Development of a novel angiotensin converting enzyme 2 stimulator with broad implications in SARS-CoV2 and type 1 diabetes

Angiotensin-converting enzyme 2 (ACE2) is protective in cardiovascular disease, lung injury and diabetes yet paradoxically underlies our susceptibility to SARs-CoV2 infection and the fatal heart and lung disease it can induce. Furthermore, diabetic patients have chronic, systemic inflammation and altered ACE2 expression resulting in increased risk of severe COVID-19 and the associated mortality. A drug that could increase ACE2 activity and inhibit cellular uptake of severe acute respiratory syndrome coronavirus 2 (SARs-CoV2), thus decrease infection, would be of high relevance to cardiovascular disease, diabetes and SARs-CoV2 infection. While the need for such a drug lead was highlighted over a decade ago receiving over 600 citations,1 to date, no such drugs are available.2 Here, we report the development of a novel ACE2 stimulator, designated ‘2A’(international PCT filed), which is a 10 amino acid peptide derived from a snake venom, and demonstrate its in vitro and in vivo efficacy against SARs-CoV2 infection and associated lung inflammation. Peptide 2A also provides remarkable protection against glycaemic dysregulation, weight loss and disease severity in a mouse model of type 1 diabetes. No untoward effects of 2A were observed in these pre-clinical models suggesting its strong clinical translation potential.

ACE2-dependent conversion of natural substrate Ang II to Ang 1-7 was monitored using liquid chromatography mass spectrometry (LCMS). Ang II levels were reduced by 83 ± 5 and 58 ± 4% over a 24h period in the presence of ACE2 + 2A and ACE2 alone, respectively (P=0.008; Figure 1E). Consistent with this, Ang 1-7 levels increased in the presence of ACE2 + 2A by 165 ± 12% compared to ACE2 alone (P=0.003; Figure 1E) further supporting the notion that 2A stimulates ACE2 activity.
2A can be detected in multiple organs when administered to normal mice We next examined the tissue distribution of 2A (1mg/kg) upon administration to normal wild-type C57BL/6J mice via intravenous (i.v), subcutaneous (s.c) or intranasal (i.n) routes. 2A was rapidly cleared from the circulation over the rst 4h, which could reduce untoward off target effects of the peptide, however a small amount of peptide was still detected in plasma 24 h post administration ( Figure 1F).
Interestingly, irrespective of the route of administration, 2A was detectable at low concentrations in the heart, lung, spleen, kidney, brain and spinal cord 24-h post administration ( Figure 1G). We next examined the mechanism of action of 2A, and since ACE2 is the receptor for SARS-CoV-2 4 , we also examined how 2A affects the interaction between SARS-CoV-2 spike protein and ACE2.
Molecular Docking predicts 2A weakens the binding between ACE2 and the SARS-CoV-2 spike protein Molecular docking was employed to understand the mechanism of action of 2A. Binding studies indicate that 2A is buried deep inside the ACE2 structure ( Figure 2A) and results in a clear change in conformation ( Figure 2B). The two helices anking the ACE2 active site (Glu22 -Leu91) change conformation to accommodate 2A ( Figure 2C and supplementary gure 2A). Residues 22-53 comprise the binding site for the receptor binding domain (RBD) of the spike protein, and conformational changes here are likely to lead to disruptions in ACE2 binding. We propose that 2A binding induces a novel ACE2 conformation, distinct from other known structures, which may correspond to a SARS-CoV-2-resistant state.
