Diminazene Aceturate Inhibits The Sars-Cov-2 Spike Protein-Induced Inflammation

Diminazene aceturate inhibits the SARS-CoV-2 spike protein-induced
inflammation involving leukocyte migration and DNA extracellular traps
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formation
Gean C. Pereira-Silva1,‡, Cassia K. C. A. Cornélio2,‡, Gabriella Pacheco3,‡, Natalia C.
Rochael1, Isaac A. B. Gomes2, Aurilene G. Cajado4, Katriane C. Silva2, Barbara
Simonson Gonçalves5, Jairo R. Temerozo5,6, Ruan S. Bastos2, Jefferson A. Rocha2,
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Leonardo P. Souza2, Marcellus H. L. P. Souza4, Roberto C. P. Lima-Júnior4, Jand V. R.
Medeiros2,3, Marcelo C. Filgueiras2, Dumith Chequer Bou-Habib5,6, Elvira M.
Saraiva1,*, Lucas A. D. Nicolau2,3,*
1 Laboratory on Innate Immunity, Department of Immunology, Institute of
Microbiology Paulo de Góes, Universidade Federal do Rio de Janeiro (UFRJ), Rio de
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Janeiro, RJ, Brazil;
2 Biotechnology and Biodiversity Center Research, Laboratory of Inflammation and
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Translational Gastroenterology, Universidade Federal do Delta do Parnaíba (UFDPar),
Parnaíba, PI, Brazil;
3 Department of Biochemistry and Pharmacology, Health Sciences Center,
Universidade Federal do Piauí (UFPI), Teresina, PI, Brazil;
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4 Department of Physiology and Pharmacology, Universidade Federal do Ceará (UFC),
Fortaleza, CE, Brazil.
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5 Laboratory on Thymus Research, Oswaldo Cruz Institute (Fiocruz) Rio de Janeiro,
RJ, Brazil.
6 National Institute of Science and Technology on Neuroimmunemodulation, Rio de
Janeiro, Brazil
‡ These authors contributed equally.
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*Corresponding authors: Elvira Saraiva (esaraiva@micro.ufrj.br); Lucas Nicolau

(lucasnicolau@ufpi.edu.br)
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This preprint research paper has not been peer reviewed. Electronic copy available at: https://ssrn.com/abstract=4797164
Highlights
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 SARS-CoV-2 Spike (Spk) protein induces acute inflammatory responses in a novel
murine model of peritonitis.
 Diminazene aceturate (DIZE) exerts anti-inflammatory effects on Spk-induced
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inflammation in mice.
 DIZE mitigates DNA extracellular traps (DETs) in human leukocytes: neutrophils,
monocytes and macrophages.
Abstract
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Due to the severity of COVID-19, potential therapies are explored to prevent
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hyperinflammatory complications. Here, we aimed to investigate the inflammatory
response induced by SARS-CoV-2 Spike protein (Spk) and its downmodulation by
diminazene aceturate (DIZE). Through inducing Spk inflammation in murine models,
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leukocyte migration to the peritoneum, levels of myeloperoxidase (MPO),
malondialdehyde (MDA), rolling and adhesion of mesenteric leukocytes, and vascular
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permeability were investigated. Using human neutrophils, monocytes, and
macrophages, extracellular DNA traps (DETs) induced by Spk and the production of
IL-6 and TNF-α were analyzed. In silico assays assessed the molecular interaction
between DIZE and molecules related to leukocyte migration and DETs induction. Spk
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triggered acute inflammation, demonstrated by increasing leukocyte migration.

Oxidative stress was evidenced by elevating levels of MPO and MDA in the peritoneal
liquid. DIZE attenuated cell migration, rolling, and leukocyte adhesion, improved
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vascular barrier function, mitigated DETs, and reduced the production of pro-
inflammatory cytokines induced by Spk. Computational studies supported our findings,
showing the molecular interaction of DIZE with targets such as β2 integrin, PI3K, and
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PAD2 due to its intermolecular coupling. Our results outline a novel role of DIZE as a
potential therapeutic agent for mitigating the inflammation induced by Spk.
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Keywords: COVID-19, Leukocyte, NETs, DETs, DIZE, Innate immunity.

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1. Introduction
Since 2002, there have been three major outbreaks of the human coronavirus, the most
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recent of them caused by the severe acute respiratory syndrome coronavirus 2
(SARS‐CoV‐2), the etiological agent of the coronavirus disease 2019 (COVID‐19).
Infection by SARS-CoV-2 causes lung inflammation and acute respiratory distress
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syndrome, but aside from the respiratory system, other organs in the body can also be
affected by this virus. The SARS-CoV-2 uses the angiotensin-converting enzyme 2
(ACE2) as a receptor to enter host cells [1]. Once inside, the downregulation of ACE2 is
believed to make individuals more susceptible to lung and systemic inflammatory
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events, which can lead to severe COVID-19 and even death [2,3]. Besides ACE2,
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different receptors have been involved in the SARS-CoV-2 interaction with the host
cells and may contribute to the COVID-19 pathogenesis [4-7].
The hyperinflammatory syndrome triggered by SARS-CoV-2 significantly contributes
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to the severity and mortality of COVID-19 and can be triggered, among others, by
SARS-CoV-2 spike protein interaction with different Toll like receptors [8-12]. The
damaging inflammatory reaction arises from an imbalanced immune response, leading
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to pathological changes such as cytokine storm, lymphopenia, macrophage activation,
monocyte death, and neutrophil abnormalities in severe cases of COVID-19 [13,14].
Endotheliopathy, neutrophil extracellular traps (NETs) formation, and elevated serum
levels of proinflammatory mediators indicate disease severity following SARS-CoV-2
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infection [15,16].
Diminazene (DIZE) is a widely used antiparasitic drug that can prevent protozoan
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diseases [17]. Chemically, it is a small molecule with a structural similarity to

xanthenone, a well-known ACE2 activator [18]. Interestingly, DIZE has been suggested
as a potential therapeutic agent for preventing tissue injuries similar to those related to
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SARS-CoV-2 infection, including pulmonary inflammation, cardiac damage, and gut

disorders, presenting notable anti-inflammatory properties [19-23]. Computational and
in vitro assays suggest that DIZE partially inhibits the binding of the Spike protein to
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ACE2 and a replication step targeting the Mpro of SARS-CoV-2 [24]. Another study
demonstrated that DIZE acts as a dual inhibitor of the human host proteases TMPRSS2
and furin, both involved in SARS-CoV-2 infection [25]. Furthermore, DIZE suppresses
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certain signalling pathways linked to cytokine production, suggesting that it may

