Synthetic Glycolipids as Molecular Vaccine Adjuvants: Mechanism of Action in Human Cells and in Vivo Activity

Article abstract of DOI:10.1021/acs.jmedchem.1c00896

Modern adjuvants for vaccine formulations are immunostimulating agents whose action is based on the activation of pattern recognition receptors (PRRs) by well-defined ligands to boost innate and adaptive immune responses. Monophosphoryl lipid A (MPLA), a detoxified analogue of lipid A, is a clinically approved adjuvant that stimulates toll-like receptor 4 (TLR4). The synthesis of MPLA poses manufacturing and quality assessment challenges. Bridging this gap, we report here the development and preclinical testing of chemically simplified TLR4 agonists that could sustainably be produced in high purity and on a large scale. Underpinned by computational and biological experiments, we show that synthetic monosaccharide-based molecules (FP compounds) bind to the TLR4/MD-2 dimer with submicromolar affinities stabilizing the active receptor conformation. This results in the activation of MyD88- and TRIF-dependent TLR4 signaling and the NLRP3 inflammasome. FP compounds lack in vivo toxicity and exhibit adjuvant activity by stimulating antibody responses with a potency comparable to MPLA.

Full text of DOI:10.1021/acs.jmedchem.1c00896

pubs.acs.org/jmc
Article
Synthetic Glycolipids as Molecular Vaccine Adjuvants: Mechanism of
Action in Human Cells and In Vivo Activity
Fabio A. Facchini, Alberto Minotti, Andrea Luraghi, Alessio Romerio, Nicole Gotri,
Alejandra Matamoros-Recio, Andrea Iannucci, Charys Palmer, Guanbo Wang, Rebecca Ingram,
Sonsoles Martin-Santamaria, Grisha Pirianov, Marco De Andrea, Miguel A. Valvano, and Francesco Peri*
Cite This: J. Med. Chem. 2021, 64, 12261−12272
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ABSTRACT: Modern adjuvants for vaccine formulations are immunosti-
mulating agents whose action is based on the activation of pattern
recognition receptors (PRRs) by well-defined ligands to boost innate and
adaptive immune responses. Monophosphoryl lipid A (MPLA), a detoxified
analogue of lipid A, is a clinically approved adjuvant that stimulates toll-like
receptor 4 (TLR4). The synthesis of MPLA poses manufacturing and
quality assessment challenges. Bridging this gap, we report here the
development and preclinical testing of chemically simplified TLR4 agonists
that could sustainably be produced in high purity and on a large scale.
Underpinned by computational and biological experiments, we show that
synthetic monosaccharide-based molecules (FP compounds) bind to the
TLR4/MD-2 dimer with submicromolar affinities stabilizing the active
receptor conformation. This results in the activation of MyD88- and TRIF-dependent TLR4 signaling and the NLRP3
inflammasome. FP compounds lack in vivo toxicity and exhibit adjuvant activity by stimulating antibody responses with a potency
comparable to MPLA.
arm via the endocytic pathway activates interferon regulatory
factors and secondary NF-κB activation,13 playing an important
role in the stimulation of early T-cell responses.14 TRIF-
dependent type I IFN is a central mechanism for TLR4-
mediated adjuvant effects on T cell priming by TLR4 agonists.15
Moreover, the activation of the NLRP3 inflammasome and
subsequent caspase-1 activation and release of interleukin-1β
(IL-1β) is associated with the potent adjuvant effect by
particulate adjuvants, such as alum, chitosan, and QuilA/
saponin.16,17 IL-1β exerts multiple effects on the immune
system,18 which include promoting the differentiation of Th17
cells.19
Lipid A, the component of LPS that directly binds TLR4/
MD-2 is one of the most potent immune-stimulating agents
known. The toxicity of lipid A makes it unsuitable for safe use in
humans but the monophosphoryl lipid A (MPLA, Figure 1),20 a
lipid A derivative in which the anomeric phosphate has been
removed, is an effective adjuvant used in various approved
vaccines.21,22 MPLA and aminoalkyl glucosaminide phosphates
INTRODUCTION
■
Modern subunit vaccines, based on purified or synthetic
antigens that are often poorly immunogenic, require combina-
tion with adjuvants for optimal immune responses.1 Molecular
adjuvants are single-molecule innate immune stimulants that
enhance the adaptive immune response against antigens.
Adjuvants activate antigen-presenting cells, such as dendritic
cells and macrophages. These cells express pathogen recognition
receptors that, when activated, initiate immune responses
leading to the priming of T cells.2 Pathogen-associated
molecular patterns (PAMPs), the ligands of pathogen
recognition receptors, can be exploited as molecular adjuvants.
Many well-defined PAMPs have been explored, most of them
targeting the toll-like receptors (TLRs),3 C-type lectins,4 and
nucleotide-binding oligomerization domain (NOD)-like recep-
tors.5 Since TLR4 stimulation plays a key role in initiating rapid
innate immune responses, TLR4 agonists are promising
candidates to develop vaccine adjuvants6−10 and cancer
immunotherapeutics.11 Lipopolysaccharide (LPS)-stimulated
activation of TLR4 promotes the formation of the [TLR4/
MD-2/LPS]2 membrane dimer,12 which interacts with two
important adaptor protein molecules: MyD88 and TRIF.