Analysis of 2A-ACE2 interactions indicates that Lys7 in 2A forms a multitude of contacts with ACE2 (supplementary gure 2A -2B), including hydrogen bonding with the backbone of Leu391, Asn394, and cation-p interactions with Phe40 and Phe390 (supplementary gure 2B). Leu10 in 2A also forms a hydrogen bond with Glu375. Alanine scanning mutagenesis studies indicate that residues 7 and 10 are critical for ACE2 stimulation. Hydrogen bond pairing plays an important role in the regulation of proteinligand binding a nity 5 . In contrast, molecular docking of the control scrambled peptide suggests that it is unlikely to change ACE2 conformation (supplementary gure 2C). The scrambled peptide also forms fewer hydrogen bonds compared to 2A, with any given sidechain forming no more than one hydrogen bond with ACE2 (supplementary gure 2D). Subsequent dockings of apo and 2A-modi ed ACE2 to the spike protein RBD using HADDOCK 6,7 predict that apo ACE2 binds more strongly to the RBD ( Figure 2D), with close resemblance to the ACE2-RBD crystal structure 8 , while the 2A-modi ed ACE2 binds more weakly ( Figure 2E), with substantially fewer interactions. This result suggests that conformational changes induced by 2A leads to a weaker association between ACE2 and the virus, conferring resistance to infection by SARS-CoV-2. This may be particularly important for Variants of Concern (VOC) that more tightly bind to ACE2 9 . To corroborate our ndings from molecular docking studies, we next examined whether 2A attenuates SARS-CoV-2 infection in primary human nasal epithelial cells. We selected these cells as ACE2 in primary human nasal epithelial cells plays a substantial role in SARS-CoV-2 infection. 10,11 Pre-treatment with 2A completely prevents SARS-CoV-2 infection in primary human nasal epithelial cells To determine the appropriate dose of 2A to use in cell culture, a preliminary experiment was performed in post-infected Vero cells ( Figure 3A). Sixteen hours post-infection cells treated with 350 µM of peptide 2A had the lowest titres of SARS-CoV-2 in cell culture supernatant although none of the 2A concentrations tested were signi cantly different from that of the PBS control. We therefore elected to use 350 µM of peptide 2A on primary human nasal epithelial cells ( Figure 3B). To examine the potential of 2A as a prophylactic, we pre-treated the cells with 2A. Primary human nasal epithelial cells were differentiated at an air-liquid interface. Cells were then pre-treated apically and basolaterally with PBS, peptide 2A, scrambled peptide or DIZE for 1 h prior to infecting the cells with SARS-CoV-2. 2A, DIZE, PBS or scrambled peptide was added every 12 h for a period of 48 h. Viral titres produced by epithelial cells was subsequently assessed at 48 hours post-infection. Strikingly, no infectious virus was detected in the nasal epithelial cells treated with peptide 2A indicating that anti-viral effects of 2A can last up to 12 h. In contrast, viral titres in epithelial cells treated with scrambled peptide or DIZE were not signi cantly different to cells treated with PBS ( Figure 3B). These ndings in primary human nasal epithelial cells indicate 2A (350 µM) is effective in preventing infection when replaced at 12-h intervals and provide direct support for the vast potential of 2A as being used as a prophylactic. We next examined whether 2A can exert anti-in ammatory effects post-infection.
Administration of 2A post SARS-CoV-2 infection improves the clinical score and lung in ammation in K18-hACE2 mice Data in primary human nasal epithelial cells provided a strong impetus for the further evaluation of 2A. We therefore examined the effect of 2A in a mouse model of SARS-CoV-2 infection. Murine ACE2 is incompatible with the spike protein of SARS-CoV-2. 12 Transgenic mice which possess the human ACE2 receptor under the control of the K18 promoter (K18-hACE2 mice) are susceptible to infection. 12,13 We infected K18-hACE2 mice with 10 3 PFU of SARS-CoV-2 (Wuhan strain) or sham (PBS), and treated mice with 2A or vehicle from 3 days post-infection (dpi) (i.n; daily). Infected vehicle treated mice dramatically lost weight from 4 dpi, and developed severe clinical signs including laboured breathing, lethargy, hunching and ru ed fur requiring euthanasia (Figure 4 A-B). Infected mice treated with 2A (i.n) were protected against severe clinical signs with reductions in laboured breathing and lethargy. They also had fewer in ammatory cells in bronchoalveolar lavage uid and in the airways under histological examination, as well as improved in ammatory scoring in the lung architecture ( Figure 4 C-H). This provides evidence that 2A can improve lung in ammation when administered at 24-h intervals which is consistent with our tissue distribution data indicating 2A can be detected in lungs 24-h post intranasal delivery. Interestingly, we did not observe any signi cant differences in viral loads in bronchoalveolar lavage uid or lung homogenates between mice treated with vehicle or 2A, suggesting a complex interplay between ACE2 stimulation and SARS-CoV-2 infection (Figure 4 I-J). Docking experiments predict 2A prevents infection by inducing a conformational change in ACE2 structure. Since we administered 2A to mice post-infection, it is not surprising that 2A did not reduce viral loads in these mice. In contrast, 2A was able to completely prevent viral infection in primary human nasal epithelial cells as 2A was administered pre-infection in this experiment.
