A potent leukocyte transmigration blocker: Gt-73 showed a protective effect against lps-induced ards in mice

Article abstract of DOI:10.3390/molecules26154583

We recently developed a molecule (GT-73) that blocked leukocyte transendothelial migration from blood to the peripheral tissues, supposedly by affecting the platelet endothelial cell adhesion molecule (PECAM-1) function. GT-73 was tested in an LPS-induced acute respiratory distress syndrome (ARDS) mouse model. The rationale for this is based on the finding that the mortality of COVID-19 patients is partly caused by ARDS induced by a massive migration of leukocytes to the lungs. In addition, the role of tert-butyl and methyl ester moieties in the biological effect of GT-73 was investigated. A human leukocyte, transendothelial migration assay was applied to validate the blocking effect of GT-73 derivatives. Finally, a mouse model of LPS-induced ARDS was used to evaluate the histological and biochemical effects of GT-73. The obtained results showed that GT-73 has a unique structure that is responsible for its biological activity; two of its chemical moieties (tert-butyl and a methyl ester) are critical for this effect. GT-73 is a prodrug, and its lipophilic tail covalently binds to PECAM-1 via Lys536. GT-73 significantly decreased the number of infiltrating leukocytes in the lungs and reduced the inflammation level. Finally, GT-73 reduced the levels of IL-1β, IL-6, and MCP-1 in bronchoalveolar lavage fluid (BALF). In summary, we concluded that GT-73, a blocker of white blood cell transendothelial migration, has a favorable profile as a drug candidate for the treatment of ARDS in COVID-19 patients.

Full text of DOI:10.3390/molecules26154583

molecules
Article
A Potent Leukocyte Transmigration Blocker: GT-73 Showed a
Protective Effect against LPS-Induced ARDS in Mice
Eliav Blum 1 , Raanan Margalit 2, Laura Levy 1, Tamar Getter 1, Ron Lahav 3, Sofia Zilber 4, Paul Bradfield 5,
Beat A. Imhof 6 , Evgenia Alpert 3,* and Arie Gruzman 1,
*
1
Department of Chemistry, Faculty of Exact Sciences, Campus Ramat-Gan, Bar-Ilan University,
Ramat-Gan 5290002, Israel; eliavblum@gmail.com (E.B.); levylaura55@hotmail.com (L.L.);
tamargetter25@gmail.com (T.G.)
2
3
4
5
6
Science in Action, 3 Pinchas Sapir Street, Weizmann Science Park, Ness-Ziona 7403650, Israel;
raanan.margalit@gmail.com
AltA-ZuZ Therapeutics, 3 Pinchas Sapir Street, Weizmann Science Park, Ness-Ziona 7403650, Israel;
lahav.r@gmail.com
Department of Pathology, Shaare Zedek Medical Center, 12 Shmuel Bait Street, Jerusalem 9103102, Israel;
sofiazilber@szmc.org.il
MesenFlow Technologies, Chemin des Aulx, 14, CH-1228 Geneva, Switzerland;
paul.bradfield@mesenflow.com
Department of Pathology and Immunology, University of Geneva, Rue Michel-Servet, CH-1211 Geneva,
Switzerland; beat.imhof@unige.ch
*
Correspondence: genia.alp@gmail.com (E.A.); gruzmaa@biu.ac.il (A.G.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Abstract: We recently developed a molecule (GT-73) that blocked leukocyte transendothelial mi-
gration from blood to the peripheral tissues, supposedly by affecting the platelet endothelial cell
adhesion molecule (PECAM-1) function. GT-73 was tested in an LPS-induced acute respiratory dis-
tress syndrome (ARDS) mouse model. The rationale for this is based on the finding that the mortality
of COVID-19 patients is partly caused by ARDS induced by a massive migration of leukocytes to the
lungs. In addition, the role of tert-butyl and methyl ester moieties in the biological effect of GT-73
was investigated. A human leukocyte, transendothelial migration assay was applied to validate
the blocking effect of GT-73 derivatives. Finally, a mouse model of LPS-induced ARDS was used to
evaluate the histological and biochemical effects of GT-73. The obtained results showed that GT-73
has a unique structure that is responsible for its biological activity; two of its chemical moieties
(tert-butyl and a methyl ester) are critical for this effect. GT-73 is a prodrug, and its lipophilic tail
covalently binds to PECAM-1 via Lys536. GT-73 significantly decreased the number of infiltrating
leukocytes in the lungs and reduced the inflammation level. Finally, GT-73 reduced the levels of
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Blum, E.; Margalit, R.; Levy,
L.; Getter, T.; Lahav, R.; Zilber, S.;
Bradfield, P.; Imhof, B.A.; Alpert, E.;
Gruzman, A. A Potent Leukocyte
Transmigration Blocker: GT-73
Showed a Protective Effect against
LPS-Induced ARDS in Mice.