Signaling through the MyD88-dependent pathway results in
rapid activation of NF-κB and mitogen-activated protein kinase
(MAPK), both of which drive pro-inflammatory gene expression
and cytokine production. Stimulation of the TRIF-dependent
Received: May 18, 2021
Published: August 12, 2021
© 2021 The Authors. Published by
American Chemical Society
https://doi.org/10.1021/acs.jmedchem.1c00896
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present here a preclinical study on the new synthetic TLR4
agonists FP11 and FP18 (compound FP111 turning out to be
inactive), their synthesis, computational studies, in vitro binding
studies with the TLR4/MD-2 dimer, cell studies on the
mechanism of action and TLR4 pathways activation, and in
vivo assessment of their adjuvant potency, compared to FDA-
approved MPLA, in an ovalbumin (OVA) vaccination model.
RESULTS AND DISCUSSION
■
Synthesis of FP Molecules. Compounds FP11, FP18, and
FP111 were synthesized through a protocol previously
developed and optimized for the preparation of TLR4
antagonists of the FP series.32 The synthesis for FP11 and
FP18 (Scheme 1) was carried out starting from the
commercially available D-glucosamine hydrochloride, following,
in part, a previously published protocol consisting of the
transformation of the amine at C2 into azide (2), followed by
protection of sugar’s C4−C6 positions as p-methoxybenzylidene
(3), silylation of the anomeric position (4), and Staudinger
hydrolysis of azide into amine (5).32 Compound 5 was acylated
with myristoyl chloride or lauroyl chloride to obtain,
respectively, compound 6 or 7. Regioselective opening of the
p-methoxybenzylidene by means of sodium cyanoboronhydride
(NaBH3CN) in trifluoroacetic acid (TFA) gave C6 p-
methoxybenzyl (PMB)-protected compounds 8 and 9, whose
acylation with myristoyl chloride or lauroyl chloride, respec-
tively, afforded triacylated sugars 10 and 11. Cleavage of the
anomeric silane with tetrabutylammonium fluoride (TBAF),
followed by the reaction with phosphoramidite and one-pot
oxidation with m-chloroperbenzoic acid (m-CPBA) gave
compounds 14 and 15. Catalytic hydrogenation, followed by
the treatment with sodium cation exchange resin gave the final
compounds FP11 and FP18. Similarly, FP111 was synthesized
starting from the intermediate compound 13 by removal of the
PMB group at C6 through catalytic hydrogenation (16),
simultaneous phosphorylation of sugar’s C6 and C1 (17), and
final removal of benzyl groups again by catalytic hydrogenation.
In Vitro Binding Tests: FP11 and FP18 Bind to Human
MD-2. Direct interaction of FP11 and FP18 with human TLR4/
MD-2 dimer was investigated by surface plasmon resonance
(SPR) analysis. The recombinant human TLR4/MD-2 receptor
complex was directly immobilized on a nitrilotriacetic (NTA)
sensor chip by amine coupling and probed with increasing
amounts of FP11 or FP18. The resulting SPR sensorgrams
(Figure 2B) showed a direct interaction between FP molecules
and the TLR4/MD-2 receptor with similar equilibrium
dissociation constants (Kd), 0.18 μM for FP11 and 0.57 μM
for FP18. Moreover, these data indicated that both FP11 and
FP18 bind to the TLR4/MD-2 receptor with fast association
(Ka) and slow dissociation (Kd) rates, as reported for LPS and
other FP molecules.32,35
Figure 1. Chemical structure of TLR4 agonists: MPLA, ONO4007,
SDZ MRL 953, and FP molecules.
(AGPs)20 are well-studied nontoxic TLR4 ligand adjuvants that
promote Th1 (cellular)-biased immune responses. MPLA also
stimulates the TLR4-mediated activation of the TRIF cascade,14
which also explains the reduced toxicity of MPLA.23 However,
the production of MPLA is challenging. MPLA derived from the
modification of a bacterial lipid A is not chemically
homogeneous, making it difficult to assess its quality. Total
synthesis of MPLA has been developed to obtain a
homogeneous compound, which is named glucopyranosyl
lipid A (GLA).24−26 However, the total synthesis of MPLA is
complex (it involves around 24 chemical steps) and expensive.
In contrast, easier synthetic access to monosaccharides bearing
lipid chains and phosphate groups makes this class of molecules
suitable to develop novel TLR4 agonists with simpler and
scalable production methods. Synthetic monosaccharide mim-
etics of lipid X, a monosaccharide biosynthetic precursor of lipid
A, were developed as TLR4 modulators. Compound SDZ MRL
953 (Figure 1) showed powerful immunostimulatory activity
both in mice and humans27 and was tested in a phase I trial as a
tumor immunotherapeutic.28 Also, the compound ONO 4007
(Figure 1) is a powerful immunostimulant for antitumor
therapy.29,30 Our group developed synthetic monosaccharides,
named FP compounds, which bind to the MD-2 coreceptor and
block the TLR4 pathway in cells and in animal models.31−34
Guided by detailed structure−activity information, we predicted
new compounds switching from antagonism to agonism by
altering the ratio of fatty acid chains and phosphates.