Together, these ndings indicate the vast potential of 2A as both a prophylactic and as an-antiin ammatory drug post-infection. In this context, chronic in ammation together with altered ACE2 expression are hallmarks of diabetes which increase the risk of SARS-CoV2 infection. 14 Therefore, we next examined the effects of 2A in type 1 diabetes.
2A treatment prevented the increase in blood glucose levels and loss of body weight in STZ-induced diabetic mice Streptozotocin (STZ; 150 mg/kg; i.p) was used to induce type 1 diabetes in male C57BI/6J mice. Blood glucose was measured 1 week post STZ injection to con rm the presence of diabetes (BG levels > 15 mmol/L at 1 week post STZ injection was included in this study). After establishment of diabetes (T0), mice were randomly allocated to receive 2A (1 mg/kg/ day; 10% DMSO in saline) or vehicle (10% DMSO in saline) via subcutaneous minipumps for a period 3 months. Non fasting blood glucose levels were measured every 2 weeks until study end (T3), and body weight was measured daily, until study end. 24-h urine samples were collected at 1 month intervals since commencement of 2A or its vehicle. (Detailed Methods section is provided at the end of the Discussion).
Blood glucose levels increased by 43 ± 16% (P = 0.03; paired t-test) at study end, compared to respective pre-treatment levels, in diabetic mice administered vehicle ( Figure 5A; Supplementary Figure 3A). In contrast, blood glucose levels did not signi cantly change (P=0.93; paired t-test) in diabetic mice administered 2A (1 mg/kg/day; 3 months; s.c mini-pumps; Figure 5A; Supplementary Figure 3A). Urinary glucose excretion in diabetic mice receiving 2A or vehicle was not signi cantly different at study end (T3), compared to respective levels immediately prior to 2A or vehicle treatment (T0) (P ≥ 0.21; paired t-test; Figure 5B). This suggests that 2A does not dampen the increase in blood glucose levels in diabetes via increasing its excretion.
Interestingly, 24-h urine volume at study end (T3) increased by 2 and 3 folds, respectively, in diabetic mice receiving vehicle or 2A, when compared with respective baseline levels immediately prior to administration of these respective treatments (T0; P <0.001; paired t-test; Supplementary Figure 3B). Further analysis indicate that 43% of diabetic mice receiving 2A had no change in 24-h urine volume from T0 to T3 (P = 0.52; paired t-test; Supplementary Figure 4A), with a parallel 28 ± 3% reduction in blood glucose content (P <0.001; paired t-test; Supplementary Figure 4B) and no signi cant change in urinary glucose excretion (P = 0.09; paired t-test; Supplementary Figure 4C). In 57% of diabetic mice administered 2A, 24-h urine volume increased by 3-fold from T0 to T3 (P <0.001; paired t-test; Supplementary Figure 4D). In this subset of mice, neither blood glucose nor urinary glucose changed from T0 to T3 (P ≥ 0.17; Supplementary Figure 4E -3F). Of particular note, the cohort of diabetic mice administered 2A with a 3 fold increase in urine volume from T0 to T3, had 30% higher baseline (T0) blood glucose levels than the cohort of diabetic mice administered 2A with no change in urine volume from T0 to T3 (P = 0.02; unpaired t-test; Supplementary Figure 4B, 4E) suggesting that 2A is more effective in dampening the increase in blood glucose levels at early stages of diabetes.