effectively treat conditions caused by excessive cytokine production [19]. However, no
in vivo studies have provided insights into whether DIZE could control the
inflammatory response induced during SARS-CoV-2 infection.
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The present study was designed to evaluate the phlogistic properties of SARS-CoV-2
Spike protein by different methods, encompassing in vivo, ex vivo, in vitro, and in
silico approaches, and to investigate whether DIZE mitigates the inflammatory response
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triggered by the Spike protein.
2. Methods
2.1. Reagents and chemicals
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Diminazene aceturate (DIZE), carrageenan (CG), dexamethasone (DEXA), o-
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dianisidine, penicillin, streptomycin, RPMI-1640 medium and human serum were
purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents used in the
study were purchased from standard commercial suppliers and were of high analytical
grade.
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2.2. SARS-CoV-2 Spike protein synthesis
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The trimeric spike protein of SARS-CoV-2 was kindly provided by Dr. Leda Castilho
(Cell Culture Engineering Laboratory, Universidade Federal do Rio de Janeiro, Brazil),
purified according Alvim et al [25]. In brief, SARS-CoV-2 spike glycoprotein was
produced in HEK293-3F6 (NRC) cells that stably express the soluble ectodomain
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(amino acids 1-1208) of the spike protein in the trimeric prefusion conformation [26].
The cells were cultivated in HEK-GM medium (Xell AG), at 37oC, 5% CO2 under
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shaking (180 rpm, shaker with 5 cm stroke). The protein was purified on a StrepTrap
XT affinity chromatography column (Cytiva), following the manufacturer's instructions.
Protein concentration, purity, and identity in eluted fractions were confirmed by
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Nanodrop (Thermo Fisher), silver-stained SDS-PAGE, and Western blot analyses,

respectively (Alvim et al., 2022). The absence of endotoxin in our samples of Spike
protein was determined using the PierceTM Chromogenic Endotoxin Quant Kit. Spk
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protein was used dissolved in PBS, which was also endotoxin-free.
2.3. Animal experiments

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BALB/c mice of both genders (weight: 25-30 g) obtained from the rodent bioterium of
UFDPar Research Center were used throughout. They were housed in cages at 23 ± 1
℃, and 12 h light/dark cycle, with unrestricted access to a standard pellet diet and tap
water throughout the experiments. Mice were randomly assigned to groups of six and
underwent an 18-h fast prior to the experiments, while still having unrestricted access to
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water.
2.4. Peritoneal model of acute inflammation
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Neutrophil migration into the peritoneal cavity was conducted based on Chaves,
Nicolau et al. [27] with modifications. Briefly, mice received intraperitoneal (i.p.)
injections of 250 µL sterile saline, dexamethasone (1 mg/kg), or DIZE (10 mg/kg).
After 30 min, they received 250 µL of carrageenan i.p. (500 μg/cavity) or SARS-CoV-2
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Spike protein (Spk) at different concentrations (0.3, 1, and 5 µg/cavity). Mice were
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euthanized 4 h later, and peritoneal cells recovered with 1.5 mL of phosphate-buffered
saline (PBS) containing a 0.2% solution of sodium heparin (Cristália, SP, Brazil). The
volumes recovered (approximately 95% of the injected volume) were similar in all
experimental groups. Total cells were counted in a Neubauer chamber, and the
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differential counts after cytocentrifugation and staining with hematoxylin and eosin.
The results are presented as the number of neutrophils/ml of peritoneal exudate.
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Aliquots of the peritoneal exudates were stored at -80 ℃ to analyze malondialdehyde
(MDA) levels, and myeloperoxidase (MPO) activity.
2.5. Resident macrophages depletion

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Peritoneal macrophages were removed according to Ribeiro, Souza-Filho et al. [28]. In

brief, mice were subcutaneously (s.c.) anaesthetized using xylazine hydrochloride (10
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mg/kg, Konig®, Mairinque, Brazil) and ketamine (80 mg/kg, Syntec®, Santana de
Parnaíba, Brazil). Next, sterile saline (3 mL) was injected into the peritoneal cavity,
followed by a gentle massage, and the whole procedure was repeated 5 times, which
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reduced the peritoneal macrophage population by around 80%. The same procedure was
performed for control (sham) mice, including impalement and manipulation, but no
fluid was injected or withdrawn. After that, each group (sham and lavage) received PBS
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(250 µL/cavity) or SARS-CoV-2 Spk (5 µg/cavity), and exudate was collected 4 h later.
2.6. Myeloperoxidase (MPO) activity

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MPO is an enzyme predominantly found in the azurophilic granules of neutrophils, and

it has been widely utilised as a biochemical marker for the infiltration of granulocytes
into tissues and cavities, including the peritoneum. The enzymatic activity of MPO in
the peritoneal exudate was assessed as described [29]. Aliquots of the peritoneal
exudates were subjected to centrifugation at 4000 rpm for 7 min at 4 ℃. The resulting
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pellet was resuspended, and MPO activity was determined by measuring the change in
absorbance at 450 nm using o-dianisidine dihydrochloride (3,3′-dimethoxybenzidine)
and 1% hydrogen peroxide (H2O2). The results are reported as units of MPO per
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millilitre of peritoneal exudate (UMPO/mL).
2.7. Malondialdehyde (MDA) levels

Peritoneal exudates were combined with 1.5 mL of 1% phosphoric acid (H3PO4) and 0.5
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mL of 0.6% tert-butyl alcohol (aqueous solution). The mixture was stirred and heated in
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a boiling water bath for 45 min, followed by promptly cooled in an ice bath, and 4 mL
of n-butanol was added. The resulting mixture was shaken, and the butanol layer was
separated by centrifugation at 1200g for 10 min. The reaction was measured at 535 and
520 nm, and the difference between the two readings was calculated as the tert-butyl
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alcohol value [30]. The concentration of malondialdehyde (MDA) is reported as
mmoles/ml of peritoneal fluid.
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2.8. Intravital microscopy to assess mesenteric leukocytes rolling and adhesion
The rolling and adhesion of leukocytes in the mesenteric microcirculation was done by
intravital microscopy [31,32]. Mice were treated with DIZE (10 mg/kg, p.o.) 30 min
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prior to Spk (5 μg/cavity) inoculation. After 4 h, mice were anesthetized with ketamine
(100 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.), and their mesenteric tissue was
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exteriorized and placed on a heated pad at 37 ℃. Based on their location within the
microvascular network, third-order venules corresponding to postcapillary venules with
a 10-15 μm diameter were selected for analysis. Leukocyte rolling, defined as the
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movement of white blood cells at a slower velocity than erythrocytes in the same
stream, was recorded at 10 min intervals. These leukocytes moved slowly, allowing for
individual visibility, and were counted as they rolled past a 10 μm segment of the
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vessel. A leukocyte was considered adherent to the endothelium if it remained

