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. Author manuscript; available in PMC: 2022 Nov 15.
Published in final edited form as: J Immunol. 2021 Oct 13;207(10):2521–2533. doi: 10.4049/jimmunol.2100144

Selective inhibition of the interaction between SARS-CoV-2 spike S1 and ACE2 by spike S1-interacting domain of ACE2 receptor (SPIDAR) peptide induces anti-inflammatory therapeutic responses

Ramesh K Paidi 1,*, Malabendu Jana 1,*, Rama K Mishra 2, Debashis Dutta 1, Kalipada Pahan 1,3
PMCID: PMC8664124  NIHMSID: NIHMS1741850  PMID: 34645689

Abstract

Many COVID-19 patients in intensive care units suffer from cytokine storm. Although anti-inflammatory therapies are available to treat the problem, very often these treatments cause immunosuppression. Since angiotensin-converting enzyme 2 (ACE2) on host cells serves as the receptor for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), in order to delineate SARS-CoV-2-specific anti-inflammatory molecule, we designed a hexapeptide corresponding to the Spike S1-Interacting Domain of ACE2 Receptor (SPIDAR) that inhibited the expression of proinflammatory molecules in human A549 lung cells induced by pseudotyped SARS-CoV-2, but not vesicular stomatitis virus (VSV). Accordingly, wild type (wt), but not mutated (m), SPIDAR inhibited SARS-CoV-2 spike S1-induced activation of NF-κB and expression of IL-6 and IL-1β in human lungs cells. However, wtSPIDAR remained unable to reduce activation of NF-κB and expression of proinflammatory molecules in lungs cells induced by TNFα, HIV-1 Tat, and viral double-stranded RNA mimic poly IC, indicating the specificity of the effect. The wtSPIDAR, but not mSPIDAR, also hindered the association between ACE2 and spike S1 of SARS-CoV-2 and inhibited the entry of pseudotyped SARS-CoV-2, but not VSV, into human ACE2-expressing HEK293 cells. Moreover, intranasal treatment with wtSPIDAR, but not mSPIDAR, inhibited lung activation of NF-κB, protected lungs, reduced fever, improved heart function, and enhanced locomotor activities in SARS-CoV-2 spike S1-intoxicated mice. Therefore, selective targeting of SARS-CoV-2 spike S1-to-ACE2 interaction by wtSPIDAR may be beneficial for COVID-19.

Keywords: COVID-19, SPIDAR, Inflammation, Spike S1, Fever, Lung pathology

Introduction

Since December 2019, the novel severe acute respiratory syndrome coronavirus 2 is causing a worldwide pandemic leading to a highly morbid and potentially fatal coronavirus disease 2019 (COVID-19). Common symptoms of COVID-19 are fever, cough, and shortness of breath (1, 2) and until now, more than 4 million people died worldwide from COVID-19. While anyone is susceptible to COVID-19, the ones over 60 or with preexisting conditions, such as hypertension, obesity, asthma, or diabetes, are more vulnerable to severe symptoms. It appears that COVID-19 is more lethal in men than women (3). It has been suggested that in severely ill COVID-19 patients, cytokine storm may be responsible for lung injury and multi-organ failure (4). Although about 50% people in USA have been fully vaccinated and a number of drugs (e.g. remdesivir, dexamethasone, etc.) have been approved for repurposing for COVID-19, it is always important to describe a specific antiviral and anti-inflammatory agent for treating COVID-19.

Angiotensin-converting enzyme 2 (ACE2) being the main effector of the classical renin-angiotensin system is an important molecule for the regulation of blood pressure and hypertension as the prototype function of ACE2 is to convert angiotensin II (AngII), a vasoconstrictor, to Ang1–7, a vasodilator (5, 6). However, SARS-CoV-2 requires ACE2 to enter into human cells. The spike protein on the surface of SARS-CoV-2 binds to ACE2 (2, 7) and during infection, the spike protein is cleaved into S1 and S2 subunits. The spike S1 subunit harbors the receptor-binding domain (RBD). Since ACE2 is predominant in lung, heart and kidney (8), SARS-CoV-2 easily infects these organs causing multi-organ failure in severe COVID-19 cases.

Therefore, inhibition of ACE2 should reduce the SARS-CoV-2 infection and associated inflammation. However, either inhibition or knock down of ACE2 may not be a valid therapeutic option for COVID-19 as it is a beneficial molecule. Therefore, to target COVID-19 from therapeutic angle, we took an out-of-the-box approach and designed a hexapeptide corresponding to Spike S1-interacting domain of ACE2 receptor (SPIDAR). Interestingly, wtSPIDAR, but not mSPIDAR, inhibited the association between receptor-binding domain-containing spike S1 and ACE-2 and suppressed spike S1-induced activation of NF-κB and expression of IL-6 in human lungs cells. Accordingly, the wtSPIDAR also inhibited the entry of pseudotyped SARS-CoV-2, but not VSV, into human ACE2-expressing HEK293 cells. Moreover, after intranasal administration, wtSPIDAR, but not mSPIDAR, reduced lung inflammation, decreased lung neutrophil infiltration, reduced fever, inhibited arrhythmias, and enhanced locomotor activities in an animal model of COVID-19. Therefore, intranasal SPIDAR may be beneficial for COVID-19.

Materials and methods

Reagents

SARS-CoV-2 spike-pseudotyped lentiviral particles, VSV-G-pseudotyped particles and human ACE2-expressing HEK293 cells were purchased from GeneCopoeia, Rockville, MD. Recombinant COVID-19 Spike protein S1 was purchased from Abeomics, San Diego, CA. While anti-SARS-CoV-2 Spike S1 antibody was bought from BioVision (Milpitas, CA), anti-human ACE2 antibody was purchased from R&D Systems (Minneapolis, MN). Anti-human MyD88 antibody was bought from Millipore (Burlington, MA). Human A549 lung carcinoma cell line and F-12K medium were obtained from ATCC, Manassas, VA. Hank’s balanced salt solution, 0.05% trypsin, and antibiotic-antimycotic mixture were bought from Mediatech (Washington, DC). Fetal bovine serum (FBS) was obtained from Atlas Biologicals, Fort Collins, CO. ACE2:SARS-CoV-2 Spike Inhibitor Screening Assay Kit was purchased from BPS Bioscience, San Diego, CA. Anti-human IL-6 antibody and human IL-1β and IL-6 ELISA kits were bought from ThermoFisher, Waltham, MA.

In silico structural analysis

To study the interaction between human ACE2 and SARS-CoV-2 spike S1, we performed in silico structural analysis was performed as described earlier (911). Briefly, by utilizing the protein preparation tools from the Schrodinger, Inc. platform, at first, we evaluated the quality of the crystal structure of human ACE2 and SARS-CoV-2 spike S1 followed by addition of hydrogens to the hydrogen bond orientation, charges, missing atoms, and side chains of the different residues of both the proteins. Finally, the complex structure was subjected to energy minimization in the Optimized Potential for Liquid Simulations (OPLS3) force field to make it torsion free. Then the spike protein was extracted out from the ACE2 in order to apply the dynamic hydrogen bonding module for finding potential hydrogen bonds between the two structures. We also evaluated other interactions such as hydrophobic interactions between the two structures.