Molecules 2021, 26, 4583. https://
doi.org/10.3390/molecules26154583
Academic Editor: Kenneth H. Pearce
Received: 13 June 2021
Accepted: 22 July 2021
Published: 29 July 2021
IL-1β, IL-6, and MCP-1 in bronchoalveolar lavage fluid (BALF). In summary, we concluded that
GT-73, a blocker of white blood cell transendothelial migration, has a favorable profile as a drug
candidate for the treatment of ARDS in COVID-19 patients.
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Keywords: ARDS; leukocyte transmigration; cytokine storm; PECAM-1; covalent inhibitor
1. Introduction
COVID-19 is a new viral disease affecting many human organs, predominantly tar-
geting the respiratory system and the lungs. All of us are currently trying to survive this
deadly global pandemic that seriously challenges and limits public health systems around
the world. In about 20% of patients, severe symptoms occur after a mean incubation period
of five days. Furthermore, 5% of patients need intensive care therapy, including ventilation
support [1–3].
The main way of obtaining COVID-19 treatment is based on established recommen-
dations for treating patients with acute lung injury (ARDS) and “cytokine storm”. The
IL-6 level, in particular, should be reduced dramatically to block the severe results of
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Molecules 2021, 26, 4583. https://doi.org/10.3390/molecules26154583
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 4583
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the “cytokine storm” [
4]. Lung ventilation (protective and functional), anti-inflammatory
therapy, diuretics, and an adequate management of organ failures constitute the mile-
stones of therapy. In case of significant lung failure, venovenous extracorporeal membrane
oxygenation may also be used [5].
ARDS is a major reason for mortality in COVID-19 patients [6,7]. In advanced and
especially in the terminal stages of the disease, leukocytes accumulate in the lungs and oc-
cupy mainly the alveolar space, including the interstitium due to inflammation induced by
viral infection. This first leads to a “cytokine storm”, followed by a devastating destruction
of the alveolar tissues [
tissue; the adaptive immune cells are not yet effective in fighting the virus by antibodies or
the specific killing of infected host cells [ ]. Thus, blocking this process may successfully
8]. During this stage, the immune system mainly attacks the host
9
treat and reverse the effects of ARDS in COVID-19 patients, similar to the results obtained
with our in vivo animal model.
We recently developed a potent inhibitor of leukocyte transmigration from the blood
to the tissues (GT-73). This compound was also able to increase the reverse transmigration
of monocytes [10]. Compound GT-73, based on the scaffolding of barbituric acid, has
already shown highly promising results as a drug candidate for the treatment of other
inflammatory and autoimmune disorders (e.g., Crohn’s disease, multiple sclerosis, fatty
liver disease, inflammatory bowel disease, and rheumatoid arthritis). This was achieved
without any toxic effects on human endothelial cells in vitro. In addition, GT-73 was not
toxic in vivo, even when it was used at suprapharmacological doses.
Despite these promising preliminary results, GT-73’s suggested mechanism of action
(blocking PECAM-1 activity) has not been elucidated. We previously reported that accord-
ing to in silico calculations, GT-73 might affect PECAM-1 activity. PECAM-1 is one of the
vascular adhesion molecules that play an important role in leukocyte transendothelial mi-
gration during inflammation and cancer [11–13]. PECAM-1 not only resides at endothelial
junctions, but it is also a constituent of a recycling compartment of endothelial cells, termed
the lateral border recycling compartment (LBRC), which most likely provides PECAM-1
molecules to junctions during leukocyte transmigration [14,15]. It was shown that high
levels of PECAM-1 lead to increased transendothelial migration of immune cells from
blood to organs [16–19].
Here, we describe the substantial therapeutic effect of GT-73 in LPS-induced ARDS
in mice. GT-73 was administered intranasally and had a significant positive effect on the
severity of the “cytokine storm” and on lung morphology. We also found that PECAM-1 is
indeed a primary target of GT-73 activity.
Finally, two critical residues for the biological effect of moieties on GT-73 were identi-
fied: tert-butyl and methyl ester. The absence of these functional groups in the structure of
GT-73 completely abolishes its biological activity.
2. Results
2.1. Structure–Activity Relationships (SARs) and a Possible Mechanism Underlying GT-73 Action
In our previous publication, we reported the screening results (the blocking of leuko-
cyte transendothelial migration) of a series of lipophilic compounds [10]. We carefully
studied the structural differences between the nonblocking (GT-77) and the completely
blocking (GT-73) compounds [10]. There are two differences between these molecules:
(1) the addition of the tert-butyl group on the central aromatic ring and (2) the replacement
of carboxylic acid with an ester moiety by the addition of a methoxy group. Thus, the
importance of both of these chemical moieties in enhancing the biological activity was
investigated.