stationary for 30 sec. The number of adherent cells (termed "stickers") was reported as
the count per 10 μm segment of the venule. Cell counts were conducted using recorded
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images, with 3-4 fields analyzed per animal to minimize sampling variability. The data
were presented as the mean ± SEM of 5 animals per group.
2.9. Evaluation of vascular permeability
Vascular permeability was analysed in mice by assessing Evans blue extravasation, a
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substance that strongly binds to serum albumin. Using the same experimental model of
acute peritonitis, mice were administered DIZE (10 mg/kg, p.o.) 30 min prior to the
administration of SARS-CoV-2 Spk (5 μg/cavity). Thirty minutes before euthanasia,
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mice were intravenously injected with 50 μL of Evans blue (Sigma-Aldrich) at 25
mg/kg in the retro-orbital plexus. Evans blue extravasation in the peritoneal exudate was
quantified using a spectrophotometer at 620 nm after constructing a standard curve. The
control group received sterile saline 0.9% (w/v) as the vehicle [33].
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2.10. Isolation of human peripheral blood monocytes cells (PBMCs) and neutrophils
PBMCs were collected from blood of healthy donors by density gradient (Histopaque,
Sigma-Aldrich), at 400 g, 30 min, 21 ℃, according to Arteaga-Blanco et al. [34].
PBMCs were resuspended in RPMI medium (Gibco) supplemented with 10% heat-
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inactivated fetal bovine serum (FBS, Cultilab, SP, Brazil) and incubated at 37 ℃, 5%
CO2 for 2 h. Afterward, non-adherent cells were removed, and the adhered monocytes
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were washed 2 to 3 times with PBS and assayed in RPMI medium without serum.
Neutrophils were purified from the same gradient as described by Silva-Oliveira,
Linhares-Lacerda, et al. [34] (2022) resuspended in RPMI-medium and used
immediately after isolation.
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2.11. Culture of human monocyte-derived macrophages (HMDM)

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To obtain monocyte-derived macrophages, PBMCs were plated at 2x105 cells/well in

96-well plates and then incubated in RPMI medium, supplemented with 10% heat-
inactivated human serum (Sigma-Aldrich) and 1% penicillin/streptomycin (Sigma-
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Aldrich) at 37 °C, 5% CO2. After 7-10 days, non-adhered cells were washed out, and
the macrophages were maintained in RPMI with 5% human serum and 1%
penicillin/streptomycin for 2 days [35]. HMDM were then washed twice in PBS and
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assayed in RPMI without serum.
2.12. NETs/DETs inhibition and quantification assays

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Neutrophils, monocytes, and macrophages (2x105/well) in RPMI were incubated for 30

min in the absence or presence of various concentrations of DIZE (10, 50, and 100
µg/mL), at 37 °C with 5% CO2. Later on, the cells were stimulated or left unstimulated
with PMA (100 nM; Merck, Darmstadt, Germany) and SARS-CoV-2 Spike protein
(Spk; 2 µg/mL) in 96-well plates, with a final volume of 100 µL, for 3 h at 37 ℃ with
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5% CO2. Unstimulated cells were used as a control. The levels of double-stranded DNA
(NETs/DETs-DNA) were measured in the culture supernatant using the Quant-iT
PicoGreen dsDNA assay (Invitrogen, Waltham, MA, USA), following the
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manufacturer's instructions. The assay was performed in opaque 96-well plates, and the
fluorescence intensity was measured at 485/538 nm excitation/emission in a
SpectraMax Paradigm Fluorimeter (Molecular Devices, San Jose, CA, USA), as
previously described by Guimarães-Costa, Nascimento, et al. [36].
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2.13. Cell viability assay
Cell viability was carried out by assessing the activity of the enzyme lactate
dehydrogenase (LDH) in culture supernatants using the CytoTox96 Non-Radioactive
Cytotoxicity Assay (Promega, WI, USA), following the manufacturer's instructions.
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Lysed cells were used as a positive control and reaction read at 490 nm.
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2.14. NETs/DETs Immunofluorescence characterization
Neutrophils, monocytes, and macrophages (6x105/well) were adhered to 13 mm glass
coverslips (Knittel Glass, Germany) coated with 0.001% poly-L-lysine (Sigma-Aldrich)
in 24-well plates. Cells were incubated in the presence or absence of the spike protein (2
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µg/mL) for 3 h at 37 °C, 5% CO2. Afterwards, the cells were fixed with 4%
formaldehyde (Sigma-Aldrich) for 30 min, washed with PBS and incubated with
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blocking solution (PBS/3% BSA; Sigma-Aldrich) for 1 h at room temperature. The cells
were incubated overnight, at room temperature, with antibodies anti-SARS-CoV-2
(1:200; serum from a COVID-19 positive patient), and anti-elastase (1:500; Rabbit;
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Calbiochem, La Jolla, CA, USA). They were washed with PBS and incubated with
secondary antibodies Alexa Fluor 546 anti-Human IgG (1:500, Invitrogen), Alexa Fluor
488 anti-Rabbit (1:500, Invitrogen), for 2 h at room temperature. Samples were
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counterstained with DAPI (1 μg/mL; Sigma-Aldrich) for DNA staining. Coverslips

were again washed and mounted on slides with ProLong Gold antifade with DAPI (10
μg/mL; Thermo Fisher Scientific, Waltham, MA, USA). Images were obtained in a
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Zeiss Elyra PS.1 confocal microscope (Zeiss, Oberkochen, Germany).
2.15. Cytokines measurements
The levels of IL-6 and TNF-α were quantified by ELISA in cell-free supernatants from
monocytes and macrophages pre-treated or not for 30 min with DIZE (100 μg/mL) and
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then incubated or not with the spike protein (Spk 2 µg/mL) at 37 °C, 5% CO2,
performed according to manufacturer’s instructions (R&D Systems, MN, USA).
Supernatants from monocytes were harvested 4 h after cell stimulation, and for
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macrophages, the supernatants were harvested at 4 h and 24 h after cell stimulation.
2.16. Molecular docking

In this study, molecular targets involved in the process of rolling and adhesion of
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leukocytes on the endothelium were selected. They are P-Selectin (PDB ID: 1G1S), L-
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Selectin (PDB ID: 5VC1), E-Selectin (PDB ID: 1G1T), and β2 integrin (PDB ID:
5E6U). Molecular targets involved in the release of DNA extracellular traps, known as
NETs and DETs (other leukocytes DNA extracellular traps), were also analysed. They
are: Caspase 1 (PDB ID: 6BZ9), CDK6 (PDB ID: 6OQO), Gasdermin D (PDB ID:
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5NH1), MPO (PDB ID: 4C1M; PDB ID: 5FIW), NADPH (PDB ID: 1JA9; PDB ID:
7CFZ), NADPH Domain (PDB ID: 3A1F), Neutrophil Elastase Human (PDB ID:
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3Q76), PAD2 (PDB ID: 4N2K; PDB ID: 4N2M; PDB ID: 4N20), PAD4 (PDB ID:
2DEW; PDB ID: 2DEX) and PI3k (PDB ID: 2WXH; PDB ID: 2WXF). All molecular
targets were acquired from the Protein Data Bank (https://www.rcsb.org/). To select the
biological targets, the inclusion criterion was based on structures resolved by
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crystallography with a resolution below 2 angstroms. However, exceptions were made