Spike S1-interacting domain of ACE2 receptor (SPIDAR) peptides

Wild type (wt) SPIDAR: EDLFYQ

Mutated (m) SPIDAR: EKLFYG

SPIDAR peptides (>98% pure) were synthesized in Genscript (Piscataway, NJ).

ACE2:SARS-CoV-2 Spike binding assay

The efficacy of wtSPIDAR and mSPIDAR to dissociate the binding of SARS-CoV-2 spike S1 with ACE2 was investigated using the ACE2:SARS-CoV-2 Spike inhibitor screening assay kit (BPS Bioscience, San Diego, CA) according to manufacturer’s instructions as described by us recently (11). Briefly, 96-well nickel-treated plate provided by the manufacturer was coated with ACE2 solution. After washing with immuno buffer and treatment with blocking buffer, different concentrations of wtSPIDAR and mSPIDAR were added to each well followed by addition of SARS-CoV-2 Spike (RBD)-Fc. After washing and incubation with blocking buffer, plates were treated with anti-mouse Fc-HRP followed by addition of an HRP substrate. Resultant chemiluminescence was monitored in a Perkin Elmer multimode microplate reader, Victor X5.

Infection with pseudotyped lentiviral particles

Human ACE2-expressing HEK293 cells were plated in 24-well plates at 70–80% confluence followed by infection with lentiviral particles pseudotyped with either SARS-CoV-2 spike S1 or VSV-G at an MOI of 0.5. Cells were also infected with empty lentiviral (lenti-naked) particles as control. After 48 h of infection, the entry of pseudovirus into HEK293 cells was checked by either assaying luciferase activity in total cell extracts using a TD-20/20 Luminometer (Turner Designs) or monitoring GFP fluorescence in an Olympus BX-41 fluorescence microscope.

Semi-quantitative RT-PCR analysis

Total RNA was isolated from mouse lungs and human A549 lung cells using Ultraspec-II RNA reagent (Biotecx Laboratories, Inc., Houston, TX), and RNAeasy Mini kit (Qiagen, Germantown, MD), respectively. To remove any contaminating genomic DNA, total RNA was digested with DNase. RT-PCR was carried out as described earlier (12, 13) using a RT-PCR kit (Clontech, Mountain View, CA) and following primers of human proinflammatory molecules: tumor necrosis factor α (TNFα), sense, 5’-CTG AGT CGG TCA CCC TTC TCC AGC T-3’; antisense, 5’-CCC GAG TGA CAA GCC TGT AGC CCA T-3’; IL-1β, sense, 5’-GGA TAT GGA GCA AC A AGT GG-3’; antisense, 5’-ATG TAC CAG TTG GGG AAC T-3’; IL-6, sense, 5’-TTT TGG AGT TTG AGG TAT ACC TAG-3’; antisense, 5’-GCT GCG CAG AAT GAG ATG AGT TGT-3’; GAPDH, sense, 5’-GGT GAA GGT CGG AGT CAA CG-3’; antisense, 5’-GTG AAG ACG CCA GTG GAC TC-3’.

GAPDH gene was used to confirm the synthesis of an equivalent amount of cDNA from different samples. Amplified products were electrophoresed on a 1.8% agarose gels and visualized by ethidium bromide staining.

Quantitative real-time PCR analysis

Real-time PCR was performed in the ABI-Prism7700 sequence detection system (Applied Biosystems, Foster City, CA) as described earlier (12, 13).

CRISPR editing of MyD88 gene in human A549 lung cells

Cells were plated in 6-well plates at a density of 1.8 ×106 cells per well with DMEM/F-12 containing FBS (10%) and antibiotic-antimycotic mixture at 370C with 5% CO2. After 24 h, cells were infected with CRISPR scrambled sgRNA all-in-one lentivirus and human MyD88 sgRNA CRISPR all-in-one lentivirus (Viral Titer=9×106 IU/ml) according to the protocol provided by the manufacturer (Applied Biological Materials Inc., Richmond, Canada). Following the infection, cells were incubated at 370C with 5% CO2. After overnight incubation, culture medium was replaced with 2 ml of complete medium and cells were incubated for 48 h followed by monitoring genomic editing by Western blot.

Assay of NF-κB-driven luciferase activity

A549 cells plated at 60–70% confluence in 12-well plates were transfected with 0.25 μg of pNF-κB-Luc using Lipofectamine Plus (Invitrogen). After 24 h of transfection, cells were stimulated with different stimuli under serum free condition for 4 h. Firefly luciferase activities were analyzed in cell extracts using the luciferase assay system kit (Promega) in a TD-20/20 Luminometer (Turner Designs) as described earlier (14, 15).

Animals and intoxication of C57/BL6 mice with recombinant SARS-CoV-2 spike S1

Mice were maintained and experiments conducted in accordance with National Institute of Health guidelines and were approved by the Rush University Medical Center IACUC. C57/BL6 mice (6–8 week old; Envigo) of both sexes were intoxicated with recombinant SARS-CoV-2 spike S1 (50 ng/mouse/d) intranasally as described by us recently (11). Briefly, recombinant spike S1 was dissolved in 2 μl normal saline, mice were hold in supine position and 1 μl volume was delivered into each nostril using a pipetman. Control mice received only 2 μl saline.

Intranasal Delivery of SPIDAR

Starting from 5 d of SARS-CoV-2 spike S1 intoxication, C57/BL6 mice (6–8 week old; Envigo) of both sexes were treated intranasally with wtSPIDAR or mSPIDAR (100 ng/mouse/d) for 7d. Briefly, SPIDAR peptides were dissolved in 2 μl normal saline, mice were hold in supine position and 1 μl volume was delivered into each nostril using a pipetman.

Non-invasive ECG recording

ECG recording was performed as described by us recently (11). Briefly, mice were acclimatized to the ECG pulse transducer pad (AD instruments TN 012/ST, USA) and the experimental housing conditions prior to ECG recording. ECG pulse transducer pad was placed around the heart of each animal and ECG recording was carried out for 120s. For ECG analysis, electrocardiography data were exported from the Labchart pro, version 8.0 (Power Lab 4/35 model) as raw data format and the digital signal processing was performed using this software. The recording was conducted for 120 s and the ECG signals were recorded at a sampling range of 20 mV with 4 beats/s sampling rate and different ECG parameters were calculated as described (11). In the detection setting, typical QRS width was kept at 10 ms and R wave reserved at least 60 ms apart with the alignment sustained at QRS maximum. During analysis, pre-baseline was kept at 10 ms with maximum at 50 ms. We selected for rodent waves to measure ST segment height at 10 ms. Moreover, the recording and analysis settings were kept same for all the experimental mice included in this study.