Two novel compounds (EB-237 and EB-269) were synthesized to determine the im-
portance of the two structural moieties of GT-73 (methyl ester and tert-butyl) in enhanc-
ing the biological effect. The synthesis was initiated from the commercial materials 4-
(chloromethyl) benzoic acid and 4-(bromomethyl) benzoic acid, respectively, using Kno-
evenagel condensation [20] to obtain EB-237 and EB-269, respectively, as described in

Molecules 2021, 26, 4583
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Supplementary Schemes S1 and S2. Both compounds were tested for their ability to bridge
the gap between GT-77 and GT-73 [10]. Therefore, we added to the basic scaffold (GT-77)
only one of the supposed functional groups at that time to obtain compounds EB-237 and
EB-269. Next, the effect of both compounds GT-73 and its fluorinated version termed EB-
251 (Chart 1 and Supplementary Scheme S2) on the transendothelial migration of primary,
human monocytes was investigated under flow conditions using inflammatory, TNF-α-
activated HUVECs. GT-73 and EB-251 exhibited a significant time-dependent inhibition of
monocyte transmigration from two independent donors (Figure 1). In contrast, the novel
modified compounds EB-237 and EB-269 did not block this process (Figure 1).
O
O
HN
NH
HN
NH
O
O
O
O
O
O
O
O
O
OH
Compound GT-73
Compound GT-77
EB-251
Compound 6
EB-237
Compound 3
EB-269
Compound 8
Chart 1. Structures of the synthesized compounds.
Next, GT-73’s mechanism of action was investigated. According to in silico calcula-
tions, the protein affected by GT-73 during monocyte transmigration may be PECAM-1 [10].
Furthermore, we hypothesized that GT-73 could act as a prodrug that binds covalently
to PECAM-1. Therefore, the nonstable bond between barbituric acid and the lipophilic
moiety might be cleaved by a nucleophilic attack of the primary amine of a lysine located
in PECAM-1. Such an attack will result in the formation of a covalent imine bond between
the amino acid of the protein and the lipophilic moiety of GT-73 (Supplementary Chart S1)
.
To test this hypothesis, we used mass spectroscopy. To this end, recombinant human
PECAM-1 was incubated with GT-73. However, under these conditions, the imine bond
would be cleaved, and identifying a covalently attached fragment to PECAM-1 would have
been impossible. To avoid this scenario, we added sodium borohydride to the reaction mix
before trypsinization, which induces the reduction of the imine bond to a secondary amine,
as shown in Supplementary Chart S1. Secondary amines are stable under acidic pH condi-
tions when PECAM-1 is trypsinized as a part of the sample preparation for the mass spec
analysis. When inspecting the protein sequence of PECAM-1, we could identify a lipophilic
tail of GT-73 as a covalent adduct to Lys536, as shown in Supplementary Figure S1.

Molecules 2021, 26, 4583
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A
1400
1200
1000
800
600
400
200
0
1600
1400
1200
1000
800
600
400
200
0
*
*
*
DMSO:
EB-237:
*
DMSO:
*
GT-73:
0
10
20
30
40
0
10
20
30
40
Time (min)
Time (min)
1400
1200
1000
800
600
400
200
0
1400
1200
1000
800
600
400
200
0
*
DMSO:
EB-269:
DMSO:
* *
*
EB-251:
0
10
20
30
40
0
10
20
30
40
Time (min)
Time (min)
B
1400
1200
1000
800
600
400
200
0
DMSO:
EB-237:
1400
1200
1000
800
600
400
200
0
DMSO:
GT-73:
*
30
*
20
*
40
*
*
0
10
0
10
20
30
40
Time (min)
Time (min)
1400
1200
1000
800
600
400
200
0
DMSO:
1400
1200
1000
800
600
400
200
0
DMSO:
EB-251:
EB-269:
*
*
*
*
*
0
10
20
30
40
0
10
20
Time (min)
30
40
Time (min)
Figure 1. Monocyte transmigration (absolute number) on the surface of the activated HUVECs under flow. Primary human
monocytes were cocultured on TNF- -activated HUVECs. Test compounds at 30 M or DMSO (0.1%) were added to cells
α
µ
for 45 min. Cocultures were kept under a flow of 0.05 Pa. Mean ± SE, * p ≤ 0.05. (A) First donor. (B) Second donor.