for targets that were not accessible on the platform at the specified resolution. The
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UCSF Chimera software was utilised to eliminate water molecules and other residues
that could potentially disrupt the ligand-macromolecule interaction, resulting from
crystallography [37].
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Molecular docking simulations were conducted using AutoDock 1.5.6 software [38]. To
prepare the ligand and the macromolecule, hydrogen atoms were added using Gasteiger
charges and non-polar hydrogens were merged. The active site grid was determined
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based on the RMSD of the targets or the wells for targets lacking crystallographic
ligands (Table 1 and Table 2), employing a cubic box in two different dimensions: 60 x
60 x 60 for cell adhesion molecules and 30 x 30 x 30 for NETs/DETs release,
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considering the size of the ligands. This selection was based on the molecular size of
cell adhesion molecules, such as carbohydrates. The Lamarckian GA algorithm was
employed during the simulation, with 100 runs, 150 populations, and many evaluations.
Finally, conformations and molecular clusters were analysed to compare chemical
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similarities between DIZE and known antagonists of events comprising leukocyte
migration and NETs/DETs development [39,40].
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2.17. Animal and human ethical statements
All animal experiments and protocols adhered to the National Institutes of Health
Guidelines for the Care and Use of Laboratory Animals and received approval from the
Animal Care and Use Committee of the Universidade Federal do Delta do Parnaíba –
UFDPar (Protocol #004/2022). All procedures involving human cells were reviewed
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and approved by the Ethics Committee for Human Studies at Hospital Clementino
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Fraga Filho, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil,
under the protocol No. 4261015400005257. Blood collection was performed after the
volunteers signed the informed consent form.
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2.18. Statistical analysis
The data are presented as the mean ± SEM of n mice per group, and statistical
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differences were assessed using one-way analysis of variance (ANOVA) after
confirming the homogeneity of variances with Bartlett's test. Post-hoc comparisons
were performed using Bonferroni's test. Statistical analysis of cytokines measurement
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was performed using the Friedman test with Dunn's post-test. All the data were
uploaded on the daw GraphPad Prism 8.0.1 (GraphPad Software Inc., La Jolla, CA,
USA) running on a MacBook Pro. Cytokine production of monocytes and macrophages
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was analyzed by ONE way ANOVA and Holm-Sidak post-test. A significance

threshold of p<0.05 was considered.
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3. Results
3.1. Spike protein (Spk) induces leukocyte migration on acute peritonitis in mice
Peritonitis was induced in mice by injecting Spk or carrageenan and after 4 h, peritoneal
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exudate was collected to determine leukocyte migration and myeloperoxidase (MPO)

and malondyaldehyde (MDA, Fig. 1 A). Our results shown that 5 µg Spk increased the
total number of leukocytes recruited to the peritoneum (21428 ± 3482 × 103 cells/mL),
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similarly to carrageenan (Fig. 1 B). Neutrophils were the majority cells that migrated to
the peritoneal cavity compared to the sham group (2358 ± 888.2 × 103 cells/mL and
57.31 ± 34.89 × 103 cells/mL, respectively, Fig. 1 C). However, there was no significant
difference between sham group and Spk groups at the concentrations 0.3 and 1 µg in
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leukocyte migration. No significant difference was found between 5 µg Spk and
carrageenan, the reference phlogistic agent, in total and differential leukocyte count
(Cg: 19817 ± 2,076 × 103 cells/mL and 1924 ± 149.8 × 103 cells/mL, respectively, Fig.
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1 B, C).
Intraperitoneal inoculation of 5 µg Spk increased MPO activity (2.963 ± 0.96 U/mL of
peritoneal exudate; Fig. 1 D) and MDA levels (57.02 ± 10.56 nmol/mL of exudate; Fig.
1. E) compared to the sham group (MPO: 0.2616 ± 0.1352 U/mL of peritoneal exudate;
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MDA: 14.12 nmol/mL of peritoneal exudate). Besides, at the concentrations 0.3 and 1
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µg, no significant difference in MPO activity was. Spk 1 µg increased MDA levels
(46.30 ± 5.338 nmol/mL of peritoneal exudate), while no significant difference was
observed in the Spk 0.3 µg group (25.03 ± 4.849 nmol/mL of peritoneal exudate).
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3.2. Spk-induced acute peritonitis depends on macrophage involvement
In order to verify the role of macrophages in the Spk-mediated leukocyte recruitment,
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we depleted these cells before Spk injection (Fig. 1 F). Thus, resident macrophages
were depleted by 5 PBS washes of the peritoneal cavity followed by the injection of 5
µg Spk. After 4 h the exudate was collected and leukocyte number determined. Sham
animals were only injected with Spk. Our results show that macrophage numbers were
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reduced by 78% by this washing protocol (Fig. 1 G). Leukocyte migration induced by
Spk (5 µg) in the macrophage-depleted mice was 65% lower (3642 ± 618.8 × 103
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cells/mL of exudate) relative to the non-depleted ones (10371 ± 1,522 U/mL of exudate)
(Figure 1 G).
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3.3 Spk-induced acute peritonitis was mitigated by Diminazene aceturate (DIZE)