Compounds FP11 and FP18 (Figure 1), with a triacylated
monophosphoryl glucosamine core and one phosphate group at
C1, were designed and synthesized along with compound
FP111, which has an additional phosphate group at C6. We
TLR4 Binding of FP11, FP18, and FP111: Computa-
tional Studies. To provide a three-dimensional (3D)
perspective of the TLR4 binding, FP11 and FP18 were subjected
to computational studies to predict their binding modes at the
atomic level; FP111 was also studied. Compounds FP11, FP18,
and FP111 were docked into the human TLR4/MD-2
heterodimer in the agonist activated conformation (Figure 2A,
and the Supporting Information). Preliminary binding poses,
obtained with AutoDock Vina, were used as starting geometries
for redocking calculations with AutoDock. The calculations
resulted in favorable predicted binding energies for FP11 and
FP18 ligands (ranged from −3.8 to −2.7 kcal mol−1, for the best-
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a
Scheme 1. Synthesis of FP11, FP18 and FP111
a
Conditions: (i) NaN3, Tf2O, copper sulfate, Py, H2O, quant. (ii) Anisaldehyde dimethyl acetal, camphorsulfonic acid (CSA), dimethylformamide
(DMF), 50 °C, 68%. (iii) TBDMSCl, imidazole, dichloromethane (DCM), 62%. (iv) PPh3, tetrahydrofuran (THF), H2O, quant. In the following
steps, the first yield refers to FP11 synthesis and the second to FP18. (v) RCOCl, 4-dimethylaminopyridine (DMAP), triethylamine (TEA), DCM,
75%, 85%. (vi) NaBH3CN, 4 Å molecular sieves, THF, and then TFA, 83%, 85%. (vii) RCOCl, DMAP, TEA, DCM, 97%, 98%. (viii) TBAF,
AcOH, THF, 87%, 90%. (ix) P(OBn)2N(i-Pr)2, imidazolium triflate, DCM and then m-CPBA, 50%, 55%. (x) H2/Pd−C, MeOH, and then IRA78-
Na+, 80%, 87%. (xi) H2, Pd−C 10%, MeOH, 95%.
ranked poses), and unfavorable binding energy for compound
FP111 (values greater than 3.0 kcal mol−1). FP11 and FP18
showed the predicted binding poses with their fatty acid chains
buried inside the MD-2 pocket, interacting with many
hydrophobic and aromatic residues, and with the saccharide
moiety located at the MD-2 rim, establishing polar interactions
(Figure 2A). The docking calculations suggested a strong affinity
and plausible binding modes for compounds FP11 and FP18
with the TLR4/MD-2 system but indicated nonefficient binding
for ligand FP111 (Figure 2A). The stability of the best FP11 and
FP18 predicted binding modes was confirmed by molecular
dynamics (MD) simulations. Starting from the best docked
TLR4/MD-2/ligand complexes, we constructed full [TLR4/
MD-2/ligand]2 models (Figure S1) that were submitted to 50 ns
MD simulations (Supporting Information). The complexes
were stable during the simulations (Figure S2) and none of the
ligands underwent orientation flip, all remaining in the agonist
orientation predicted from docking calculations (Figure S3).
Further, MD-2 Phe126 retained the agonist conformation along
the MD simulations (Figure S4). We, therefore, suggest these
complexes as plausible binding modes for FP11 and FP18,
accounting for their agonist activity in the TLR4/MD-2 system.
TLR4 Activation by Synthetic Agonists. The ability of FP
molecules to activate human TLR4 was first assessed using
HEK-Blue hTLR4 cells. These are a HEK293-derived cell line
stably transfected with the LPS receptors CD14, TLR4, and
MD-2 and a reporter gene, secreted embryonic alkaline
phosphatase (SEAP) placed under the control of two TLR4-
dependent transcription factors (NF-κB and AP-1). The HEK-
Blue hTLR4 cells were treated with increasing concentrations
(0.1−25 μM) of FP11, FP18, and FP111 over 18 h. Stimulation
with LPS (smooth chemotype, S-LPS) served as a positive
control for the activation of the TLR4-mediated pathway.
The molecules FP11 and FP18 induced the release of the
SEAP reporter protein in the medium in a concentration-
dependent manner, indicating that both compounds activate
NF-κB and AP-1, while FP111 was inactive (Figure 3A). The
three compounds did not inhibit LPS-induced SEAP produc-
tion, suggesting they lack a TLR4 antagonistic activity (Figure
3B). A lack of activity on HEK-Blue Null cells, which carry the
same SEAP reporter gene but lack the LPS receptors, confirmed
that both FP11 and FP18 act via TLR4 (Figure 3C). To confirm
the selectivity on TLR4 over TLR2, the molecules were also
tested on the HEK-Blue cells expressing hTLR2, and no agonist
activity was detected (Figure 3D). These data confirm the
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Figure 2. (A) Docking studies of compounds FP11, FP18, and FP111. (Top) Best AutoDock predicted binding modes for ligands FP11 and FP18 to
the human TLR4/MD-2 heterodimer. On the right are details of the interactions between FP11 (magenta and cyan sticks) and FP18 (yellow sticks)
and MD-2 protein. (Bottom) Binding of compound FP111 by means of the AutoDock program to the human TLR4/MD-2 system. On the right are
represented detailed poses of FP111 to the TLR4/MD-2 heterodimer, with details of the interactions between FP111 type A antagonist-like (purple
sticks) and type B agonist-like (salmon pink sticks) orientations. (B) Surface plasmon resonance (SPR) analyses of FP11 and FP18 binding to
immobilized TLR4/MD2. Increasing concentrations of FP11 (4.34−69.50 μM, left panel) or FP18 (5.34−85.50 μM, right panel) diluted in running
buffer were injected over a TLR4/MD2-immobilized NTA sensor chip. FP11 and FP18 bind to TLR4/MD2 with equilibrium dissociation constant
(Kd) values of 0.18 and 0.57 μM, respectively. Data are representative of three independent experiments. Binding kinetics are also shown. Ka,
association rate constant; M, molarity; s, seconds; Kd, dissociation rate constant; KD, equilibrium dissociation constant; Rmax, maximum response; RU,
response units; Chi2, average squared residual.
binding data with purified TLR4/MD-2 and suggest that FP11
and FP18 are specific TLR4 agonists that directly bind to TLR4/
MD-2.