Monitoring lung infiltration and pathology

After treatment, animals were anesthetized with ketamine/xylazine injectable followed by transcardial perfusion (16). The lungs were collected and processed for histological studies. Hematoxylin-eosin (HE) (Sigma, St Louis, MO) staining was performed from 4 μm thick paraffin embedded sections and used for studying the general lung tissue morphology. Number of epithelial cells, and the number infiltrated neutrophils in alveolar spaces and interstitial space were analyzed by NIH Image J. At least ten 40x fields from each group were chosen for the counting of the epithelial and infiltrated neutrophils. Lung injury score was measured as described by Matute-Bello et al (17) using a scale (11) in a blinded manner.

Immunostaining

It was performed as described before (18, 19). Briefly, paraffin embedded sections were deparaffinized, rehydrated and blocked with PBS containing 4% normal horse serum and 2% BSA for 1 h. For double-labeling, sections were incubated overnight at 4°C with antibodies for ACE2 (1:100) and IL-6 (1:500). Following washings, sections were incubated with Cy2 or Cy5-labelled secondary antibodies and imaged under an Olympus BX41 fluorescence microscope. Mean fluorescence intensity (MFI) of target proteins was measured using ImageJ. Intensity of tau and phospho-tau DAB staining was quantified using Fiji (ImageJ2).

In situ chromatin immunoprecipitation (ChIP)

Recruitment of NF-κB to the IL-6 promoter in vivo in the lung of mice was examined by in situ ChIP analysis as described before (13). Briefly, after formaldehyde fixation, lungs were kept in 4% paraformaldehyde for overnight followed by washing with PBS and then homogenization in Tris-EDTA buffer (pH 7.6). The homogenates were kept in 500 μL lysis buffer at 52°C for overnight until tissue fragments were dissolved completely. Then the genomic DNA was isolated and sonicated followed by immunoprecipitation with antibodies against p65, p50, transcriptional coactivator p300, and RNA polymerase according to standard protocol as described by us (20, 21). Immunoprecipitated DNA was analyzed by PCR and real-time PCR using following primers:

Sense 5’-CCAATCAGCCCCACCCACTCTGGCCCC-3’

Anti-sense 5’-GGAATTGACTATCGTTCTTGGTGGGCT-3’.

ELISA for IL-6 and C-reactive protein (CRP)

IL-6 ELISA was performed in lung homogenates and mouse serum as described earlier (22) using an assay kit (eBioscience) according to manufacturer’s instruction. CRP ELISA was performed in mouse serum using a kit from Abcam.

Statistical Analysis

Statistical analyses were performed using GraphPad Prism 9.0.0 (GraphPad Software, Inc., La Jolla, CA). Results were examined by one-way ANOVA followed by Tukey’s multiple comparison post-hoc test. Data were expressed as means ± SEM. Statistical differences between means were calculated by the Student’s t-test (two-tailed) while comparing between two groups. A p- value of less than 0.05 (p<0.05) was considered statistically significant.

Results

Designing of a peptide corresponding to the Spike S1-Interacting Domain of ACE2 Receptor (SPIDAR):

Since the interaction of SARS-CoV-2 with its receptor ACE2 is critical for the infection of host cells and the pathogenesis of COVID-19, we decided to target the interaction between ACE2 and the receptor-binding domain (RBD) of SARS-CoV-2 spike S1 (23). Therefore, we applied rigid-body protein-protein interaction tool to model the interaction between ACE2 and RBD of spike S1 and found that Glu37 to Gln42 region of ACE2 is responsible for binding with spike S1 (Fig. 1A). Therefore, we designed a small peptide (Fig. 1B) corresponding to the Spike S1-interacting domain of ACE2 receptor (SPIDAR) to perturb the communication between ACE2 and SARS-CoV-2 Spike S1:

Figure 1. Designing of Spike S1-Interacting Domain of ACE2 Receptor (SPIDAR) peptide for disruption of ACE2 and SARS-CoV-2 interaction.

Figure 1.

(A) A rigid-body in silico docked pose of human ACE2 (green) and SARS-CoV-2 spike S1 (magenta). B) Sequence of wild type and mutated SPIDAR peptides. Positions of mutations are underlined. C) Inhibition of ACE2 to SARS-CoV-2 spike S1 binding by wtSPIDAR, but not mSPIDAR, peptide. **p < 0.01; ***p < 0.001 vs spike S1. HEK293 cells expressing hACE2 plated at 70–80% confluence in 24-well plates were infected with lenti-naked or lentiviral particles pseudotyped with either SARS-CoV-2 spike S1 at different MOI. After 2 d of infection, entry of pseudovirus was monitored by luciferase activity (D). ***p < 0.001. After infection with lentiviral particles, cells were treated with either anti-ACE2 antibody or control IgG. After 48h, entry of pseudovirus was monitored by luciferase activity (E). **p < 0.01; ***p < 0.001. Cells pretreated with different concentrations of SPIDAR and ACE2-interacting domain of SARS-CoV-2 (AIDS) for 10 min were infected with lenti-SARS-CoV-2 pseudovirus (MOI = 0.5). After 2 d of infection, entry of pseudovirus was monitored by luciferase activity (F). ***p < 0.001 vs lenti-SARS-2. Cells pretreated with different concentrations of SPIDAR and AIDS for 10 min were infected with lenti-VSV-G pseudovirus (MOI = 0.5). After 2 d of infection, entry of pseudovirus was monitored by luciferase activity (G). Results represent three independent experiments. NS, not significant.

Wild type (wt) SPIDAR: 37EDLFYQ42

Mutated (m) SPIDAR: 37EKLFYG42

In these peptides, we have underlined the position of mutations. Next, we investigated whether wtSPIDAR inhibited the binding of SARS-CoV-2 spike S1 with ACE2 receptor. Therefore, we employed a chemiluminescence-based ACE2:SARS-CoV-2 spike S1 binding assay (catalog# 79936; BPS Bioscience). We found that SARS-CoV-2 spike S1 binding to immobilized ACE2 was strongly inhibited by wtSPIDAR (Fig. 1C). As expected, we did not see any inhibition in binding with mSPIDAR (Fig. 1C), indicating the specificity of the effect.