47% of mice in the vehicle group were euthanized due to severe weight loss as per the ethics requirements. Only 12% of mice were culled in the cohort of diabetic mice receiving 2A ( Figure 5C; Supplementary Figure 5A). Diabetic mice in the latter cohort were culled 60 days post-STZ injection or later, in the 90-day study protocol, while the majority of diabetic mice in the vehicle group were euthanized prior to 60 days post-STZ injection due to severe weight loss as per the ethics requirements ( Figure 5C). Endpoint blood glucose in mice culled before study end are shown in supplementary gure 5B. Mice in non-diabetic groups survived until study end (data not shown).
2A had minimal effects on organ weight (Supplementary Table 1).
While the precise mechanisms by which 2A prevents the elevation in blood glucose levels and prevents loss of body weight remain to be determined, our observations nonetheless provide solid evidence that 2A has great promise in the treatment of type 1 diabetes. Protective arm of the RAS improves pancreatic beta cell function by improving the function of islet microvascular endothelial cells, 15 a source which increasing evidence demonstrates is critical for the function of beta cells. 16 In this context, IL-6 is a key in ammatory mediator in type 1 diabetes as well as in SARS-CoV2 related in ammation. 17-19 19,20 Therefore, we next examined whether 2A can reduce RAS induced increase in IL-6 expression in endothelial cells.
Anti-in ammatory effects of 2A: We used a straight channel micro uidic device mimicking the physiological microvascular environment to study the anti-in ammatory effects of 2A. IL-6 expression in cells lining the device was greater with AngII treatment compared with control. IL-6 expression was a near 14-fold less with AngII + 2A compared to Ang II alone. Near 3-fold reduction in IL-6 was also observed in the presence of Ang II and 2A compared with control ( Figure 6A), suggesting decreased IL-6 expression and increased anti-in ammatory effect. Fluorescence images clearly showed the different levels of IL-6 expression in the three groups based on the red colour, and merged images proved the colocalization of IL-6 expression in the cells (Figure 6 C-D).

Discussion
Here, we report the development of a novel drug lead '2A' which stimulates ACE2 enzyme activity and has strong protective effects in SARS-CoV2 and type 1 diabetes.
2A provides a unique and rst of its kind approach to simultaneously weaken the interaction of SARS-CoV-2 with ACE2 while also increasing the catalytic and hence the anti-in ammatory effects of ACE2. Our data indicate that 2A completely prevents SARS-CoV-2 infection in primary human nasal epithelial cells, and attenuates lung in ammation improving clinical presentation in transgenic K18-hACE2 mice infected with SARS-CoV-2. Consistent with this, molecular docking experiments predict that 2A reduces the binding a nity of SARS-CoV-2 spike protein to ACE2 by changing its conformation. These data are in line with previous ndings indicating impaired ACE2 activity contributes to lung in ammation and disease severity. 13,18,21 Of particular interest, 2A has strong clinical translational potential. Since nasal epithelial ACE2 plays a critical role in SARS-CoV-2 infection in humans, 11 our observation that 2A can completely prevent SARS-CoV-2 infection indicates its vast potential in being used as a prophylactic. In addition, 2A reduces lung in ammation and improves clinical presentation in transgenic K18-hACE2 mice infected with SARS-CoV-2. While there are anti-in ammatory drugs which provide bene t to patients with COVID-19 to varying degrees, there is an unmet need to develop new therapeutic approaches to more effectively target lung in ammation in SARS-CoV-2, 22-25 which contributes substantially to patient mortality. 2A represents a new class of anti-in ammatory drugs, which not only weakens the interaction between SAR-CoV-2 and ACE2 but also exerts anti-in ammatory effects via stimulating ACE2 enzyme activity.