Mice were treated orally with dexamethasone, Spk or saline and after 4 h the peritoneal
exudate was analysed (Figure 1 H). Our results show that leukocyte recruitment by Spk
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(5 µg) was reduced by 76% by DEXA and 63% by DIZE (Fig. 1 I).
3.4. DIZE attenuated mesenteric leukocyte rolling, adhesion, and vascular permeability
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Mice were treated with DIZE, and 4 h after Spk i.p. injection, they were inoculated with
Evans blue 30 min before euthanasia, and the mesenteric vessels were analyzed by
intravital microscopy (Fig. 2 A). Representative images of mesenteric vessels of the
three animal groups are shown in Fig. 2 B.
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The number of leukocyte rolling induced by Spk increased 7 times compared to sham
(440.2 ± 33.68 leukocyte rolling/min versus 62.43 ± 3.154 leukocyte rolling/min), and
DIZE reduced the leukocyte rolling induced by Spk by 3 times (Fig. 2 C). A similar
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effect was obtained in the leukocyte adhesion analysis, as Spk increased by 23-fold
relative to sham mice, while DIZE decreased by 8-fold the adhesion promoted by Spk
(Fig. 2 C).
Vascular permeability was analysed by Evans blue after mice treatment as above (Fig. 2
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D). Spk caused an increase in vascular permeability, measured by the concentration of
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Evans blue in the peritoneal exudate (Fig. 2 E). The concentration of Evans blue was
49.89 ± 7.482 µg/mL in the Spk group, and only 3.401 ± 0.5622 µg/mL in the sham
group. DIZE pre-treatment reduced the vascular permeability to 16.20 ± 1.459 µg/mL of
exudate.
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3.5. DIZE inhibits the release of NETs/DETs (DNA extracellular traps) release in
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human cells
Because NETs are inflammatory, produced in high amounts by COVID-19 patients, and
correlate with the severity of the infection, we tested DIZE's capacity to modulate the
release of these structures by neutrophils, monocytes, and macrophages (Fig. 3A). We
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found that pre-treatment with DIZE inhibited NET release induced by Spk and PMA
(Fig. 3 B). Neutrophils incubated with DIZE alone did not release NETs, and DIZE was
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not toxic for those cells (Fig. 3 C). NETs released by Spk-stimulated neutrophils stained
with anti-elastase, DAPI for DNA, and anti-Spk depicted a co-localization of these three
molecules, that were absent in the DIZE treated neutrophils (Fig. 3 D).
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Monocytes also released DETs when stimulated by Spk and PMA, and DIZE effectively
decreased DET formation when these cells were exposed to either stimulus (Fig. 3 E).
Furthermore, cell viability assays showed that DIZE was not toxic for monocytes (Fig.
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3F). DETs extruded by monocytes were visualized and labeled with DAPI and Spk,
which were co-localized (Fig. 3 G).
We also tested whether DIZE could inhibit DET formation by macrophages stimulated
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with PMA and Spk. Similarly, we observed that pre-incubation of macrophages with
DIZE mitigated DET release induced by PMA and Spk (Fig. 3 H). Moreover, DIZE was
not toxic for macrophages at the tested concentrations (Fig. 3 I). DET induced by Spk in
macrophages was labeled with elastase, DAPI, and Spk; and DIZE inhibited DET
extrusion (Fig. 3 J).
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3.6. DIZE reduces inflammatory cytokine levels
The cytokine storm that affects COVID-19 patients prompted us to investigate whether
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DIZE could control the production of proinflammatory cytokines by monocytes and
macrophages stimulated by Spk (Fig. 4 A). Thus, the levels of IL-6 and TNF-α were
evaluated in the same culture supernatants in which we measured DET formation. We
found that Spk potently induced IL-6 and TNF-α production by both cells (for
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macrophages, measured after either 4 h or 24 h), and that DIZE abrogated this
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production, reducing the cytokine amounts to the control levels (Fig. 4 B). These
results, associated with the other findings reported above, indicate that DIZE is
endowed with a powerful anti-inflammatory activity, as demonstrated in our assays
using SARS-CoV-2 spike protein.
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3.7. Computational studies with DIZE upon inflammation targets
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The exploration of molecular docking encompassed various crucial enzymes in
leukocyte migration and classical pathways associated with NETs/DETs formation.
Hence, we aimed to comprehend the potential mechanisms of action of DIZE, as
revealed by binding energy values documented in Tables S-1 and S-2 (Supplemental
ot
Information). To facilitate comparisons, structural simulations of aceturate were

conducted, confirming that the binding energy is indeed attributed to the DIZE
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structure. All these data are expressed on Fig. 5.
For adhesion targets, namely P-Selectin, E-Selectin, L-Selectin, and β-2 integrin
proteins (Table S-1, Supplemental Information), DIZE exhibited optimal binding energy
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with β-2 integrin PDB 5E6U, recording a value of -6.96 kcal.mol-1 and an inhibitory
constant of 7.9µM. Hydrogen bonds formed in the nitrogenous regions involving six
amino acids (ARG119, GLN117, GLN360, GLY62, LYS280, and PHE122).
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Additionally, Alkyl-π interactions occurred on residues ARG119, LYS280, and

PRO281, accompanied by Attractive charge interactions with ASP362 (Fig. 5, Panel B).
Various PDB targets of Protein Arginine Deiminase 2 (PAD2) underwent scrutiny
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(4N2K, 4N2M, and 4N20), with DIZE showcasing a binding energy of -7.17 kcal.mol-1
for 4N20 and an inhibitory constant of 5.51µM. Five hydrogen bonds were established
between amino acids (ASP634, GLU412, GLY599, MET476, THR649) in the
nitrogenous regions of the ligand. Noteworthy interactions included salt bridge types,
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Attractive charge types, lone-pair–π bonding with the amino acid ASN590, and Alkyl-π
interactions with residues (PRO601 and ILE592) involving the aromatic ring (Fig. 5,
Panel B).
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Regarding the phosphatidyl-inositol 3-kinase (PI3K) target, two PDB elements (2WXF
and 2WXH) were examined, revealing an inhibitory constant of 21.18µM for DIZE and
a binding energy of -6.38 kcal.mol-1. Hydrogen bonds formed with amino acids
(ASP787, MET900, and PRO758) in the nitrogenous region of the ligand. Additional
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interactions encompassed salt bridges, π-π T-shaped interactions with the TRP760
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residue in the aromatic ring region, Alkyl-π interactions (ILE777 and ILE825), and
Sigma-π interactions with amino acids (ILE910 and MET752) (Fig. 5, Panel B).
Simulation results demonstrated energies that elucidate the binding of DIZE with the
substrate of the targets. In comparison with PAD2 inhibitors, DIZE outperformed the
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standard enzyme inhibitor and approached the value of Cl-Amidine (Figure 5, Panel C).
In contrast, when compared to PI3K inhibitors like GSK and Buparlisib, DIZE didn't
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exhibit superiority but showed a comparable value to Taselisib. While DIZE didn't
surpass MK and Cilengitide ligands for β-2 integrin, its high binding energy positions it
as a promising candidate (Fig. 5, Panel C).
To assess structural similarity, a study of molecular superimposition (overlay) was
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conducted, evaluating steric field (ste) and electrostatic (ele) similarities (Table 1). The
analysis revealed approximately 80% steric field similarity between DIZE and Cl-
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amidine. Cilengitide exhibited a 92% electrostatic similarity, whereas PAD2-IN

demonstrated 62%. Notably, the structural comparison shown in Fig. 5, Panel D
highlights DIZE's unique features in relation to other structures.
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4. Discussion
Here, we identified significant findings about the pathobiology of translational COVID-
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19 and the involvement of DIZE as a pharmacological tool that mitigates inflammation