Since wtSPIDAR inhibited the association between ACE2 and SARS-CoV-2 spike S1, next, we examined whether wtSPIDAR inhibited viral entry. Pseudoviruses are appropriate for virus entry assays, as they permit viral entry to be distinguished from other virus life-cycle stages. Therefore, we used lentiviral particles pseudotyped with the SARS-CoV-2 Spike S1 protein. Since human embryonic kidney 293 (HEK293) cells do not have any detectable ACE2, we employed human ACE2 expressing HEK293 cells for entry assay. In pseudovirus luciferase assay, viral entry into cells is correlated to the levels of luciferase signals in the cells. While lenti-naked infection did not increase luciferase signals in hACE2-expressing HEK293 cells, marked increase in luciferase activity was seen in pseudo-SARS-CoV-2-infected cells (Fig. 1D), indicating the entry of pseudo-SARS-CoV-2 into hACE2-HEK293 cells. As evident from Figure 1E, neutralizing antibodies against human ACE2, but not control IgG, markedly inhibited the admission of pseudo-SARS-CoV-2 into HEK293 cells, confirming that the entry of pseudo-SARS-CoV-2 into hACE2-HEK293 cells is dependent on ACE2. Although wtSPIDAR at a concentration of 0.1 μM did not inhibit pseudo-SARS-CoV-2-induced luciferase activity, marked reduction of luciferase activity was seen at 0.5 and 1.0 μM wtSPIDAR (Fig. 1F), suggesting that wtSPIDAR inhibits the entry of pseudo-SARS-CoV-2 into hACE2-HEK293 cells. This result was specific as mSPIDAR at same concentration remained unable to inhibit luciferase activity (Fig. 1F). Recently, we have described that a peptide corresponding to ACE2-interacting domain of SARS-CoV-2 (AIDS) also inhibits the interaction between spike S1 and ACE2 (11). Therefore, here, we wanted to determine which one between AIDS and SPIDAR is more potent in inhibiting the entry of SARS-CoV-2 pseudovirus in hACE2-HEK293 cells. Similar to wtSPIDAR, wtAIDS also reduced luciferase activity in pseudo-SARS-CoV-2-infected hACE2-HEK293 cells (Fig. 1F). However, wtSPIDAR was more effective than wtAIDS (Fig. 1F) in inhibiting luciferase activity in pseudo-SARS-CoV-2-infected hACE2-HEK293 cells. Moreover, similar to pseudo-SARS-CoV-2, infection with pseudo-VSV also led to marked increase in luciferase activity in hACE2-HEK293 cells (Fig. 1G). However, either SPIDAR or AIDS remained unable to modulate pseudo-VSV-induced luciferase activity in hACE2-HEK293 cells (Fig. 1G), indicating the specificity of both SPIDAR and AIDS peptides.

To further confirm inhibition of viral entry by wtSPIDAR, we monitored GFP fluorescence. While no GFP expression was seen in the cells infected with lenti-naked viral particles (Fig. 2AB), marked GFP expression was found in cells infected with both SARS-CoV-2 pseudovirus (Fig. 2A) and VSV pseudovirus (Fig. 2B). However, wtSPIDAR, but not mSPIDAR, markedly inhibited GFP expression induced by pseudo-SARS-CoV-2 (Fig. 2A & 2C). On the other hand, either wtSPIDAR or mSPIDAR had no effect on VSV pseudovirus-induced GFP expression in hACE2-HEK293 cells (Fig. 2B & 2D). Together, these results suggest that wtSPIDAR is capable of inhibiting the entry of pseudo-SARS-CoV-2, but not pseudo-VSV, into hACE2-HEK293 cells.

Figure 2. Effect of SPIDAR on the entry of pseudotyped SARS-CoV-2 and VSV into hACE2-expressing HEK293 cells.

Figure 2.

Cells plated at 70–80% confluence in 24-well plates were treated with 2 μM wtSPIDAR or mSPIDAR for 10 min followed by infection with lentiviral particles pseudotyped with either SARS-CoV-2 spike S1 (A) or VSV-G (B) at an MOI of 0.5. After 2 d of infection, cells were fixed and GFP fluorescence was recorded in an Olympus BX-41 fluorescence microscope. Results represent three independent experiments. GFP-positive cells (C, SARS-CoV2; D, VSV-G) were counted in 10 different fields of each of different treatment groups and expressed as mean GFP-positive cells per field. ***p < 0.001. NS, not significant.

The wtSPIDAR inhibits the expression of proinflammatory molecules in SARS-CoV-2 pseudovirus-infected human A549 lung cells:

Some COVID-19 patients display a severe symptom of acute respiratory distress syndrome (ARDS) with high mortality. This high severity is dependent on pulmonary inflammation induced by a cytokine storm (4) that is most likely mediated by interleukin-6 (IL-6) and other proinflammatory cytokines. Therefore, at first, we investigated if SARS-CoV-2 pseudovirus infection leads to the expression of proinflammatory cytokines in A549 lung cells. Marked upregulation of IL-6 (Fig. 3A) and IL-1β (Fig. 3B) mRNA expression in lenti-SARS-CoV-2-infected, but not lenti-naked-infected, cells suggest that SARS-CoV-2 pseudovirus infection is capable of upregulating proinflammatory cytokines in human lung cells. Abrogation of SARS-CoV-2 pseudovirus-induced mRNA expression of IL-6 and IL-1β by neutralizing antibodies against SARS-CoV-2 spike S1 indicate that the induction of proinflammatory cytokine gene expression in A549 cells may be due to SARS-CoV-2 spike S1. It could be also due to the fact that the entry of the virus is blocked and therefore, there is a lack of intracellular pathogen-associated molecular patterns (PAMP) detection. Next, we examined if SPIDAR was capable of suppressing the expression of IL-6 and IL-1β in SARS-CoV-2 pseudovirus-infected A549 lung cells. Inhibition of pseudo SARS-CoV-2-induced expression of IL-6 (Fig. 3C) and IL-1β (Fig. 3D) by wtSPIDAR, but not mSPIDAR, suggest that wtSPIDAR is capable of knocking down the expression of proinflammatory molecules in SARS-CoV-2 pseudovirus-infected A549 cells. Alternatively, these results also suggest that wtSPIDAR, but not mSPIDAR, inhibits pseudoviral entry thus preventing PAMP-mediated expression of IL-6 and IL-1β. This result was specific as either wtSPIDAR or mSPIDAR remained unable to inhibit the mRNA expression of IL-6 and IL-1β in VSV pseudovirus-infected A549 cells.

Figure 3. Effect of wtSPIDAR and mSPIDAR peptides on pseudotyped SARS-CoV-2- and VSV-induced expression of proinflammatory molecules in human A549 lung cells.

Figure 3.

A549 cells plated at 70–80% confluence in 12-well plates were infected with either lenti-naked or lentiviral particles pseudotyped with SARS-CoV-2 spike S1. After 24 h of infection, the mRNA expression of IL-6 (A) and IL-1β (B) was monitored by real-time PCR. Cells treated with different concentrations of wtSPIDAR or mSPIDAR for 10 min were infected with lentiviral particles pseudotyped with SARS-CoV-2 spike S1 at an MOI of 0.5. In parallel experiment, infected cells also received neutralizing antibodies against spike S1 at a concentration of 0.5 μg/ml. After 24 h of infection, the mRNA expression of IL-6 (C) and IL-1β (D) was monitored by real-time PCR. ***p < 0.001 vs lenti-SARS-2. Cells treated with wtSPIDAR or mSPIDAR for 10 min were infected with lentiviral particles pseudotyped with VSV-G at an MOI of 0.5. After 24 h of infection, the mRNA expression of IL-6 (E) and IL-1β (F) was monitored by real-time PCR. Results are mean ± SEM of three independent experiments. NS, not significant.