We next examined the effects of 2A in type 1 diabetes, a pathological condition characterized by systemic in ammation and altered ACE2 expression resulting in increased risk of severe COVID-19. Reduced ACE2 expression is reported in patients with type 1 diabetes which contributes substantially to disease pathology. 15,26 Our data indicate 2A completely prevents the increase in blood glucose levels in mice with STZ induced type 1 diabetes and ameliorates the related loss of body weight. 2A also completely prevents RAS induced IL-6 expression in endothelial cells in a micro uidic device mimicking the physiological environment, indicating its vast potential in exerting anti-in ammatory effects in the setting of SARS-CoV2 and type 1 diabetes.
We did not observe any untoward effects in our animal models suggesting 2A has strong clinical translation potential. Our observations provide a solid basis to develop a novel class of anti-in ammatory drugs with broad implications in both non-communicable and communicable diseases.

Discovery of 2A from a snake venom
We previously reported that a synthetic peptide corresponding to the rst 20 amino acids of Bothrops asper myotoxin II increases the activity of several Zn 2+ dependent metalloproteases including ACE-2 3 . We then made a peptide library (Genic Bio Ltd, Shanghai, China) by deleting two amino acids at a time from the C or N-termini, and both termini simultaneously. These peptides were screened using the QFS based ACE-2 assay described below. The selected peptide 2A was C-terminally amidated to improve stability.
Crude pepsets consisting of Alanine substituted analogues of 2A were synthesized (Genic Bio Ltd, Shanghai, China) for initial screening using the QFS based ACE-2 assay. Thereafter the Alanine substituted analogues of interest were synthesised at >95% purity for further testing.
Effects of 2A on ACE2 catalytic activity Measurement of recombinant human ACE2 enzyme activity using quenched uorescence substratebased assay.
All QFS-based assays were conducted in 96-well format. In each of the QFS-based assays described below, uorescence was measured using λ ex = 320 nm and λ em = 405 nm at 37 ˚C. Enzymes were incubated with 2A or truncated peptides for 1 h at 37 C prior to adding appropriate QFS (40 µM) indicated in Table 1. The reaction rate was calculated from the linear portion of the uorescence curve. Speci c enzyme activity was calculated using a standard curve of known concentrations of 7-methoxycoumarin-4-acetic acid ( uorophore). All synthetic peptides were synthesised by GeniBio Ltd (Shanghai, China). All recombinant human enzymes were purchased from R&D systems (Minneapolis, USA). Effect of alanine scan analogues on rhACE2 activity A library of 2A analogs were synthesised where one amino acid residue at a time was replaced by an Ala residue. These analogs were initially synthesised as crude pepsets and were screened for their effects on rhACE2 activity. From these pepsets, 7 analogs that induced an increase in ACE2 activity were identi ed and resynthesised at 95% purity. These 7 peptides (2.6 µM) were then re-screened for their effects on ACE2 activity using the QFS-based assay. Each peptide was incubated with rhACE2 at 37 ˚C for 1 h before adding QFS.
The effects of 2A on ACE2 dependent breakdown of angiotensin II and formation of angiotensin 1-7 The effect of 2A on ACE2-mediated cleavage of angiotensin II was assessed using LCMS. rhACE2 (0.1 ng/μL) was incubated with 2A (1.7 μM) for 1 h at 37 ˚C. Angiotensin II (0.02 μg/μL) was then added. Aliquots of equal volumes were collected at T = 0, 3, 6 and 24-h. Aliquots were immediately acidi ed with tri uoroacetic acid (0.1% nal). Samples were snap frozen in dry ice and lyophilized for analysis by LCMS.