MyD88-dependent intracellular TLR4 signaling in human
macrophages.
To investigate whether FP11 and FP18 also trigger the TRIF-
dependent pathway, four readouts in TDM were selected: the
activation of IRF3, the expression and release of IFNβ, the
activation of STAT1, and the expression of interferon-
stimulated genes (ISGs). Stimulation with rough chemotype
of LPS (R-LPS) was also included as reference in this part of the
study.
Activation of MyD88 and TRIF Pathways in Human
Macrophages. We investigated whether the FP compounds
are able to induce the same signaling pattern observed with S-
LPS and MPLA stimulation in vitro. First, we evaluated whether
FP11 and FP18 activate the MyD88-dependent pathway in
THP-1-derived macrophages (TDM). The recruitment of
MyD88 adaptor protein by the TLR4 cytosolic TIR domain
promotes NF-κB and the mitogen-activated protein kinase
(MAPK) activation.36 Therefore, the phosphorylation status of
NF-κB (p65 subunit) and MAPK p38 in response to FP11,
FP18, S-LPS, and MPLA treatment was assessed over time. The
stimulation with S-LPS, and with FP11 and FP18, triggered p65
phosphorylation after 1 h, while for MPLA the activation of p65
occurred earlier (Figure 4A). The kinetics of p38 activation by
FP11 and FP18 was similar to p65 NF-κB phosphorylation
(Figure 4A), with the peak of phosphorylation after 1 h, as also
observed upon S-LPS stimulation. In contrast, MPLA showed an
earlier peak of phosphorylation at 30 min. The quantity of pro-
inflammatory cytokines (TNFα, IL-1β, and IL-6) released
following the treatment with increasing concentrations of FP11
and FP18 was compared with MPLA and S-LPS. S-LPS
triggered the release of TNFα, IL-1β, and IL-6 (Figure 4B), as
MPLA stimulation, albeit in reduced amounts. The effect of
MPLA was dose-independent, suggesting that the concen-
trations used were sufficient to reach receptor saturation. FP11
treatment only triggered a dose-dependent secretion of IL-1β,
while FP18 induced the production of all the three cytokines at
levels exceeding those observed with MPLA stimulation (Figure
4B). These data clearly show that FP11 and FP18 activate
S-LPS strongly induced IRF3 phosphorylation 1 h after
treatment but the effect of FP18 was delayed, with a peak of
IRF3 activation observed after 2.5 h (Figure 5A). The peak of
IRF3 phosphorylation following R-LPS and MPLA stimulation
was of a similar magnitude to that induced by FP18, appearing
between 1 and 1.5 h post-treatment (Figure 5A). In contrast,
FP11 did not induce IRF3 phosphorylation (Figure 5A). Next,
we evaluated whether IFNβ induction followed IRF3 activation
by examining gene expression and cytokine release. Consistent
with previous results, increased IFNβ transcription was observed
2 h poststimulation with S-LPS, R-LPS, and MPLA in
comparison with untreated cells, with 600-, 250- and 60-fold
increase, respectively, followed by a rapid decline at 6 h post-
treatment (Figure 5B, left panel). FP18 showed limited ability to
induce the IFNβ mRNA expression, with a 20-fold increase at 2
h post-treatment in comparison with untreated cells. In
agreement with the IRF3 phosphorylation status, IFNβ
mRNA expression was unaffected by FP11 stimulation. The
levels of IFNβ secreted corroborated the mRNA results,
whereby significantly higher levels were detected in the culture
supernatants of cells treated with S-LPS and R-LPS than from
untreated cells at both 3 and 6 h post-treatment (Figure 5B, right
panel). In contrast, induction of IFNβ was lower in MPLA- and
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reaching a maximum peak 6 h post-treatment (Figure 5D). A
similar pattern of activation was observed for FP18, R-LPS, and
MPLA treatments causing increased RSAD2 expression after 6
h, with a potency similar to S-LPS. In contrast, very low RSAD2
transcription was detected in cells stimulated with FP11 (Figure
5D). These results suggest that FP18 triggers both the MyD88-
and the TRIF-dependent pathways, while FP11 preferentially
activates the MyD88 pathway.
FP18 Induces Caspase-1 Activation and Release of
Mature IL-1β in a NLRP3-Dependent Manner. IL-1β is the
predominant pro-inflammatory cytokine induced by FP
molecules and its release is normally associated with the
activation of NLRP3 inflammasome. The ability of FP11 and
FP18 to trigger caspase-1 activation and IL-1β maturation was
investigated using TDM. We first analyzed IL-1β production by
a cell-associated ELISA, which allowed us to compare precursor
and mature forms of the cytokine in cell lysates and
supernatants, respectively.