2.2. In Vivo Investigation
Knowing that GT-73 can covalently bind to PECAM-1 and that it most likely affects
its function, we tested GT-73 in a pathology model that leads to severe/terminal stages
of COVID-19: ARDS. During the development of ARDS, leukocytes migrate into the
lungs, which is accompanied by a “cytokine storm”. We used BALB/c female mice and
induced ARDS with intratracheal instillation of LPS. Treatment with GT-73 and vehicle,
also administered intratracheally, began 3 h after LPS application. The experiment was
then terminated at 72 h. The effect of GT-73 on the number of infiltrating leukocytes in the
lungs and the cytokine levels in bronchoalveolar lavage fluid (BALF) were analyzed, as

Molecules 2021, 26, 4583
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shown in Figure 2A,B, respectively. GT-73 significantly reduced the number of infiltrating
leukocytes and significantly reduced the levels of IL-1β, IL-6, and MCP-1 in BALF.
Figure 2. In vivo effect of GT-73. (
Female BALB/c mice were anesthetized and orally intubated with a catheter and challenged with
intratracheal instillation LPS (800 g) in PBS. Mice were euthanized at 3 d after LPS challenge to
A) Effect of GT-73 on the number of leukocytes in the lungs.
µ
collect tissues for analysis. All animals from the treatment group were administered GT-73 (5 mg/kg
in 1% methyl cellulose) for 3 h after LPS application by the intranasal route 3 times a day. Lungs were
collected in 10% buffered formalin, embedded in paraffin, and cut into sections. Sections were stained
with hematoxylin/eosin, and images were taken with an Olympus BX53 microscope. (
B) Samples of
BALF were centrifuged. The supernatant was flash-frozen, and the cytokine levels were measured.
n = 7, MEAN
±
SD, significant difference between treated by GT-73 and vehicle groups, #
0.0001. Significant difference between treated by GT-73 and model control
p
≤
0.004,
##
p
≤
0.003, and ###
p
≤
groups ** p ≤ 0.003, and *** p ≤ 0.0001.
To evaluate the potential role of GT-73 on the histopathological changes in the lungs,
we produced paraffin-embedded tissue sections and stained them with hematoxylin/eosin.
The inflammatory infiltrates, interalveolar septal thickening, and interstitial edema were
evaluated (Figure 3A–D). Administration of GT-73 efficiently reduced the airspace inflam-
mation. Furthermore, the severity of lung injury was also assessed using a semiquantitative
histopathology score system, which evaluates lung injury according to four categories:
alveolar septae, alveolar hemorrhage, intra-alveolar fibrin, and intra-alveolar infiltrates.
Vehicle and LPS control groups exhibited a prominent neutrophilic infiltrate in the
alveolar space and the interstitial septa, but the extent was variable both within and
across lobes and in neutrophilic debris deposits, numerous lymphocytes, and macrophages
(Figure 3E–K). The ARDS induction affected the air–blood barrier, leading to a progressive
breakdown of the capillary walls and the epithelial integrity, which permitted leakage
of protein-rich edema fluid (interstitial fluid and plasma proteins) into the alveoli. This
proteinaceous fluid presented itself as a lightly eosinophilic material that ranges from ho-
mogeneous to fibrillar (i.e., fibrin strands) in appearance in the H&E-stained sections and is
present to a variable extent in all specimens. Reactive pneumocytes showed desquamation
into alveoli. All specimens collected from the GT-73-treated group showed a diffuse distri-

Molecules 2021, 26, 4583
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bution of ill-defined low-density inflammatory lesions, with mild to moderate neutrophilic
infiltrate into the alveolar space and interstitial septae, but without neutrophilic debris
deposits or lymphocytes; however, there was a higher number of foaming macrophages (in-
dicating the start of the repair phase). In contrast, specimens collected from nontreated mice
exhibited huge amounts of neutrophilic debris, high numbers of lymphocytes, and almost
no macrophages. In addition, in the treated mice we detected minimal numbers of focal
reactive pneumocytes with desquamation into alveoli. Overall, we found that treatment
with GT-73 significantly reduced the number of these lung injury scores (Figure 3K).
Figure 3. Histological evaluation of the lung inflammation level. Slides were prepared and processed as described in
Figure 2. Images of whole-mount lungs were taken at low magnification ( 20): ( ) naive mice; ( ) untreated model control
(LPS); ( ) vehicle-treated lungs; ( ) GT-73-treated lungs. Images were taken at high magnification ( 400). Upper panel,
vehicle-treated lungs: ( ) fibrin deposits; ( ) septal and alveolar neutrophils infiltration. ( ) neutrophilic debris deposits.
Lower panel, GT-73-treated lungs: ( ) alveolar neutrophil infiltration and foaming macrophages; ( ) septal neutrophil
infiltration. ( ) normal lungs (naïve); ( ) Lung injury histological score, n = 7, mean SD, significant difference between
0.003. Significant difference between treated by GT-73 and the model control
×
A
B
C
D
×
E
F
G
H
I
J
K
±
treated by GT-73 and the vehicle groups, ## p
groups ** p ≤ 0.003.