Prior to loading, samples were reconstituted in 100 μL loading buffer. Samples were analysed using a quadrupole TOF mass spectrometer (MicroTOFq, Bruker Daltonics, Bremen, Germany) coupled online with a 1200 series nano HPLC (Agilent Technologies). Samples were loaded onto a zorbax 300SB reversed-phase trap column equilibrated with 95% buffer A (0.1% formic acid). The ow rate was set to 10 μL/min. The components were eluted over a 10 min gradient to 70% buffer B (80% acetonitrile, 0.1% formic acid) and peptides were separated on zorbax 300SB-C18 nano column (75 μm×15 cm, 3.5 μm).   lethargy, hunching, ru ing and early laboured breathing, and Cat 3 based on exacerbations of Cat 2 signs accompanied by complete immobility. At day 6 post-infection, mice were euthanised by intraperitoneal overdose with pentobarbitone (Virbac, Australia). Blood was collected via heart bleed, and serum collected (10,000 x g, 10 minutes) at room temperature. Multi-lobe lungs were tied off and bronchoalveolar lavage uid collected from the single lobe lung in 1 mL HANKS solution (Sigma-Aldrich, USA) using a blunted 19G needled inserted into the trachea. Bronchoalveolar lavage uid was centrifuged (300 x g, 7 minutes) and the supernatant collected and placed at -80 ˚C for further analysis. The cell pellet was resuspended in 200 µL of Red Blood Cell Lysis Buffer (ThermoFisher, Australia) and incubated for 5 minutes, followed by addition of 700 µL of HANKS solution and centrifuged as previously outlined.
Following centrifugation, the supernatant was discarded and the cell pellet was resuspended in 160 µL of HANKS solution. Total cell counts were enumerated using a disposable haemocytometer (Sigma-Aldrich, USA). Differential counts were performed by loading the cell suspension into a disposable cytospin funnel and pelleting cells (300 x g, 7 minutes) onto glass slides. Cytospin slides were then stained using QuickDip Stain Kit (Modi ed Giemsa Stain) protocol as per manufacturer's instructions (POCD Scienti c, Australia) and cells differentiated using an inverted light microscope. Multi-lobe lungs were collected and either frozen and stored at -80 ˚C for further analysis, or homogenised in 2 mL HANKS solution using a GentleMACS tissue homogeniser. Lung homogenate was centrifuged (300 x g, 7 min) to pellet cells, followed by collection of supernatants for plaque assays. The single lobe lung was perfused with 0.9% NaCl 2 via the heart, followed by in ation through the trachea with 0.5 mL 10% neutral buffered formalin and then submerged in formalin solution. Following 2 week xation, lungs were transported to a PC2 facility where they were para n-embedded, cut to 3 µm thick sections using a Leica microtome (Leica, Germany), and then stained using Quick Dip Stain Kit. In ammatory cells in the single lobe lung sections were enumerated using a Zeiss Axio Imager.Z2 microscope with a 40X objective (Zeiss, Germany).
In ammatory scoring was applied to lung architecture based on an unbiased scoring system encompassing in ammation in the parenchyma, vasculature and the airways, with 3 being the lowest score possible representing little or no in ammation, while a score of 13 was attributed to the highest in ammatory score representing severe and excessive in ammation.

Docking study methods
The HADDOCK webserver was used to predict the binding of the two conformations of ACE2 to the spike protein RBD. The 2A-free ACE2 and the spike protein RBD structures employed for HADDOCK calculations were those obtained from PDBID: 7KJ2 30 . We utilized the MDockPep program 5 to perform the peptideprotein docking simulation, which is accessible at https://zougrouptoolkit.missouri.edu/mdockpep/.
MDockPep is a state-of-the-art method for predicting the protein-peptide complex structures and is developed for addressing the challenge of predicting all-atom structures of protein-peptide complexes without any prior knowledge about the peptide binding site and the bound peptide conformation. It provides signi cantly better performance better than existing docking methods. In benchmarking tests, it successfully generated near-native peptide binding modes in 95.0% of the bound docking cases and in 92.2% of the unbound docking cases 5 . The 2A-modi ed ACE2 structure employed was that predicted using MDockPep. The active residues for both ACE2 conformations were speci ed as residues 27-38, while those for the spike protein RBD were speci ed as residues 489-496. Default values for all other parameters were used. Schroedinger Maestro (18) was used to produce 2D ligand-receptor interaction diagrams.