The accumulation of IL-1β precursor in cell lysates was
induced by FP11 and FP18, as well as the other treatments
tested, after 3, 6, and 18 h (Figure 6A). This induction was,
however, only significant in the FP18-, S-LPS- and R-LPS-
treated cells. Similarly, the analysis of supernatants revealed that
although IL-1β release was triggered by all molecules, only FP18
and S-LPS caused significant secretion of the IL-1β mature form.
The lower level of activity of FP11 is consistent with the previous
results (Figure 6A). Moreover, IL-1β levels were higher in cell
lysates, suggesting a partial activation of the inflammasome with
a limited release of the mature cytokine. For this reason, we
evaluated the canonical inflammasome activation by monitoring
caspase-1 activation and mature IL-1β release in the extracellular
compartment through western blotting. Caspase-1 was con-
stitutively expressed in differentiated THP-1 cells, while, in
agreement with the cell-associated ELISA assay, the IL-1β
precursor was induced after 6 h by all compounds (Figure 6B). It
was also demonstrated that only FP18 and S-LPS induced high
levels of caspase-1 cleavage and IL-1β maturation and release,
while MPLA and R-LPS stimulation resulted in weaker signaling
(Figure 6B). To evaluate NLRP3 contribution in FP18-
triggered IL-1β release, TDM were pretreated for 1 h with
increasing concentration of the NLRP3 inhibitor MCC950
(0.01−10 μM) and then stimulated with FP18 or S-LPS for 6 h.
After treatment, cell supernatants were checked for IL-1β levels
by ELISA assay and western blot. MCC950 pretreatment
significantly inhibited IL-1β release, in a concentration-depend-
ent manner, in both FP18- and S-LPS-treated cells (Figure
6C,D). Collectively, these data demonstrated that FP18 triggers
NLRP3 canonical inflammasome inducing caspase-1 activation
and IL-1β release.
Adjuvant Activity of FP11 and FP18 and In Vivo
Toxicity: OVA Immunization Experiments. The ability of
FP11 and FP18 to induce immune responses in vivo was
compared to MPLA by evaluating antibody production in
C57Bl/6 mice immunized with chicken ovalbumin (OVA) as a
model antigen. We first evaluated the toxicity of FPs in a pilot
experiment in which mice were injected subcutaneously with 10
μg of FP11 and FP18. The results showed that the two test
adjuvants had no obvious adverse effect on mice, as assessed by
the local response at the injection site and by determining the
animal weight and state of alertness over 7 days (Figure 7A).
Next, mice were immunized with the tested adjuvants mixed
with ovalbumin (OVA). The induction of antibody was
evaluated 21 days postimmunization. The results showed that
Figure 3. HEK-Blue hTLR4, HEK-Blue Null, and HEK-Blue hTLR2
cells were treated as indicated and incubated for 18 h. Supernatants
were collected and SEAP levels were quantified by the QUANTI-blue
method. Data were normalized to stimulation with S-LPS (A, B), IL-1β
(C), or PAM2CSK4 (D) and expressed as the mean percentage
standard deviation (SD) of three independent experiments (treated
versus untreated (-): **p < 0.01 and ***p < 0.001).
FP18-treated cells, and not observed with FP11 stimulation.
Despite the diminished induction of IFNβ, downstream STAT1
phosphorylation was detected starting from 2.5 h upon FP18
and S-LPS stimulation, indicating that IFNβ release mediated by
FP18 was sufficient to induce ISGs. By comparison, R-LPS and
MPLA triggered STAT1 phosphorylation after 1.5 and 2 h upon
stimulation, respectively (Figure 5A). The phosphorylation of
STAT1 due to IFNβ binding to its receptor was confirmed using
a specific blocking antibody that prevents IFNAR activation. As
predicted, S-LPS and FP18 exposure or direct exogenous IFNβ
stimulation triggered a significant increase in STAT1 phosphor-
ylation (Figure 5C), which was completely abolished by
blocking IFNAR in all of the three cases, indicating that FP18
can induce type I IFNs release (Figure 5C). Finally, STAT1-
mediated expression of an ISG (namely, Viperin/RSAD2) was
also evaluated. S-LPS induced Viperin/RSAD2 gene expression,
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Figure 4. (A) TDM were treated with S-LPS (100 ng/mL), MPLA (1 μg/mL), FP11 (10 μM), FP18 (10 μM), or left untreated (NT) and collected
after the indicated time. The levels of phospho-p65, phospho-p38, and actin were detected by immunoblotting. (B) In vitro cytokines released from
TDM measured by enzyme-linked immunosorbent assay (ELISA). Cells were treated with S-LPS or with increasing concentrations of MPLA, FP11,
and FP18 (0.1−1 to 10−25 μM) for 18 h. Error bars represent the SD of the mean. TNFα (n = 9), IL-1β (n = 9), and IL-6 (n = 7), where *p < 0.05, **p
< 0.01, and ***p < 0.001. Statistical analysis is between treated vs untreated (-) (one-way analysis of variance (ANOVA) test).