≤
3. Discussion
The presented results substantially enhance our knowledge about GT-73’s mechanism
of action and elucidate its function in a COVID-19-relevant inflammatory pathology. It
was important to confirm that the chemical structure of GT-73 has two functional moieties:
tert-butyl and methyl ester, both of which are essential for maintaining the biological
activity. Only the replacement of hydrogen by a fluorine atom in the second aromatic
ring left the activity intact. The absence of both functional groups in the GT-73 structure
led to the complete loss of biological activity. These results might indicate that these two
lipophilic groups are required for the binding between GT-73 and the targeted protein.
This hypothesis will be investigated in future work.

Molecules 2021, 26, 4583
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We determined, as an adduct to the PECAM-1 sequence, only part of GT-73. Therefore,
GT-73 is a classical prodrug and disintegrates after the nucleophilic attack of lys536 in
PECAM-1 into two molecules: one is a leaving group (barbituric acid), and the other is the
lipophilic alkyl tail, which covalently binds to the lysine by forming an imine bond. Imine
bonds can undergo hydrolysis; thus, the covalent inhibition of PECAM-1 is reversible. This
may be of importance for therapy and it explains the total absence of toxicity, which was
previously reported by our laboratories [10]. The covalent addition of a lipophilic moiety
to the extracellular domain of PECAM-1 (lys536 is located extracellularly) may be critical
for its function, and it should seriously interrupt the function of PECAM-1, an important
adhesion molecule in inflammation-driven leukocyte migration. It might be the basis for
the therapeutic effect of GT-73, which we observed in the treatment of ARDS and in other
in vivo autoimmune disease models [10].
ARDS contributes to COVID-19-induced patient death. Older age and additional
comorbidities increase the rate of mortality from ARDS [21]. ARDS in patients begins with
an augmented permeability of alveolar capillaries for proteins such as albumin, fibrinogen,
proinflammatory cytokines, and coagulation factors [22]. The next step is the massive
transendothelial migration of inflammatory leukocytes, including neutrophils to the lungs.
Such a massive accumulation of white blood cells leads to a severe local inflammatory
response, a “cytokine storm”, or fibrin deposition; all these factors induce the progression
of respiratory dysfunction and heart failure [23]. Recently, suppression of the accumulation
of white blood cells in the lungs of COVID-19 patients by dexamethasone (a glucocorticoid
that was recently shown to be the first life-saving drug in this disease) was recommended as
a mandatory part of the treatment because no other therapies have shown a positive effect
during the late or terminal stages of the disease [24]. Here and in our previous publication,
we showed that GT-73 inhibits the transmigration of white blood cells [10]. This led to
a significantly improved lung morphology after induction of ARDS by LPS. In treated
mice, the lungs were cleared from neutrophils and cell debris. GT-73 caused a significant
reduction in the number of infiltrated leukocytes but increased the number of macrophages.
The latter indicates that the recovery stage in the local inflammatory response had already
started. It is known that macrophages play different roles in inflammation, depending on
their differentiation stages. In the first (the acute) phase, alveolar macrophages resemble
an activated, inflammatory phenotype (M1 in humans); they secrete proinflammatory
mediators and factors. In the later phase, the M1 phenotype changes to a rather M2 anti-
inflammatory phenotype, and this change allows them to eliminate apoptotic cells, cell
debris, and induces the replacement of the dead alveoli by connective tissues [25,26].
Cytokines are deeply involved in the development of inflammation [27,28]. During
the onset of an antiviral response, cytokines such as interferons play an important role
in the induction of an anti-inflammatory response, but at later stages, other types of
cytokines in critical-condition patients can lead to severe tissue damage, as they induce
the release of toxic reagents by leukocytes such as cytotoxic reactive oxygen species [29].
Therefore, reducing the extent of the “cytokine storm” is a mandatory step in the treatment
of COVID-19 patients. It was recently reported that in patients with severe COVID-19
the levels of IL-2, IL-7, IL-10, GSCF, IP10, MCP-1, MIP1A, and TNF-
elevated [30]. In addition, the proinflammatory cytokines IL-6 and IL-1
α
are dramatically
are also elevated
β
in ARDS patients. This leads to a higher expression of cell adhesion molecules by vascular
endothelium and VEGF secretion. The latter augments the vascular permeability of the
lung endothelium, allowing quick viral dissemination; the former induces a massive
transmigration of neutrophils and inflammatory monocytes from the blood to the lungs. IL-
6 and IL-1β also stimulate the proliferation and differentiation of hematopoietic progenitors
in bone marrow. As immature granulocytes, they then can go directly to the struggling
lungs and further increase local inflammation, which can lead to ARDS. This chain of
events in the lung-systemic loop is responsible for the cytokine storm syndrome [31]. GT-73
exhibited significant inhibitory effects on the cytokine level of three cytokines: IL-1β, IL-6,

Molecules 2021, 26, 4583
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and MCP-1 in the lungs. However, this effect might turn into an important advantage in
the fight against the cytokine storm associated with ARDS.