Ethics approval
All  Cell culture In this study, we used SVEC4-10, a murine endothelial cell line; from ATCC, Rockville, MD, which was cultured in DMEM (Dulbecco's Modi ed Eagle Medium) high glucose medium supplemented with 100 IU/ml of penicillin G, 100 µg/ml streptomycin, and 10% (vol/vol) FBS (fetal bovine serum). Cells were cultured in an incubator with 5% CO 2 at 37 C and subcultured when they reached 90% con uence, and passage-6 was used in this experiment.

Coating and growing SVCE-10 cells in PDMS device under ow conditions
The device was washed with 70% ethanol, sterile PBS (pH-7.4) and treated under UV for an hour to sterilize completely. The PDMS device was coated with bovine skin collagen (Sigma-Aldrich). Brie y, a 10 µL of collagen stock (3 mg/mL) was added to 90 µL of high glucose DMEM to get the nal working concentration at 300 µg/mL. A 20 µL working solution was infused through the device and incubated for an hour in the cell incubator (5% CO 2 , 37°C). The solution was taken out and the device was washed with PBS. The device was placed in the cell incubator at 37 C for 15 min for drying. In the meantime, the SVEC-10 cells were trypsinized and detached from the ask. The cells were collected and resuspended in high glucose DMEM supplemented with FBS and antibiotics. A 10 µL of cell suspension (10 million cells/mL) was infused through the channel. The device was placed in a shaker and incubated at 37°C and 200 rpm for an hour. Then, the device was transferred to the cell incubator and incubated for another two hours. Next, the device was placed upside down and incubated for two hours to ensure cell attachment in the device. After ve hours of incubation, the device was connected to a peristaltic pump, complete DMEM media was perfused at a ow rate of 12 µL/min (~400 s -1 ), and cells were allowed to continue growing under ow condition overnight. Then, the ow rate was increased to 31 µL/min, corresponding to the physiological shear rate 1000 s -1 and ran for 24 h to reach the con uent SVEC-10 monolayer. The ow rate was calculated using Newton's law of viscosity 33 .
Evaluating anti in ammatory effects of 2A peptide After reaching the con uent monolayer of SVEC-10 in the device at 1000 s -1 , the cells were treated with Angiotensin II (10 µM

Studies in Vero Cells and Primary Human Nasal Epithelial Cells
Results from viral plaque forming assay was compared against PBS using Kruskal-Wallis test and multiple comparisons test. Outliers were removed by ROUT with P<0.05.

Studies in K18-hACE2 mice
Body weight curves were analysed using a two-way ANOVA. BALF, differential counts, in ammatory cell counts and in ammatory scoring were analysed using a one-way ANOVA. Viral titres were analysed using unpaired Student's t-tests. Data shown is the mean ± SEM. *P <0.05, **P <0.01, ****P <0.0001. All data were analysed using GraphPad Prism version 9.0. Figure 1 2A increases recombinant human ACE2 activity in vitro and changes the conformation of ACE2. (A) The effects of the truncated analogues of 20 AA parent peptide of 2A (2.6 µM) on ACE2 activity (n=4-5); *signi cantly different compared to ACE2 alone, and + signi cantly different compared to ACE2 + amidated 2A; one-way ANOVA followed by Tukey's post-hoc test; P < 0.05. (B) The effects of increasing concentrations of 2A (0.9-26 µM) on ACE2 activity (n=10; SEM are too small to be displayed); *signi cantly different compared to ACE2 alone; one-way ANOVA followed by Tukey's post-hoc test; P < 0.001. (C) The effects of 2A (2.6 µM) on the enzyme activity of Zn 2+ metalloproteases closely related to ACE2 (n=5-10); *signi cantly different compared with respective enzyme alone; unpaired t-test; P < 0.01.     loss of ≥ 15% of the pre-diabetic body weight. All the non-diabetic mice survived until study end. Log-rank test was used to calculate the P-value of the survival curve, which indicate that there is a difference between the survival curves of the treatment groups (P = 0.03).