The wtSPIDAR inhibits lung cell inflammation induced by SARS-CoV-2 spike S1, but not TNFα, double-stranded RNA (poly IC) and HIV-1 Tat:

Recently we have demonstrated that recombinant SARS-CoV-2 spike S1 is capable of inducing the activation of NF-κB and the expression of IL-6 in human A549 lung cells (11). While boiled spike S1 does not induce the expression of proinflammatory molecules in A549 cells, anti-spike S1 neutralizing antibody (BioVision; Cat# A3000–50) neutralized the proinflammatory function of recombinant SARS-CoV-2 spike S1 (11). To further confirm that the induction of proinflammatory molecules in A549 cells by recombinant SARS-CoV-2 spike S1 is not due to any contaminant, we examined the effect of spike S1 in MyD88 depleted A549 cells. We performed CRISPR-mediated editing of MyD88 gene in human A549 cells followed by Western blotting with anti-MyD88 antibody (catalog # AB16527, Millipore). As evident from Figure 4AB, CRISPR editing markedly reduced the level of MyD88 compared with scrambled control. However, CRISPR knockdown of MyD88 remained unable to inhibit SARS-CoV-2 spike S1-induced expression of TNFα (Fig. 4C), IL-6 (Fig. 4D) and IL-1β (Fig. 4E) mRNAs in A549 cells. Since most of the known TLRs except TLR3 require MyD88 as an adapter, these results indicate that the proinflammatory function of SARS-CoV-2 spike S1 is not due to any contamination by TLR ligands.

Figure 4. CRISPR knockdown of MyD88 does not alter SARS-CoV-2 spike S1-induced expression of proinflammatory molecules in human A549 lung cells.

Figure 4.

A549 cells were infected with CRISPR scrambled sgRNA all-in-one lentivirus and human MyD88 sgRNA CRISPR all-in-one lentivirus (MOI = 5) according to manufacturer protocol and 2 d after infection, the level of MyD88 protein was monitored in cells by Western blot. Actin was run as a loading control. Bands were scanned and values (MyD88/Actin) presented as relative to lenti-scrambled CRISPR (B). Results are mean ± SD of three separate experiments. After 2 d of infection, cells were treated with 10 ng/ml recombinant SARS-CoV-2 spike S1 for 6 h followed by monitoring the mRNA expression of TNFα (C), IL-6 (D) and IL-1β (E) by real-time PCR. Results are mean ± SD of three separate experiments. ***p < 0.001; NS, not significant.

SARS-CoV-2 spike S1 is known to interact with ACE2 receptor (2). Therefore, to confirm that the proinflammatory effect of SARS-CoV-2 spike S1 in human A549 lung cells is also dependent on ACE2, we examined the effect of anti-human ACE2 antibody (R&D Systems) on SARS-CoV-2 spike S1-induced expression of proinflammatory cytokines. As expected, stimulation with SARS-CoV-2 spike S1 led to marked induction of IL-6 (Fig. 5A) and IL-1β (Fig. 5B) mRNAs in A549 cells. However, anti-ACE2 antibody, but not control IgG, at different doses tested markedly suppressed the SARS-CoV-2 spike S1-induced expression of IL-6 (Fig. 5A) and IL-1β (Fig. 5B) mRNAs, indicating an essential role of ACE2 in SARS-CoV-2 spike S1-stimulated expression of proinflammatory cytokines in A549 cells. It has been shown that human A549 cells are poor hosts for SARS-CoV (24). A key feature in the infection of host cells with live SARS-CoV-2 is the involvement of transmembrane protease serine 2 (TMPRSS2). Only ACE2 expression is not enough SARS-CoV-2 infection because TMPRSS2 is necessary for the priming of spike protein of SARS-CoV-2 (25, 26). However, once spike protein is cleaved and processed, it binds with ACE2 and does not require TMPRSS2. Similarly, recombinant spike S1 also does not need the involvement of TMPRSS2. Here, since we are stimulating A549 cells with recombinant SARS-CoV-2 spike S1, basal level of ACE2 present in A549 cells is sufficient for biological activity of spike S1.

Figure 5. Neutralization of ACE2 inhibits SARS-CoV-2 spike S1-induced expression of proinflammatory molecules in human A549 lung cells.

Figure 5.

A549 cells preincubated with different concentrations of anti-human ACE2 antibody (R&D Systems) for 15 min were stimulated with 10 ng/ml recombinant SARS-CoV-2 spike S1 for 6 h followed by monitoring the mRNA expression of IL-6 (A) and IL-1β (B) by real-time PCR. Results are mean ± SD of three separate experiments. NS, not significant; *p < 0.05; ***p < 0.001 vs spike S1 only.

Since wtSPIDAR inhibited SARS-CoV-2 pseudovirus-induced expression of IL-6 and IL-1β in A549 cells, next, we examined if SPIDAR was capable of suppressing inflammation in human A549 lung cells induced by recombinant SARS-CoV-2 spike S1. To understand the specificity of the effect, cells were also stimulated with proinflammatory cytokine TNFα, double-stranded RNA mimic poly IC and HIV-1 Tat. Recombinant spike S1 (Fig. 6A), TNFα (Fig. 6E), poly IC (Fig. 6I), and HIV-1 Tat (Fig. 6M) induced the activation of NF-κB in A549 cells as monitored by NF-κB-driven reporter (luciferase) activity. However, wtSPIDAR inhibited spike S1-induced activation of NF-κB (Fig. 6A). In contrast, wtSPIDAR remained unable to subdue the activation of NF-κB in A549 cells induced by TNFα (Fig. 6E), poly IC (Fig. 6I), and HIV-1 Tat (Fig. 6M). These results were specific as mSPIDAR had no effect on the activation of NF-κB induced by any of the stimuli. To confirm these results, we also monitored the expression of IL-6 and IL-1β, proinflammatory cytokines that are driven by activated NF-κB. Spike S1 (Fig. 6BD), TNFα (Fig. 6FH), poly IC (Fig. 6JL), and HIV-1 Tat (Fig. 6NP) increased the expression of IL-6 and IL-1β in A549 cells. However, wtSPIDAR inhibited SARS-CoV-2 spike S1-induced mRNA expression of IL-6 and IL-1β in A549 cells (Fig. 6BD). In contrast, wtSPIDAR could not inhibit the mRNA expression of IL-6 and IL-1β induced by TNFα (Fig. 6FH), poly IC (Fig. 6JL) and HIV-1 Tat (Fig. 6NP). Moreover, the inability of mSPIDAR to inhibit the mRNA expression of IL-6 and IL-β induced by any of the stimuli used further indicated the specificity of anti-inflammatory function of SPIDAR.

Figure 6. Effect of wtSPIDAR and mSPIDAR on the induction of NF-κB activation and the expression of proinflammatory molecules in human A549 lung cells.

Figure 6.