4. Materials and Methods
All chemical reagents, solvents, and acids were purchased from Acros Organic (Fair
Lawn, NJ, USA), Alfa Aesar (Ward Hill, MA, USA), Bio-Lab Ltd. (Jerusalem, Israel), and
Merck (Rehovot, Israel), and all were used as received. LPS from Salmonella enteritidis and
methyl cellulose were from Merk (Rehovot, Israel). Anhydrous tetrahydrofuran (THF) was
obtained by distillation from a boiled blue mix containing sodium (1% w/v) and benzophe-
none (0.2% w/v). Column chromatography was performed on silica gel 60 (230–400 mesh
;
Merck, Darmstadt, Germany). Analytical and preparative high-performance liquid chro-
matography (HPLC) (Young Lin Instruments, Anyang, South Korea) were performed on
LUNA C18 preparative (10
µm, 100
×
30 mm) or analytical (5
µm, 250
×
4.6 mm) columns,
both from Phenomenex, Inc. (Torrance, CA, USA). HPLC purification was carried out with
an increasing linear gradient of CH3CN in H2O. The purity of the synthesized compounds
was confirmed by HPLC analysis and elemental analysis. Analytical thin-layer chromatog-
raphy (TLC) was carried out on pre-coated silica gel 60 F254 (Merck, Darmstadt, Germany)
sheets using UV absorption and iodine physical adsorption for visualization. Mass spec-
tra were recorded on a Finnigan Model 400 instrument using a QToF microspectrometer
(Micromass, Milford, MA, USA), using electrospray ionization (ESI) in the positive ion
mode. Data were processed using mass L-ynX ver. 4.1 calculation and deconvolution
software (Waters Corp., Milford, MA, USA). High-resolution mass spectra (HRMS) were
obtained using an LTQ Orbitrap XL (Thermo Scientific, Waltham, MA, USA). Melting
points were measured with a Fisher-Johns melting point apparatus (Waltham, MA, USA).
The 1H NMR and 13C NMR were recorded at room temperature on a Bruker AVANCE
NMR spectrometer (Vernon Hills, IL, USA) operating at 300, 400, 600, and 700 MHz and
were in accordance with the assigned structures. Chemical shift values were reported
relative to tetramethylsilane (TMS), which was used as an internal standard. Chemical
shifts were expressed in
δ (ppm) and coupling constants (J) were in hertz. The splitting
pattern abbreviations are as follows: s, singlet; d, doublet; t, triplet; q, quartet; quint,
quintet; m, unresolved multiplet due to the field strength of the instrument; dd, doublet of
doublets.
4.1. In Vivo Study
4.1.1. Mice
Female BALB/c mice, 6–8 weeks old, were purchased from Envigo (Jerusalem, Israel).
4.1.2. Murine Model of LPS Induced ARDS
The murine model of LPS-induced ARDS was established as reported19. Briefly,
female BALB/c mice (n = 8 per group) were anesthetized and orally intubated with a sterile
plastic catheter and challenged with an intratracheal instillation of 800
µg of LPS (E. coli
055:B5; Merck, Rehovot, Israel) dissolved in 50 L of normal PBS. Naïve mice (without LPS
µ
instillation) were injected with the same volume of pyrogen-free PBS to serve as controls.
Mice were euthanized 3 d after LPS challenge to collect tissues for analysis.
All animals from the treatment group were administered 20
concentration in 1% methyl cellulose by intranasal route 3 times a day 3 h after LPS
application. All animals from the vehicle control group were administered 20 L of 1%
µL of GT-73 at a 10 mg/mL
µ
methyl cellulose by intranasal route 3 times a day 3 h after LPS application. Finally, animals
from the LPS control group were used as an experimental nontreated model control. Naïve
animals were used as a baseline control. The drug candidate was administered at a constant
volume dosage for 72 h following LPS administration.

Molecules 2021, 26, 4583
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4.1.3. Tissue Collection
Samples of Bronchial Alveolar Lavage fluid (BALF) were collected from each mouse
from all groups; they were centrifuged for supernatant and cell pellet separation. The
supernatant was snap-frozen to determine the cytokine levels, and cells were used for FACS
white cell differentiation. Lungs were collected and filled with 10% buffered formalin,
embedded in paraffin, and cut into 4
hematoxylin/eosin (H&E), and images were taken with an Olympus BX53 microscope
(400 ). For the lung injury score, images were evaluated by an investigator who was
µm thick sections. Sections were stained with
×
blinded to the identity of the slides. In brief, the extent of the pathological lesions was
graded from 0 to 3 (see Table 1). The score for each animal was calculated by dividing the
total score by the number of sections observed.