Figure 5. (A) TDM was treated with S-LPS (100 ng/mL), R-LPS (100 ng/mL), MPLA (1 μg/mL), FP11 (10 μM), FP18 (10 μM), or left untreated
(NT) and collected after the indicated time. The levels of phospho-IRF3, phospho-STAT1, and actin were detected by immunoblotting. (B) TDM
were treated as in panel A and collected after the indicated time. (Left) IFNβ production is expressed as fold change compared to untreated cells. Error
bars represent SD of the mean; n = 3, where *p < 0.05, **p < 0.01, and ***p < 0.001. Statistical analysis is between treated vs untreated (Wilcoxon’s
test). (Right) IFNβ release was quantified by ELISA. Error bars represent SD of the mean; n = 4 where *p < 0.05; **p < 0.01; and ***p < 0.001.
Statistical analysis is between treated vs untreated (NT) (one-way ANOVA test). (C) TDM were pretreated with IFNAR2 neutralizing antibody (1
μg/mL) 1 h prior to exposure to the indicated stimulus. Cell lysates were collected after 3 h and p-STAT1 was measured via immunoblotting. Error
bars represent SD of the mean; n = 2 where *p < 0.05 and ***p < 0.001 (one-way ANOVA test). (D) TDM were treated as in panel B and collected
after the indicated time. RSAD2 expression is expressed as fold change compared to untreated cells. Error bars represent SD of the mean; n = 3, where
*p < 0.05, **p < 0.01, and ***p < 0.001. Statistical analysis is between treated vs untreated (Wilcoxon’s test).
mice immunized with the test adjuvants exhibited marginally
higher levels of anti-OVA total IgG after prime immunization
compared to OVA-immunized control and significantly lower
levels compared to MPLA-OVA-immunized animals (Figure
7B, prime immunization). In contrast, after a boost immuniza-
tion given on day 22 and examined for ova-specific antibody
titers 14 days later, the IgG levels in the FP18-immunized mice
were higher than those in the FP11-immunized group (Figure
7B, booster immunization). These data indicate that, in
agreement with in vitro and in cell results, FP18 is a more
effective adjuvant in vivo than FP11 and has a potency
comparable or even greater than MPLA.
CONCLUSIONS
■
By engaging the TLR4/MD2 endotoxin receptor system, natural
adjuvants such as LPS and the LPS-derived MPLA modulate
adaptive immune responses by influencing early T-cell clonal
expansion and the cytokine milieu expressed during antigen-
dependent proliferation. In addition, inflammasome activation
has been associated with the adjuvant efficiency of the first
clinically approved combination adjuvants, AS01, and AS04,
which contain MPLA, the saponin QS-21, alum,17,37 and the
FDA-approved squalene-based oil-in-water emulsion adjuvants
MF59 and AS03.1,38,39 AS04 and AS01 were used in U.S. vaccine
(Cervarix) and in the recombinant Varicella zoster vaccine
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Figure 6. (A) TDM were treated with S-LPS (100 ng/mL), R-LPS (100 ng/mL), MPLA (1 μg/mL), FP11 (20 μM), FP18 (20 μM), or left untreated.
IL-1β levels were measured in both cell lysates and supernatants by ELISA. Error bars represent SD of the mean; n = 3 where *p < 0.05, **p < 0.01, and
***p < 0.001. Statistical analysis is between treated vs untreated (NT) (one-way ANOVA test). (B) TDM were treated as in panel A for 6 h. Levels of
pro-casp-1/pro-IL-1β, and cleaved casp-1/cleaved-IL-1β were detected by immunoblot in cell lysates and supernatants, respectively. (C, D) TDM
were pretreated for 1 h with an increasing concentration of MCC950 and then stimulated with S-LPS (100 ng/mL) or FP18 (20 μM) for 6 h. The effect
of MCC950 on LPS- and FP18-triggered IL-1β secretion was evaluated by ELISA (C) and immunoblot (D). In (C), error bars represent SD of the
mean; n = 5 where *p < 0.05, **p < 0.01, and ***p < 0.001 (one-way ANOVA test). In (D), the percentage of IL-1β release was normalized to
stimulation with only S-LPS or only FP18, and error bars represent SD of the mean (*p < 0.05, **p < 0.01, and ***p < 0.001, Wilcoxon’s test).
Shingrix, respectively. Despite showing reassuring overall safety
profiles in both vaccines, the lack of chemical homogeneity of
MPLA derived from chemical modification of bacterial LPS
poses manufacturing and quality control challenges at the
industrial level, potentially limiting its use. On the other hand,
the chemical synthesis of MPLA, consisting of 24 steps, is
expensive and its upscale is challenging. Druggable, chemically
simplified MPLA substitutes, such as monosaccharide-based
TLR4 agonists, could be more attractive compounds to develop
adjuvants that can be sustainably produced on a global scale.