A549 cells plated at 60–70% confluence in 12-well plates were transfected with pNF-κB-Luc (0.25 μg/well) using Lipofectamine Plus (Invitrogen). After 24 h of transfection, cells were incubated with either wtSPIDAR or mSPIDAR for 10 min followed by stimulation with 10 ng/ml recombinant SARS-CoV-2 spike S1 (A), 10 ng/ml TNFα (E), 50 μg/ml polyIC (I), and 150 ng/ml HIV-1 Tat (M) under serum-free conditions. Firefly luciferase activity was measured in total cell extracts after 4 h of stimulation. Results are mean ± SD of three separate experiments. Cells preincubated with either wtSPIDAR or mSPIDAR peptides for 10 min were stimulated with SARS-CoV-2 spike S1 (B–D), TNFα (F-H), polyIC (J-L), and HIV-1 Tat (N–P) under serum-free conditions. After 4 hours of stimulation, the mRNA expression of IL-6 (B, C, F, G, J, K, N, O) and IL-1β (B, D, F, H, J, L, N, P) was monitored by semi-quantitative RT-PCR (B, F, J, N) and quantitative real-time PCR (C, D, G, H, K, L, O, P). Results are mean ± SEM of three independent experiments. ***p < 0.001 vs spike S1. NS, not significant.

Intranasal wtSPIDAR decreases lung inflammation and reduces fever in SARS-CoV-2 spike S1-intoxicated mice:

Although SARS-CoV-2 does not easily bind to ACE2 and infect normal mice, recently we have seen that intranasal intoxication of SARS-CoV-2 spike S1 induces fever and important cardiac and respiratory symptoms of COVID-19 in normal C57/BL6 mice (11). To further characterize this SARS-CoV-2 spike S1-mediated mouse model, we performed the following: First, we monitored the status of ACE2 in lungs of C57/BL6 mice before and after spike S1 intoxication. Lungs of normal mice expressed ACE2, which decreased upon SARS-CoV-2 spike S1 intoxication (Supplemental Fig. 1AB). The decrease in ACE2 expression in lung cells could be due to the internalization of ACE2 receptor-spike S1 complex via clathrin-dependent manner (27) and/or suppression of ACE2 expression by proinflammatory cytokines generated from spike S1 intoxication (28). Anyway, the decrease in ACE2 expression was specific as the lungs of SARS-CoV-2 spike S1-intoxicated mice, but not normal mice, expressed IL-6 (Supplemental Fig. 1A & 1C). Therefore, normally, mouse lungs express ACE2, which is decreased by spike S1 insult. Second, we examined the efficacy of heat-denatured SARS-CoV-2 spike S1 in inducing fever and locomotor abnormalities in C57/BL6 mice. SARS-CoV-2 spike S1 was boiled for 5 min for denaturation. Failure of boiled recombinant SARS-CoV-2 spike S1 to induce fever (Supplemental Fig. 2A), increase serum LDH (Supplemental Fig. 2B), cause impairment in locomotor activities (Supplemental Fig. 2C, heat map; Supplemental Fig. 2D, distance travelled; Supplemental Fig. 2E, velocity; Supplemental Fig. 2F, center zone frequency; Supplemental Fig. 2G, rotorod latency) suggest that the induction of these symptoms in mice is due to SARS-CoV-2 spike S1 protein. Third, we studied whether anti-spike S1 antibody could neutralize functions of SARS-CoV-2 spike S1 in C57/BL6 mice. Neutralization of SARS-CoV-2 spike S1-mediated increase in body temperature (Supplemental Fig. 2A), upregulation in serum LDH (Supplemental Fig. 2B) and induction of hypolocomotion (Supplemental Fig. 2CG) in mice by co-administration of anti-SARS-CoV-2 spike S1 antibody indicate the involvement of SARS-CoV-2 spike S1 protein in causing these problems in mice. According to Mossel et al (24), mouse ACE2 could be a poor receptor for SARS-CoV-2. However, here, we have challenged mice with recombinant SARS-CoV-2 spike S1, not live SARS-CoV-2.

Therefore, next, we examined if wtSPIDAR was capable of controlling these symptoms in mice. Since COVID-19 patients are and/or will be treated with drugs usually after the diagnosis of the disease, we examined whether wtSPIDAR administered 5 d after initiation of the disease (Fig. 7A) was still capable of protecting mice from COVID-19 related complications. We selected the 5-day window as all SARS-CoV-2 spike S1-intoxicated mice exhibited a body temperature of around 1000F on 5 d of intoxication (Fig. 7B). Parallel to that observed in human lung cells, intranasal insult with recombinant SARS-CoV-2 spike S1 (Fig. 7A) led to the activation of NF-κB in vivo in the lung of C57/BL6 mice (Fig. 7C), which was strongly inhibited by intranasal treatment with wtSPIDAR, but not mSPIDAR (Fig. 7C). In situ chromatin immunoprecipitation (ChIP) analysis showed the recruitment of classical NF-κB subunits (p65 and p50), RNA polymerase II and transcriptional co-activator p300 to the IL-6 gene promoter in vivo in the lung of SARS-CoV-2 spike S1-intoxicated mice that was markedly inhibited by intranasal treatment with wtSPIDAR, but not mSPIDAR (Fig. 8AG). Consistently, wtSPIDAR, but not mSPIDAR, also inhibited the expression of IL-6 mRNA (Fig. 7D) and protein (Fig. 7F) as well as another proinflammatory cytokine (IL-1β) mRNA (Fig. 7E) in the lungs of SARS-CoV-2 spike S1-insulted mice. Similarly, wtSPIDAR treatment also suppressed the serum level of IL-6, an important contributor of the so-called cytokine storm-related illness (29), and C-reactive protein (CRP), an important biomarker for monitoring COVID-19-associated lethality (30), in SARS-CoV-2 spike S1-insulted mice (Fig. 7GH).

Figure 7. Intranasal delivery of wtSPIDAR decreases lung inflammation and reduces fever in a mouse model of COVID-19.

Figure 7.

Six-eight week old C57/BL6 mice (n=7) of both sexes were intoxicated with recombinant SARS-CoV-2 spike S1 (50 ng/mouse/d) via intranasal route. A) Schematic presentation of experiments. After 5 d of treatment, when spike S1-intoxicated mice displayed a body temperature of around 1000F (B), mice were treated intranasally with wtSPIDAR or mSPIDAR (100 ng/mouse/d). C) After 7d of SPIDAR treatment, the activation of NF-κB was checked in the heart by EMSA. The mRNA expression of IL-6 (D) and IL-1β (E) was monitored in lung by real-time PCR. IL-6 protein was measured in lung tissue homogenates by ELISA (F). Levels of IL-6 (G) and CRP (H) were also quantified in serum by ELISA. Body temperature (H) was monitored by Cardinal Health Dual Scale digital rectal thermometer. Results are mean ± SEM of 7 mice per group. ***p < 0.001; NS, not significant.

Figure 8. Intranasal wtSPIDAR inhibits the recruitment of NF-κB to the IL-6 gene promoter in vivo in the lungs of a mouse model of COVID-19.

Figure 8.