Table 1. Histological score.
Intra-Alveola
Infiltrations per Field
Score
Alveolar Septae
Alveolar Hemorrhage
Intra-Alveolar Fibrin
Less than 5
intra-alveolar cells
0
1
2
All are thin and delicate
No hemorrhage
No intra-alveolar fibrin
Congested alveolar septae in
less than 1/3 of the field
Erythrocytes per alveolus in
1 to 5 alveoli
Fibrin strands in less
than 1/3 of the field
5 to 10
intra-alveolar cells
Congested alveolar septae in
1/3 to 2/3 of the field
At least 5 erythrocytes per
alveolus in 5 to 10 alveoli
Fibrin strands in 1/3 to
2/3 of the field
10 to 20
intra-alveolar cells
At least 5 erythrocytes per
alveolus in more than
10 alveoli
Congested alveolar septae in
more than 2/3 of the field
Fibrin strands in more
than 2/3 of the field
More than 20
intra-alveolar cells
3
4.1.4. Histological Evaluation of the Lung Inflammation Level
The histological analysis was conducted based on the criteria listed in Table 1 [32].
4.2. In Vitro Human Monocyte Trafficking Assay
Leukocyte trafficking assays were conducted as described previously. [10] Briefly,
human umbilical vein endothelial cells (HUVECs) were isolated by collagenase treatment
of umbilical veins and maintained (passages 2–4) in Medium 199 containing 10% FCS,
15
µg/mL endothelial cell growth supplement (Upstate Biotechnology, Lake Placid, NY,
USA), 100
µ
g/mL heparin, and 50 M hydrocortisone. For the flow assays, HUVECs were
µ
cultured after a maximum of 3–5 passages in chamber slides (m-slide VI 0.1 ibiTreat, IBIDI),
previously coated with 0.2% gelatin (Merck, Darmstadt, Germany) and 1 mg/mL Collagen
G (Biochrom AG, Berlin, Germany) for 2–3 days and then treated for 24 h with acute
or chronic inflammation protocols using tumor necrosis factor
alone or in combination with interferon (IFN ), at 500 U/mL for chronic inflammatory
protocols. [10] The compounds to be tested and the leukocytes were added at 30 M.
α (TNF-α) at 1000 U/mL
γ
γ
µ
Monocytes were purified from EDTA blood collected from healthy donors using Ficoll
gradients and negative selection kits (Miltenyi Biotec, Bergisch Gladbach, Germany) for
those types of cells. The slides containing the cultured HUVEC monolayers were then
◦
mounted to a flow system set at 37 C, and flow was generated over the HUVEC monolayer
by perfusing a wash medium or a medium containing the leukocyte suspension using a
calibrated pump. The flow rate, expressed as a measurement of shear stress, represented
small venules/capillaries (0.05 Pascal (Pa)). The first stage of the assay was a washing
procedure, whereby the wash buffer was perfused over the HUVECs for 10 min. Wash
buffer was then perfused over the HUVECs for 10 min to remove unbound chemokines,
before purified leukocytes were perfused over the HUVECs for 5 min, followed by 45 min
with wash buffer.
Adhesion events were recorded as the total number of cells per unit field (mm2).
Transmigration events are presented as a percentage of the total leukocytes captured

Molecules 2021, 26, 4583
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from flow per unit field. All experiments were carried out using triplicate fields and are
presented as a mean value with +standard error measurements (+SEM). Statistical analyses
assume a parametric distribution and were conducted using Student’s t-test.
4.3. Cytokine Level Determination
The levels of IL-1β, IL-6, and MCP-1 were determined using commercially available
ELISA analytical kits (Merck, Rehovot, Israel). The assays were conducted according to the
protocols supplied by the manufacturer.
4.4. Synthetic Chemistry
The synthesis and the analytical data for the obtained molecules are presented in the
Supplementary Materials.
4.5. Mass Spectroscopy
Human recombinant PECAM-1(Seale, WA, USA) (25
µg) was dissolved in PBS buffer,
(Merck, Rehovot, Israel) (30 L). GT-73 was added to the solution in 0.5
µ
µL of DMSO to
reach a final concentration of 30 µM. The reaction was incubated under gentle agitation for
40 min at 37 ◦C. Then, NaBH4 in PBS (20 µL) was added to obtain a final concentration of
◦
100 mM, and the reaction mix was kept for an additional incubation time (1 h, at 37 C). The
sample was digested by trypsin, analyzed by LC MS/MS on Q-Exactive (Thermo Fisher,
Waltham, MA, USA), and identified by Discoverer software against Uniprot unspecific
and specific databases. All the identified peptides were filtered with high confidence,
top rank, and mass accuracy. High confidence peptides passed the 1% FDR threshold
(*FDR = false discovery rate; it is the estimated fraction of false positives in a list of peptides).