Therefore, we are working toward the development of new
glucosamine derivatives with phosphate at the α-anomeric
position and three lipid chains linked at positions C2, C3, and
C4 (termed FP compounds) as TLR4/MD-2 ligands. FP
compounds are similar to synthetic agonist SDZ MRL 953,
except for the absence of C3 hydroxyl groups on the three fatty
acid chains. However, the retention of biological activity after
deletion of C-3 is not obvious, and it generally causes reduced
activity, as observed with other lipid A derivatives.40 The
presence of nonhydroxylated fatty acid chains greatly simplifies
FP chemical synthesis, compared to MPLA or other
monosaccharide lipid A mimetics, making these compounds
scalable at an industrial level. Compounds FP11 and FP18 differ
only in the length of the fatty acid chains (14 and 12 carbons,
respectively). We show here that both compounds have
favorable predicted binding energies when docked to the
activated TLR4/MD-2 system. Compound FP111, with a
second phosphate on sugar’s C6, showed unfavorable predicted
binding energy. This behavior can be explained by the presence
of two phosphates in 1,6-positions that do not allow an LPS-like
binding mode at the MD-2 rim, unlike the mono (1- or 6-)
phosphate pattern. FP111 binding poses are predicted to be
anchored through one phosphate group to MD-2 and the
second phosphate to the TLR4 chain instead of being
completely inserted into the MD-2, accounting for the observed
lack of activity. Experiments to assess the ability of the three
molecules to trigger TLR4 activation in HEK-Blue cells
confirmed that FP11 and FP18 behave as selective TLR4
agonists, and are not active on TLR2, while the FP111 control
molecule was inactive. FP11 and FP18 bound TLR4 at sub-
micromolar affinities and are both capable to stimulate the
MyD88-dependent pathway in human THP-1 cells, ultimately
leading to the release of TNFα, IL-1β, and IL-6 cytokines, albeit
to different extents. FP18 showed a higher potency than FP11
and MPLA in inducing IL-1β release due to a greater ability to
activating the +NLRP3 inflammasome. This activity resembled
that of S-LPS, a known potent inflammasome inducer. However,
unlike S-LPS, the NLRP3-specific inhibitor MCC950 only
partially reversed the efficacy of FP18, as shown by the amount
of IL-1β still released even at the higher concentration of the
inhibitor, suggesting this compound causes. In addition, results
obtained showed that FP18 stimulatory activity is not limited to
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MPLA, and established the molecular mechanisms explaining
their action based on the selective TLR4 stimulation with
activation of MyD88- and TRIF-dependent pathways and
inflammasome. These observations, together with the lack of
obvious in vivo and in vitro (Figure S18, Supporting
Information) toxicity, and the straightforward synthesis
procedure compared to MPLA, support preclinical, and clinical
development of FP molecules as novel lead compounds for the
production of effective vaccine adjuvants.
EXPERIMENTAL SECTION
■
General. All reagents and solvents were purchased from commercial
sources and used without further purifications unless stated otherwise.
Reactions were carried out under a nitrogen atmosphere unless
otherwise noted and were monitored by thin-layer chromatography
(TLC) performed over Silica Gel 60 F254 plates (Merck) and revealed
using UV light or staining reagents (H2SO4 5% in EtOH), ninhydrin
(5% in EtOH), basic solution of KMnO4 (0.75% in H2O), molybdate
solution (molybdate phosphorus acid and Ce(IV) sulfate in 4%
H2SO4). Flash chromatography purifications were performed on silica
gel 60 (40−63 μm) from commercial sources. 1H and 13C NMR spectra
of compounds were recorded with a Bruker Advance 400 with TopSpin
software, or with an NMR Varian 400 with Vnmrj software. Chemical
shifts are reported in parts per million (ppm) relative to the residual
solvent; coupling constants are expressed in Hz. The multiplicity in the
13C spectra was deduced by attached proton test (APT) pulse
sequence; peaks were assigned with the help of 2D-COSY and 2D-
HSQC experiments. Exact masses were recorded with Orbitrap Fusion
Tribrid. Purity of final compounds was about 95% as assessed by
quantitative NMR analysis. Reaction conditions and compound
characterization are described in the Supporting Information.
Surface Plasmon Resonance Analysis. Real-time binding
interaction experiments were performed with a Biacore X100 (GE
Healthcare). Recombinant human TLR4/MD2 complex was cova-
lently immobilized onto the surface of a sensor chip NTA (cat #
BR100034, GE Healthcare) via amine coupling. TLR4/MD2 complex
was diluted to a concentration of 20 μg/mL in 10 mM sodium acetate at
pH 4.0, and was injected on the NTA chip at a flow rate of 10 μL/min,
upon washing with 0.35 M ethylenediaminetetraacetic acid (EDTA),
injection of NiCl2 for 60 s, a second wash with 3 mM EDTA, and
activation of the carboxyl groups on the sensor surface with 7 min
injection of a mixture of 0.2 M EDC and 0.05 M NHS. The remaining
esters were blocked with 7 min injection of ethanolamine. Based on the
ligand (TLR4/MD2) and analytes (FP11, FP18), molecular weights
(MW) of 90 kDa, and 934.39 or 850.04 Da, respectively, the
appropriate ligand density (RL) on the chip was calculated as follows:
RL = (ligand MW/analyte MW) × Rmax × (1/Sm), where Rmax is the
maximum binding signal and Sm corresponds to the binding
stoichiometry. The target capture level of the TLR4/MD2 complex
was 1933.5 response units (RUs). The other flow cell was used as a
reference and was immediately blocked after the activation. Increasing
concentrations of FP11 or FP18 were flowed over the NTA sensor chip
coated with TLR4/MD2 at a flow rate of 30 μL/min at 25 °C with an
association time of 60 s and a dissociation phase of 180 s. A single
regeneration step with 50 mM NaOH and an extra wash with phosphate
buffered saline (PBS-P) + with 50% dimethyl sulfoxide (DMSO) were
performed following each analytic cycle. All the analytes tested were
sonicated for 15 min and then diluted in the PBS-P + buffer (GE
Healthcare) with 5% DMSO. The KD values were evaluated using the
Biacore evaluation software (GE Healthcare) after solvent correction,
and the reliability of the kinetic constants calculated by assuming a 1:1
binding model supported by the quality assessment indicators values.