A) The map of mouse IL-6 promoter harboring consensus NF-κB-binding site (position −124 to −110). Six-eight week old C57/BL6 mice of both sexes were intoxicated with recombinant SARS-CoV-2 spike S1 (50 ng/mouse/d) via intranasal route. After 5 d of treatment, when all mice displayed a body temperature of at least 1000F, mice were treated intranasally with wtSPIDAR or mSPIDAR (100 ng/mouse/d). After 7d of treatment, in situ ChIP for p65 and p50 followed by semi-quantitative (B) and quantitative PCR (C, p65; D, p50; E, p300; F, RNA polymerase; G, control IgG) analyses of IL-6 promoter were performed. Results are mean ± SEM of four mice per group. ***p < 0.001.

Fever is probably one of the most common symptoms of COVID-19 (2, 31) and intranasal treatment with wtSPIDAR, but not mSPIDAR, also led to the normalization of body temperature of SARS-CoV-2 spike S1-intoxicated mice (Fig. 7I). SARS-CoV-2 spike S1-intoxicated mice also feature widespread neutrophil infiltration into the lungs (11). Therefore, we monitored the status of neutrophil infiltration upon SPIDAR treatment. As evident from Figure 9A, treatment with wtSPIDAR, but not mSPIDAR, led to marked inhibition of neutrophil infiltration into the lungs of SARS-CoV-2 spike S1-intoxicated mice. Cell counting as well as assessment of lung injury also showed that wtSPIDAR, but not mSPIDAR, reduced lung neutrophil infiltration (Fig. 9B), normalized lung epithelial cells (Fig. 9CD), and reduced overall lung injury (Fig. 9E) in SARS-CoV-2 spike S1-intoxicated mice.

Figure 9. Intranasal delivery of wtSPIDAR decreases lung infiltration in a mouse model of COVID-19.

Figure 9.

Six-eight week old C57/BL6 mice (n=6) of both sexes were intoxicated with recombinant SARS-CoV-2 spike S1 (50 ng/mouse/d) via intranasal route. After 5 d of treatment, when all mice displayed a body temperature of at least 1000F, mice were treated intranasally with wtSPIDAR or mSPIDAR (100 ng/mouse/d). After 7d of treatment, lung sections were analyzed by H&E (A, images of different magnification; B, neutrophil cell count; C, epithelial cell count; D, infiltrated cells as percent of epithelial cells; E, lung injury score). Cells were counted from two sections of each of six mice (n=6) per group. Results are mean ± SEM of 6 mice per group. ***p < 0.001.

Intranasal wtSPIDAR improves heart functions and increases locomotor performance in SARS-CoV-2 spike S1-intoxicated mice:

Since many cardiac features of COVID-19 are modeled in SARS-CoV-2 spike S1-intoxicated mice (11), we examined if wtSPIDAR was capable of improving heart functions in these mice. Non-invasive ECG showed cardiac arrhythmias in SARS-CoV-2 spike S1-intoxicated mice as compared to control untreated mice (Fig. 10AB). However, wtSPIDAR, but not mSPIDAR, normalized electrical activity of the heart as evident from ECG (Fig. 10AD). Similarly, wtSPIDAR, but not mSPIDAR, also stabilized heart rate (Fig. 10E), ORS interval (Fig. 10F), QT interval (Fig. 10G), RR interval (Fig. 10H), and heart rate variability (Fig. 10I) in SARS-CoV-2 spike S1-intoxicated mice. As expected, serum LDH level was also markedly higher in SARS-CoV-2 spike S1-intoxicated mice than normal mice (Fig. 10J). However, wtSPIDAR, but not mSPIDAR, reduced and/or normalized serum LDH in spike S1-intoxicated mice (Fig. 10J).

Figure 10. Intranasal delivery of wtSPIDAR protects heart functions in a mouse model of COVID-19.

Figure 10.

Six-eight week old C57/BL6 mice (n=6) of both sexes were intoxicated with recombinant SARS-CoV-2 spike S1 (50 ng/mouse/d) via intranasal route. After 5 d of treatment, when all mice displayed a body temperature of at least 1000F, mice were treated intranasally with wtSPIDAR or mSPIDAR (100 ng/mouse/d). After 7d of treatment, heart functions were monitored by non-invasive electrocardiography (ECG) using the PowerLab (ADInstruments) [A, chromatogram of control mice; B, chromatogram of spike S1-intoxicated mice; C, chromatogram of (spike S1 + wtSPIDAR)-treated mice; D, chromatogram of (spike S1 + mSPIDAR)-treated mice; E, heart rate; F, QRS interval; G, QT interval; H, RR interval; I, heart rate variability]. J) Serum LDH was quantified using an assay kit from Sigma. Results are mean ± SEM of 6 mice per group. *p < 0.05; **p < 0.01; ***p < 0.001.

Recently, we have seen that SARS-CoV-2 spike S1 intoxication also causes functional discrepancies in C57/BL6 mice (11). Therefore, we examined whether wtSPIDAR treatment was capable of improving such behavioral deficits. Interestingly, wtSPIDAR, but not mSPIDAR, treatment increased overall locomotor activities as evident by heat map (Fig. 11A), distance travelled (Fig. 11B), velocity (Fig. 11C), cumulative duration (Fig. 11D), center zone frequency (Fig. 11E), and rotorod performance (Fig. 11F). We did not notice any drug-related side effect (e.g. hair loss, appetite loss, weight loss, untoward infection and irritation, etc.) in any mouse upon treatment with intranasal SPIDAR.

Figure 11. Intranasal delivery of wtSPIDAR improves locomotor activities in a mouse model of COVID-19.

Figure 11.

Six-eight week old C57/BL6 mice (n=7) of both sexes were intoxicated with recombinant SARS-CoV-2 spike S1 (50 ng/mouse/d) via intranasal route. After 5 d of treatment, when all mice displayed a body temperature of at least 1000F, mice were treated intranasally with wtSPIDAR or mSPIDAR (100 ng/mouse/d). After 7d of treatment, mice were tested for general locomotor activities (A, heat map; B, distance travelled; C, velocity; D, cumulative duration; E, center zone frequency; F, rotorod latency). Results are mean ± SEM of 7 mice per group. *p < 0.05; **p < 0.01; ***p < 0.001.