in the models analyzed: 1) The SARS-CoV-2 Spk protein itself acts as an inflammatory
agent by recruiting leukocytes to the peritoneal cavity in mice, and 2) DIZE mitigates
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the inflammatory response triggered by the inflammatory agents PMA and the Spk
protein. Furthermore, our data demonstrate that DIZE potently affects the release of
NETs/DETs in vitro assays, confirmed in silico approaches. Considering the
inflammatory reactions observed in COVID-19 patients, this study employed one
existing drug to explore novel molecular aspects of medical concerns related to the
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SARS-CoV-2 infection, which still requires further investigation.
Firstly, different concentrations of Spk protein were tested in a traditional model of
acute peritonitis usually accessed with carrageenan, zymosan, fMLP, and other
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substances [27,28]. Initially, we identified that Spk protein at 5 μg/cavity increased the
migration of leukocytes into the peritoneum, with neutrophils being the predominant
cell type. Afterward, a significant decrease in leukocyte migration was observed after
depleting resident macrophages and then inoculating Spk protein, evidencing the
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essential role of macrophages in this cellular event. Macrophages appear to be critical
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cells during COVID-19 development in various tissues, including alveolar, endothelial,
and those derived from peripheral blood monocytes [41,42]. Additionally, our study
presents, for the first time, evidence that resident macrophages from the peritoneal
cavity are crucial for initiating the inflammatory response following inoculation with
Spk protein.
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SARS-CoV-2 Spk protein is implicated in classical molecular routes of inflammation,
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such as NF-κB signaling, with demonstrated ROS generation in different types of cells,
including immune cells [43-46]. Our data corroborate with free radical damage being an
event observed not only in vitro conditions, but also in a complex system using the
acute peritonitis as an in vivo model, in which MPO and MDA were augmented in the
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peritoneal milieu after Spk inoculation, both hallmarks are involved in oxidative stress
and lipid peroxidation, respectively. To our knowledge, our study is the first to
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demonstrate the potential of BALB/c mice for investigating in vivo inflammatory

aspects induced by SARS-CoV-2 Spk protein into the peritoneum, opening up new
possibilities for testing drugs that can reduce these inflammatory responses.
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During the course of COVID-19 pandemic, drug repurposing grew on a widespread

scale in order to restrain the mortality rate that plagued countries worldwide with ample
focus on antiviral strategies and consequences provoked by the SARS-CoV-2 virulence
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[47,48]. A RECOVERY collaborative study with global importance showed notable

efficacy of dexamethasone in patients hospitalized with COVID-19 patients. This drug
modulates inflammation-mediated lung injury and thereby reduces progression to
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respiratory failure and results in lower mortality [49]. Although these findings have
shed some light on the pathology development and pharmacological interventions for
COVID-19, complications associated to this viral disease still sustain inquiries about
assertive drug management, especially in the inflammatory phase where ACE2
downregulation subsidizes the most severe prognoses [50,51]. Then, not only
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suppressing inflammation by anti-inflammatory drugs, such as glucocorticoids, seems to
be the key factor for controlling the damages caused by SARS-CoV-2 infection, but the
health emergency encourages investigation of therapies modulating the RAS axis, such
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as ACE2 pharmacological modulators [52]. It is noteworthy that not only SARS-CoV-2,
but also the Spk protein, is also able to downregulate ACE2 [53]. Our data showed that
dexamethasone and DIZE decreased the global count of leucocyte recruitment spurred
by SARS-CoV-2 Spk protein inoculation into the murine peritoneal cavity.
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We found that DIZE, which is also an ACE2 activator, reduced leukocyte migration to
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the peritoneal cavity stimulated with SARS-CoV-2 Spk protein, suggesting a protective
role in this experimental model. Whereas SARS-CoV-2 infection can cause thrombosis
and endothelial damage, the weakened enzymatic activity of ACE2 may result in
increased concentrations of pro-inflammatory molecules, such as angiotensin II and
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bradykinin, contributing to SARS-CoV-2 pathobiology [54].
Patients with COVID-19 may also present increases of the adhesion molecules
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expression on endothelial cells and leukocytes [55-57], and it has been shown that Spk
protein induces leukocyte adhesion in endothelial cells [58]. We observed that SARS-
CoV-2 Spk protein increases the rolling and adhesion of immune cells on the
endothelium of mesenteric microcirculation, both prerequisite steps of leukocyte
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diapedesis. We also found that DIZE drastically reduced leukocyte rolling and adhesion
onto the peritoneal endothelium, as detected in an ex vivo intravital microscopy
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approach, suggesting its anti-inflammatory profile.

NETs are DNA extracellular traps released by several cell types, not exclusive to
neutrophils [59-61]. In moderate to severe cases of COVID-19, NETs were detected and
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found to be associated with the inflammatory response and lung injury [16,60-64].
Besides, SARS-CoV-2 Spk protein per se induces NET formation from human
neutrophils and THP1 monocytes [65,66]. Here, we have shown that Spk also induces
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extracellular DNA trap extrusion in human monocytes and macrophages, which was
inhibited by DIZE. It is noteworthy that this effect on human leukocytes occurred
without DIZE toxicity effects on these cells, suggesting its possible use for inhibiting
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NET/DET formation in COVID-19.

We and others described that NF-κB activation plays a pivotal role in the immune
response to SARS-CoV-2 infection, orchestrating the production of various cytokines
[8,67-71], including IL-6 and TNF-α [67,72]. IL-6 and TNF-α are pro-inflammatory
cytokines that contribute to tissue damage and systemic inflammation in severe
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COVID-19 [8,73-74]. By inhibiting the Spk-mediated elevated production of IL-6 and
TNF-α, besides reducing NET/DET formation, DIZE exhibits an anti-inflammatory
profile, targeting the exacerbated activation of neutrophils, monocytes, and
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macrophages. This modulation may have therapeutic implications, as it aligns with the
results obtained in an animal model of peritonitis triggered by SARS-CoV-2 Spk
protein with the pivotal role of macrophages in leukocyte recruiting, indicating a
promising avenue for controlling the inflammatory response associated with COVID-
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19.
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Regarding the rolling and adhesion of leukocytes, our findings demonstrated the in
silico spatial interaction between DIZE and selectins as well as β2 integrin. Notably, the
strongest results were observed with β2 integrin. These key adhesive molecules are
expressed on the surfaces of leukocytes and endothelium during inflammatory
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conditions. The mediation of immune cells trafficking depends on cell adhesion
molecules, particularly the receptors of selectins and integrins. The β2 integrins that are
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specific to leukocytes are the most prevalent integrins found in these cells. They are
essential for facilitating the trafficking of leukocytes and are involved in important
functions such as neutrophil phagocytosis, reactive oxygen species (ROS) production,
and T cell activation [75,76]. Furthermore, our molecular docking results pertaining to
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the release of NETs/DETs revealed potential interactions of DIZE with PI3K, and
peptidyl arginine deiminase-2 (PAD2), intracellular enzymes that participate in
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molecular routes associated with gasdermin D activation. Fattahi and colleagues [77]
discussed the PI3K/Akt/mTOR pathway as a potential target for anti-SARS-CoV-2,
including inhibition of hyperinflammation [77]. Moreover, PAD enzymes, including
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PAD2, catalyse the conversion of arginine residues to citrulline, regulating activity of

