT1 cells) , SFTPC, SFTPD (AT2 cells) , TAGLN, COL3A1 (EMT cells) , FOXJ1, CCDC78 (Ciliated cells) , MUC5B, MUC5AC (Goblet cells) . To further evaluate the expression of EDN1 in different cell types, we used the scWGCNA R package (v1.0.0) 65 to create pseudocells by performing calculate. pseudocells () to combine cells in the same cell type with default parameters.
Bioinformatic analysis of RNA-Seq data
Human Calu-3 epithelial cells that underwent mock or SARS-CoV-2 infection were sent for RNA sequencing. RNA-Seq analysis was performed using 1000 ng of total RNA. Library preparation and Illumina sequencing (Pair-End sequencing of 151bp) were done at The University of Hong Kong, LKS Faculty of Medicine, Centre for PanorOmic Sciences (CPOS) , Genomics Core, as we previously described66.
Pheatmap R package (v1.0.12) was used to quantify and visualize the expression. The differentially-expressed (DE) genes between SA and Mock were extracted for KEGG pathway enrichment analysis through ClusterProfiler R package (v 4.2.2) . The top 20 most significantly enriched terms were visualized through ggplot2. To map DE-genes onto the pathways of interest, the pathview R package was applied for mapping and visualization. All of the analyses were performed in R software v4.1.2.
Subcellular localisation tracking of ETBR after spike protein challenge
Plasmid encoding EDNRB-RFP was transiently transfected into A549-hACE2 cells by Lipofectamine 3000 (L3000001, Invitrogen) . Cells transfected with pcDNA-RFP served as a negative control. The subcellular localisation of EDNRB-RFP was observed under a fluorescence microscope. Time-lapse images were taken every 10 minutes for 3 hours after the challenge with SARS-CoV-2 S1 receptor binding domain (RBD) . Translocation of endothelin type B receptor towards the cell membrane from the nucleus was observed. Images were captured using Nikon ECLIPSE Ti2 microscope.
Statistical analysis
All data were presented as mean ± S. E. M. The comparisons of signal intensities and histomorphometric data among different groups were performed using unpaired two-tailed Student’s t-test, one-way ANOVA or Kruskal-Wallis test when deemed appropriate. Respective posthoc tests were carried out when overall significance was detected between groups. The level of significance was set at p < 0.05. Analyses and graphs were generated using Prism 8 (GraphPad) .
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Claims (13)
- A method of treating a coronavirus infection in a subject in need thereof, the method comprising administering a therapeutically effective amount of an endothelin receptor antagonist to the subject, with the proviso that the endothelin receptor antagonist is not co-administered with dapagliflozin or niclosamide.
- The method of claim 1, wherein the subject does not suffer from pulmonary hypertension.
- The method of claim 1, wherein the coronavirus infection is the result of a coronavirus selected from the group consisting of severe acute respiratory syndrome coronavirus (SARS-CoV) , severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) , Middle East respiratory syndrome-related coronavirus (MERS-CoV) , human coronavirus 229E (229E-CoV-2) , human coronavirus NL63 (NL63-CoV) , human coronavirus OC43 (OC43-CoV) , human coronavirus HKU1 (HCoV-HKU1) , and variants thereof.
- The method of claim 1, wherein the coronavirus infection is the result of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and variants thereof.
- The method of claim 1, wherein the endothelin receptor antagonist is a selective ETA receptor antagonist, a selective ETB receptor antagonist, or a dual ETA receptor and ETB receptor antagonist.
- The method of claim 1, wherein the endothelin receptor antagonist is a dual ETA receptor and ETB receptor antagonist.
- The method of claim 1, wherein the endothelin receptor antagonist is selected from the group consisting of sitaxsentan, ambrisentan, atrasentan, BQ-123, sparsentan, zibotentan, aprocitentan, edonentan, bosentan, macitentan, tezosentan, BQ-788, A192621, and mixtures thereof.
- The method of claim 1, wherein the endothelin receptor antagonist is bosentan, macitentan, ambrisentan, or a mixture thereof.
- The method of claim 1, wherein the endothelin receptor antagonist is macitentan.
- The method of claim 1, wherein the coronavirus infection is the result of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the endothelin receptor antagonist is bosentan, macitentan, ambrisentan, or a mixture thereof.
- The method of claim 1, wherein the coronavirus infection is the result of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the endothelin receptor antagonist is macitentan.
- The method of claim 10, wherein the SARS-CoV-2 is an omicron variant.
- The method of claim 1, wherein the subject is a human.
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