Discussion

Unravelling the mechanism of the disease process of COVID-19 to design an effective therapeutic approach for controlling the disease process is of paramount importance. While ACE2 plays an important role in vascular diseases (6), SARS-CoV-2 binds to ACE2 for entering into human cells. Therefore, COVID-19 infection becomes deadly in patients with underlying cardiovascular issues. Under such situations, it is expected to use inhibitors of ACE2 (32), however, such inhibitors may not be beneficial for severe COVID-19 patients in the ICU as ACE2 inhibition may further aggravate symptoms in COVID-19 patients with hypertension and cardiovascular issues. Therefore, to dissociate the interaction between ACE2 and SARS-CoV-2 spike S1 without altering the level and function of ACE2, we analyzed the interaction between ACE2 and SARS-CoV-2 spike S1 and designed a small hexapeptide corresponding to the Spike S1-interacting domain of ACE2 receptor (SPIDAR). Resembling to the Spike S1-binding domain of ACE2, wtSPIDAR dissociated the interaction between ACE2 and SARS-CoV-2 spike S1. Accordingly, in a pseudovirus cell entry assay, wtSPIDAR inhibited the entry of SARS-CoV-2 pseudovirus into hACE2-expressing HEK293 cells. On the other hand, wtSPIDAR remained unable to modulate the entry of VSV pseudovirus into hACE2-expressing HEK293 cells, indicating the functional specificity of SPIDAR. Recently, we have demonstrated that another peptide corresponding to ACE2-interacting domain of SARS-CoV-2 (AIDS) also detaches the association between ACE2 and SARS-CoV-2 spike S1 (11). Although wtAIDS also inhibited the entry of SARS-CoV-2 pseudovirus into hACE2-expressing HEK293 cells, wtSPIDAR seemed to be more effective than wtAIDS in suppressing the viral entry. Although COVID-19 is the first documented coronavirus pandemic in the history (33), several human-infecting coronaviruses are there with different degrees of virulence. However, the first step of coronavirus pathogenesis is the attachment of coronavirus to the host via viral spike glycoprotein. Therefore, in addition to COVID-19, our SPIDAR and AIDS technologies may be effective against other coronaviruses.

Inflammation plays an important role in the pathogenesis of many human disorders including COVID-19. It has been reported that COVID-19 patients with severe symptoms suffer from cytokine storm (34). We have seen that spike S1 alone is sufficient to drive the expression of proinflammatory cytokines in human lung cells (11). Therefore, spike S1 may be responsible for cytokine storm seen in COVID-19 patients. However, consistent to the inhibition of association between SARS-CoV-2 spike S1 and ACE2, wtSPIDAR decreased the activation of NF-κB and the expression of IL-6 and IL-1β in SARS-CoV-2 spike S1-intoxicated A549 lung cells. Inability of wtSPIDAR to inhibit the activation of NF-κB and associated expression of proinflammatory cytokines induced by TNFα (a prototype proinflammatory cytokine), poly IC (viral double-stranded RNA mimic) and Tat (transactivator of HIV-1 transcription) delineates the selective nature of wtSPIDAR. This is important because there are many anti-inflammatory therapies (e.g. steroids) available to take care of the cytokine storm in COVID-19 patients. However, it is a double-edged sword because very often these treatments cause immunosuppression. Therefore, it has been suggested that reducing hyperinflammation by anti-inflammatory drugs in severely-affected COVID-19 patients should be approached with caution. Since wtSPIDAR inhibits cytokines produced by only SARS-CoV-2 spike S1, but not other inflammatory stimuli, wtSPIDAR is not expected to cause immunosuppression.

Another highlight of our finding is that wtSPIDAR corresponds to peptide sequence of SARS-CoV-2 from the binding site with ACE2. Therefore, wtSPIDAR will only inhibit the binding of ACE2 with SARS-CoV-2 without affecting beneficial enzyme activities and basal level of ACE2. Similarly, our recently described wtAIDS should also specifically inhibit the association of ACE2 with SARS-CoV-2 (11). However, wtAIDS binds to ACE2 as this sequence has been designed from the ACE2-binding site of spike S1. On the other hand, wtSPIDAR should bind to spike S1 of SARS-CoV-2 as this sequence is derived from the spike S1-binding region of ACE2. Although we have not examined, since wtAIDS binds to ACE2, it may have ACE2-inhibitory activity. Under that condition, by suppressing ACE2 activity, wtAIDS may pose risks for patients with preexisting cardiovascular problems because the most important function of ACE2 is to convert a physiological vasoconstrictor (AngII) to a vasodilator (Ang1–7), which is known to function through Mas receptor to transduce anti-proliferative/vasodilatory activities (35). Moreover, since myocardial NADPH oxidase is responsible for myocardial oxidative stress and inflammation, by suppressing myocardial NADPH oxidase, Ang1–7 is protective for the heart (36). Therefore, any inhibition and/or dysfunction of ACE2 may not be beneficial for COVID-19 patients with hypertension, diabetes and preexisting vascular problems. On the other hand, SPIDAR should not cause these problems as it would bind to SARS-CoV-2 spike S1, but not ACE2. Therefore, SPIDAR will function only in the presence of SARS-CoV-2.

Although vaccination against COVID-19 started and about half the population in USA have been vaccinated, their distribution globally will take months and possibly years in some part of the world. On top of that, storage of some of the widely-used COVID-19 vaccines at −400C to −800C throughout the world is another big issue. Moreover, vaccines may not entirely prevent the spread of COVID-19. According to a recent (June 25, 2021) Wall Street Journal news, about half of adults infected in an outbreak of the Delta variant of CoVID-19 in Israel were fully inoculated with the Pfizer Inc. vaccine. We also must remember that despite flu vaccination, about 40,000 to 50,000 people die each year in United States from the flu. Therefore, a specific medicine for handling cytokine storm and taking care of respiratory and cardiac complications caused by SARS-CoV-2 infection will be necessary for better management of COVID-19 even in the post-vaccine era. Although some monoclonal antibodies against SARS-CoV-2 spike proteins (e.g. sotrovimab, casirivimab, imdevimab, etc.) and IL-6 receptor (e.g. tocilizumab) have been approved for COVID-19 treatment (37, 38), other than monoclonal antibodies, until now, no other COVID-19-specific therapy is available. Most of the non-antibody therapies being investigated and tried for COVID-19 are repurposed drugs. For example, Remdesivir is being repurposed from HIV to COVID-19 emergency use (39). Similarly, dexamethasone, a corticosteroid that is used to take care of wide range of conditions for its anti-inflammatory and immunosuppressive effects, has been proposed for COVID-19 (40). Aviptadil is a drug for the treatment of erectile dysfunction. It is being tried to alleviate cardiac problems of critically ill COVID-19 patients. Hydroxychloroquine, a prototype anti-malarial drug, was also considered for COVID-19 before clinical trials ruled it out for COVID-19 (31). Similarly, multiple sclerosis drug IFN β−1b is also being considered for lowering mortality rate in COVID-19 (41). On the other hand, SPIDAR is very selective to inhibit cellular entry of SARS-CoV-2 pseudovirus, but not VSV pseudovirus and knock down inflammation only associated with SARS-CoV-2 spike S1. Protection of lungs, normalization of heart functions, reduction of fever, decrease in serum markers, and improvement in locomotor activities in SARS-CoV-2 spike S1-intoxicated mice by nasally administered SPIDAR suggest that selective targeting of the ACE2-to-SARS-CoV-2 contact by SPIDAR may be beneficial for COVID-19.

Supplementary Material

1

Acknowledgments

Footnotes: This study was supported by grants (AG050431, AG069229, and AT010980) from NIH to KP. Moreover, KP is the recipient of a Research Career Scientist Award (1IK6 BX004982) from the Department of Veterans Affairs.

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