host immunity in cellular events such as NETs/DETs release [78].
When DIZE was compared with other inhibitors that target selectins, β2 integrin, PI3K,
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and PAD2, our results revealed a favourable Gibbs free energy, indicating that DIZE
has the potential to modulate these proteins' activity. Based on our computational study,
DIZE has the potential to interact with critical molecular targets involved in regulating
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the inflammatory response. Concerning chemical composition, DIZE exhibits structural

resemblances to the inhibitor molecules utilized as scaffold models in the in silico
methodologies. Moreover, since some clinicians have employed ACE2 modulators to
treat COVID-19, targeting the SARS-CoV-2/ACE2 interaction [79], we argue that the
direct activation of ACE2 by DIZE could hold significant benefits in the late stages of
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COVID-19, mainly in severe cases [3,80].
The present study yields noteworthy outcomes in a novel murine experimental model
involving fundamental leukocyte biology events. Nevertheless, despite these advances,
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it is essential to acknowledge the limitations of this work. Computational approaches
used to infer possible interactions between targets and ligands require cautious
interpretation. To validate in silico results, in vitro screening assays or in vivo biological
effects are highly recommended to be assessed. Moreover, verifying whether the results
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obtained from tests conducted solely on the Spk protein directly apply to the actual
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SARS-CoV-2 virus is essential. Such investigations would provide valuable insights
into the broader implications of the study.
5. Conclusion
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In summary, our study provides evidence that SARS-CoV-2 Spk protein can serve as a
phlogistic agent in a newly developed murine model, effectively mimicking key
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inflammatory aspects observed in COVID-19. These include vascular and cellular
events, accompanied by biochemical changes associated with oxidative stress.
Furthermore, our findings indicate that DIZE exhibits a multitarget anti-inflammatory
profile, effectively mitigating inflammatory hallmarks and reducing the release of
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NETs/DETs in human neutrophils, monocytes, and macrophages. These results

highlight the potential therapeutic benefits of DIZE in attenuating inflammation
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associated with COVID-19.

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Author contributions
G.C.P-S., C.K.C.A.C. and L.A.D.N. performed the experiments with assistance from
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N.C.R. (human leukocytes assays), G.P., I.A.B.G., and K.C.S. (animal care and
support), D.C.B.H., B.S.G. and J.R.T. (cytokines assay), R.S.B. and J.A.R. (in silico
assays), L.P.S. (schema design), A.G.C., R.C.P.L-J., M.H.L.P.S., J.V.R.M., and M.C.F.
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(mouse experiments). Statistical analysis: L.A.D.N., G.C.P-S. and J.R.T. Coordinated
the project and designed the experiments: L.A.D.N. and E.M.S., G.C.P-S., M.H.L.P.S.
and J.V.R.M., M.C.F. and J.A.R. Wrote the work: L.A.D.N., E.M.S. and D.C.B.H.
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Acknowledgements
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The authors thank Dr. Leda Castilho (Cell Culture Engineering Laboratory of
COPPE/UFRJ - Universidade Federal do Rio de Janeiro) for donating the SARS-COV-2
trimeric spike protein.
Conflict of interest
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The authors have no financial interests to disclose.
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Data availability statement
The raw data related to the findings of this study are available on reasonable request.
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Consent to publish
All authors read and approved the manuscript for publication. The authors affirmed that
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human research participants provided informed consent for the blood donation.
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Figure Legends
ed
Fig. 1. Spk promotes leukocyte recruitment and oxidative stress in acute
peritonitis. (A) Animals were intraperitoneally administered with Spk (0.3, 1, and 5
μg/cavity, i.p.). After 4 hours, peritoneal fluid was collected for the evaluation of
iew
leukocyte migration and biochemical markers. (B) Total leukocyte count, (C)
Differential (neutrophil) count, (D) MPO activity, and (E) MDA levels. Results are
expressed as the mean ± SEM of 5 animals per group. *P<0.05; **P<0.05; ****P<0.05.
The development of Spk-induced acute peritonitis depends on macrophage
participation. (F) Peritoneal lavage was performed 30 minutes before the administration
of Spk 5 µg. Subsequently, peritoneal lavage was collected for leukocyte migration
v
analysis. (G) Leukocyte migration after macrophage depletion. Results are expressed as
the mean ± SEM of 5 animals per group. ****P<0.05. (H) Animals received pre-
re
treatment with saline, DIZE (16 mg/kg), or DEXA (1 mg/kg) 30 minutes before being
injected with Spk (5 μg per cavity). After a 4-hour interval, leukocyte migration was
evaluated. (I) Leukocyte count after treatment. Each column represents the mean ± SEM
of 5 animals ****P<0.05. er
Fig. 2. DIZE reduced rolling and adhesion in the mesenteric circulation of mice.
(A) Mice were pre-treated with DIZE 30 min before 5 μg Spk administration. After 4 h,
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mice were anesthetized, and the circulation was exposed for analysis of leukocyte
rolling and adhesion in the mesenteric circulation. (B) Representative photomicrographs
of intravital microscopy from sham, 5 µg Spk, and 5 µg Spk + DIZE groups. Spk (5
µg/cavity) intensified leukocyte migration (arrowheads) and adhesion to vessels in the
mesenteric circulation (arrows). (C) Quantification of leukocyte rolling and adhesion.
(D) Mice were treated as before followed by Evans blue inoculation via retro-orbital
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plexus, and vascular permeability quantified in the peritoneal cavity. (E) Spk (5
µg/cavity) triggered peritoneal vascular permeability, that was reduced by DIZE.
Results of both experimental sets are expressed as the mean ± SEM of 5 animals per
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group. ****P<0.005.
Fig. 3. DIZE inhibited the release of NETs/DETs by human leukocytes stimulated with
Spk and PMA. (A) Human neutrophils, monocytes, and macrophages were pre-
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incubated or not with DIZE for 30 min and then stimulated with Spk or PMA followed
by the measurement of extracellular DNA and LDH activity. (B) Quantification of
extracellular DNA released from human neutrophils pre-treated with DIZE (10, 50, and
100 μg/mL) and stimulated by Spk or PMA. (C) Cell viability of neutrophils pre-treated
with DIZE by the LDH activity assay. (D) Representative confocal microscopy images
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of NETs induced by Spk showing labelling with DAPI (DNA, blue), anti-elastase
(green) and Spk (red), and the colocalization of these three markers (overlay).
Neutrophils pre-treated with DIZE and stimulated with Spk, stained as above did not
show NET structures. (E) Quantification of DETs released from human monocytes pre-
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treated with DIZE (10, 50, and 100 μg/mL) and stimulated by Spk or PMA. (F) Cell
viability of human monocytes pre-treated with DIZE by the LDH activity assay. (G)
Representative confocal microscopy images of DETs induced by Spk in monocytes
showing DNA labelling with DAPI (blue), and Spk (red), and the colocalization of these
markers (overlay). Monocytes pre-treated with DIZE and stimulated with Spk, similarly
ed
stained did not show DET structures. (H) Quantification of DETs released from human
macrophages pre-treated with DIZE (10, 50, and 100 μg/mL) and stimulated by Spk or
PMA. (I) Cell viability of human macrophages pre-treated with DIZE by the LDH
activity assay. (J) Representative confocal microscopy images of DETs induced by Spk
iew
in monocytes showing DNA labelling with DAPI (blue), anti-elastase (green), Spk
(red), and the colocalization of these markers (overlay). Monocytes pre-treated with
DIZE and stimulated with Spk, similarly stained did not show DET structures. Results
from 7 donors for the NETs/DETs assays, and 4-5 for the LDH tests are represented as
mean ± SEM. *P<0.05; **P<0.002; ***P<0.0001; ****P<0.00005.
v
Fig. 4. DIZE mitigated pro-inflammatory cytokines levels in human leukocytes
stimulated with Spk. (A) Human monocytes, and macrophages were pre-incubated or
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not with DIZE for 30 min and then stimulated with Spk, followed by the measurement
of TNF-α and IL-6 cytokines. (B) ELISA quantification of TNF-α and IL-6 released
from human monocytes pre-treated with DIZE (100 μg/mL) and stimulated by Spk for 4
h. (C) ELISA quantification of TNF-α and IL-6 released from human macrophages pre-
treated with DIZE (100 μg/mL) and stimulated by Spk at 4 h and 24 h. Results
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expressed as mean ± SEM from 7 donors of each leukocyte. *P<0.05; **P<0.002;
***P<0.0001; ****P<0.00005.
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Fig. 5. Computational studies involving DIZE in leukocyte trafficking events and
NETs/DETs release. (A) In silico experimental design utilized. (B) Graphical
representation of 2D and 3D binding interactions between DIZE and the targets β2-
integrin, PAD-2, and PI3K. (C) Comparison of ΔG (Gibbs Free Energy) in -Kcal/mol
exhibited by DIZE and known antagonists of β2-integrin, PAD-2, and PI3K. (D)
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Molecular predictive comparison of chemical similarities between DIZE and known