Semiquantitation was performed by calculating the peak area of each peptide.
5. Conclusions
We recently developed an inhibitor of leukocyte transmigration from the blood to
the peripheral tissues (GT-73). Here, we showed that GT-73 has a unique structure, which
is responsible for its biological activity, and two of its chemical moieties (tert-butyl and a
methyl ester) are critical for this effect. In addition, we showed that GT-73 is a prodrug,
and its lipophilic tail covalently binds to PECAM-1 via Lys536, forming an imine bond.
In an LPS-induced ARDS mouse model, GT-73 was given intranasally during the
induction phase of ARDS (3 h after the intranasal administration of LPS), with encouraging
results. Specifically, all specimens collected from treated animals by the compound group
showed a diffuse distribution of ill-defined low-density inflammatory lesions, with mild to
moderate neutrophilic infiltrate into the alveolar space and interstitial septa, but without
neutrophilic debris deposits or lymphocytic infiltrates, as well as a higher number of foam-
ing macrophages (an indication of a repair phase). In contrast, specimens collected from
untreated mice exhibited huge amounts of neutrophilic debris and lymphocytic infiltrates
and almost no macrophages. In addition, the treated mice exhibited only minimal amounts
of reactive pneumocytes with focal desquamation into the alveolar space, compared with
untreated mice, where the number of reactive pneumocytes and desquamation was much
higher. Finally, GT-73 significantly reduced the number of infiltrating leukocytes and
reduced the level of a “cytokine storm”, manifested by lower levels of IL-1
β, IL-6, and
MCP-1 in bronchial lavage fluid.
6. Patent
The GT-73 structure and its use as a potential therapeutic agent are protected by a
patent: Gruzman et al. (2019). “Novel barbituric acid-based leucocyte transmigration
inhibitors as drug candidates for treating inflammatory diseases, autoimmune diseases
and cancer”. WO2019043706A1.

Molecules 2021, 26, 4583
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Supplementary Materials: The following are available online. The synthetic chemistry part, PECAM-
1 covalent modification by GT-73, analytical chemistry data, and the purity level of the synthesized
compounds are available online, Scheme S1: Synthetic pathway of ester derivative 3 (EB-237),
Scheme S2: Synthetic pathway of fluorinated derivative 6 (EB-251), Scheme S3: Synthetic pathway of
carboxylic acid derivative 8 (EB-269), Chart S1: Schematic representation of the formation of an imine
bond between GT-73 and Lys536 in PECAM-1, Figure S1: Mass spec determination of the covalent
addition of a GT-73 fragment to PECAM-1.
Author Contributions: E.A., R.M., R.L., P.B., T.G., B.A.I. and A.G. designed the study. E.B., E.A.,
L.L., T.G. and S.Z. performed the experiments and the data analyses. A.G., B.A.I., E.B., E.A. and
P.B. prepared the manuscript. T.G., E.A., P.B. and B.A.I. brought important intellectual content. All
authors have read, reviewed, and approved the final submitted manuscript and agree to take public
responsibility for the published results.
Funding: This research was funded by a Bar-Ilan University new faculty grant (to A.G.), Grant
Number 182009.
Institutional Review Board Statement: Mice were handled according to the regulations formulated
by the Israel Animal Care and Use Committee (Israel Ministry of Health). All experiments were
approved by the Israeli Ministry of Health (the Animal Care Committee) and were conducted
according to the Israeli National Veterinary Law.
Informed Consent Statement: Two healthy donors donated several milliliters of blood for the
monocyte transmigration in vitro experiments.
Data Availability Statement: All supplemental data can be found at MDPI. In addition, we will
share milligram amounts of GT-73 for biological tests. Any other data will also be made available to
any investigator upon request.
Acknowledgments: We thank Steven Manch for the English editing and Nir London from the
Weizmann Institute of Science, Rehovot, Israel, for his advice to use sodium borohydride to stabilize
the covalent binding between GT-73 and PECAM-1. We also thank Marina Vainberg and Alexander
Vainberg for providing the space and the scientific equipment/environment for this work.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: All supplemental data can be found at MDPI. In addition, we will share
milligram amounts of GT-73 for biological tests. Any other data will also be made available to any
investigator upon request. All other samples of the synthetized compounds also are available from
the authors.
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