antagonists of β2-integrin, PAD-2, and PI3K.
Fig. 6. Schematic representation of the events involved in the inflammation

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triggered by the SARS-CoV-2 spike protein (Spk) and the beneficial effects of
DIZE on leukocytes. The blue panel represents the benefits of DIZE in a murine model
of peritonitis, mitigating events ranging from rolling and adhesion of leukocytes to
vascular endothelium, as well as increased vascular permeability and leukocyte
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migration to the peritoneal cavity triggered by Spk. Furthermore, the benefits of DIZE
in human leukocytes (neutrophils, monocytes, and macrophages) include a reduction in
levels of inflammatory cytokines, as well as the release of NETs/DETs triggered by
Spk.
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Table 1. Similarity analyses by molecular overlap of drugs with the DIZE for 100 steric
and 100 electrostatic.
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DIMINAZENE
LIGANDS
100ste 100ele
Cl-amidine 0,808292 0,0389785
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L-selectin 0,735293 0,0315065
Taselisib 0,717291 0,0240457
MK 0,689524 0,0410149
E-selectin 0,648264 0,0272845
Buparlisib 0,64144 0,02822
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PAD2-IN 0,639736 0,626963
GSK 0,564964 0,0299477
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Cilengitide 0,548475 0,928076
Imidazole 0,445545 0,0527712
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Diminazene Aceturate Inhibits The Sars-Cov-2 Spike Protein-Induced Inflammation

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Diminazene Aceturate Inhibits The Sars-Cov-2 Spike Protein-Induced Inflammation

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Diminazene aceturate inhibits the SARS-CoV-2 spike protein-induced

inflammation involving leukocyte migration and DNA extracellular traps

*Corresponding authors: Elvira Saraiva (esaraiva@micro.ufrj.br); Lucas Nicolau

triggered acute inflammation, demonstrated by increasing leukocyte migration.

Keywords: COVID-19, Leukocyte, NETs, DETs, DIZE, Innate immunity.

diseases [17]. Chemically, it is a small molecule with a structural similarity to

SARS-CoV-2 infection, including pulmonary inflammation, cardiac damage, and gut

certain signalling pathways linked to cytokine production, suggesting that it may

Nanodrop (Thermo Fisher), silver-stained SDS-PAGE, and Western blot analyses,

protein was used dissolved in PBS, which was also endotoxin-free.

2.3. Animal experiments

2.4. Peritoneal model of acute inflammation

2.5. Resident macrophages depletion

Peritoneal macrophages were removed according to Ribeiro, Souza-Filho et al. [28]. In

2.6. Myeloperoxidase (MPO) activity

MPO is an enzyme predominantly found in the azurophilic granules of neutrophils, and

2.7. Malondialdehyde (MDA) levels

vessel. A leukocyte was considered adherent to the endothelium if it remained

2.11. Culture of human monocyte-derived macrophages (HMDM)

To obtain monocyte-derived macrophages, PBMCs were plated at 2x105 cells/well in

assayed in RPMI without serum.

2.12. NETs/DETs inhibition and quantification assays

Neutrophils, monocytes, and macrophages (2x105/well) in RPMI were incubated for 30

counterstained with DAPI (1 μg/mL; Sigma-Aldrich) for DNA staining. Coverslips

Zeiss Elyra PS.1 confocal microscope (Zeiss, Oberkochen, Germany).

2.15. Cytokines measurements

2.16. Molecular docking

crystallography with a resolution below 2 angstroms. However, exceptions were made

was analyzed by ONE way ANOVA and Holm-Sidak post-test. A significance

exudate was collected to determine leukocyte migration and myeloperoxidase (MPO)

3.3 Spk-induced acute peritonitis was mitigated by Diminazene aceturate (DIZE)

Information). To facilitate comparisons, structural simulations of aceturate were

structure. All these data are expressed on Fig. 5.

Additionally, Alkyl-π interactions occurred on residues ARG119, LYS280, and

amidine. Cilengitide exhibited a 92% electrostatic similarity, whereas PAD2-IN

19 and the involvement of DIZE as a pharmacological tool that mitigates inflammation

demonstrate the potential of BALB/c mice for investigating in vivo inflammatory

During the course of COVID-19 pandemic, drug repurposing grew on a widespread

[47,48]. A RECOVERY collaborative study with global importance showed notable

approach, suggesting its anti-inflammatory profile.

NET/DET formation in COVID-19.

PAD2, catalyse the conversion of arginine residues to citrulline, regulating activity of

the inflammatory response. Concerning chemical composition, DIZE exhibits structural

NETs/DETs in human neutrophils, monocytes, and macrophages. These results

associated with COVID-19.

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