Cell Cultures. HEK-Blue cells and RAW-Blue Cells (InvivoGen)
were cultured according to the supplier’s instructions. Briefly, cells were
cultured in Dulbecco’s modified Eagle’s medium (DMEM) high
glucose medium supplemented with 10% heat-inactivated fetal bovine
serum (FBS), 2 mM glutamine, penicillin (100 U/mL), streptomycin
(100 U/mL), and supplemented with the antibiotics indicated in Table
Figure 7. (A) Body weight of mice over 7 days after administration of
adjuvants (n = 4 per treatment). (B) Antibody response to OVA
immunization using MPLA, FP18, and FP11 as adjuvants after prime
(22 days postimmunization) and booster immunization (19 days later)
(n = 8 per treatment). For statistical comparisons, the area under each
curve was examined by Brown−Forsythe and Welch one-way ANOVA
tests with an α of 0.05.
the activation of the MyD88-dependent pathway, but also
involves activation of the TRIF-dependent pathway leading to a
type I IFN signature.15 The stronger overall activity of FP18 over
FP11 is consistent with the higher polarity and solubility in
aqueous media of FP18 (calculated log P = 8.3 and 10.2 for FP11
and FP18, respectively, see Table S2), explaining higher efficacy
when using this molecule in an aqueous environment. The FP18
and FP11 differences in inflammasome activation were also
mirrored in their relative activities as immunization adjuvants.
Although both molecules had adjuvant effects in vivo, FP18 was
significantly more potent than FP11 and also displayed greater
potency than MPLA upon boost immunization. In conclusion,
we have demonstrated that synthetic FP11 and FP18 show in
vivo immunostimulatory activity with a potency similar to
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S1. Cells were detached using a cell scraper, counted, and seeded in a
96-well multiwell plate at a density indicated in Table S1. After
overnight incubation (37 °C, 5% CO2, 95% humidity), supernatants
were removed, and cell monolayers washed with warm PBS. Cells were
resuspended in a fresh medium supplemented with the molecules to be
tested and incubated for 18 h. THP-1 cells were cultured in the Roswell
Park Memorial Institute Medium (RPMI) supplemented with 10%
heat-inactivated FBS, 2 mM glutamine, penicillin (100 U/mL), and
streptomycin (100 U/mL). Cells were split three times weekly and
maintained at a density of 0.5 × 106 cells/mL. For experimental
procedures, THP-1 were seeded in multiwell plates at a density of 0.5 ×
106 cells/mL, 200 μL/well (96 wells), 1.5 mL/well (12 wells), and 3
mL/well (6 wells), and differentiated into macrophages with phorbol
12-myristate 13-acetate (PMA, Invivogen) at a final concentration of 25
ng/mL. After 72 h of differentiation, the culture medium was replaced
with a fresh medium, and cells were rested for another 24 h before
exposure to the molecules to be tested.
Cell Stimulation and Treatments. All LPS variants were
purchased from Innaxon. Unless otherwise indicated, cells were
stimulated with 100 ng/mL ultrapure Smooth-LPS from Salmonella
minnesota (S-LPS) for 18 h. Rough-LPS R595 (Re) from S. minnesota
(R-LPS) was used at 100 ng/mL, while MPLA R595 (Re) from S.
minnesota (MPLA) was used at 1 μg/mL. For TLR2 activation,
PAM2CSK4 (Invivogen) was added at 10 ng/mL for 18 h. hIL-1β
(Merk) was used as control for NF-κB activation and added at a final
concentration of 100 ng/mL. FP11, FP111, and FP18 compounds were
resuspended in ultrapure DMSO and diluted in culture medium. Anti-
human IFNAR2 neutralizing antibody (clone MMHAR-20) was
purchased from PBL Assay Science and used at 1 μg/mL. The
NLRP3 inhibitor MCC950 was purchased from Merck and added to
cells at the following concentrations: 0.01, 0.1, 1, and 10 μM.
HEK-Blue Cells Reporter Assay. After the addition of the
molecules to be tested, cells were incubated for 18 h. Supernatants
were collected and SEAP levels were quantified using QUANTI-Blue
assay according to the manufacturer’s instruction. Briefly, 20 μL of the
supernatants of SEAP-expressing cells was incubated with 200 μL of
QUANTI-Blue substrate in a 96-well plate for 0.5−4 h at room
temperature (RT). SEAP activity, as an indicator of TLR4 activation,
was assessed reading the well’s optical density (OD) at 630 nm. The
results were normalized with positive control (Smooth-LPS for HEK-
Blue hTLR4 cells, PAM2CSK4 for HEK-Blue hTLR2 cells, and IL-1β
for HEK-Blue Null cells) and expressed as the mean of percentage
standard error of the mean (SEM) of at least three independent
experiments.
RNA Extraction, cDNA Synthesis, and Real-Time Polymerase
Chain Reaction. Total RNA was extracted using Quick-RNA
MiniPrep (Zymo Research) according to the manufacturer’s
instruction. Reverse transcription was performed with 1 μg of total
RNA using LunaScript RT SuperMix Kit (New England BioLabs, MA),
and cDNA was amplified using the Luna Universal qPCR Master Mix
(New England BioLabs, MA) under the following conditions:
denaturation for 1 min at 95 °C; annealing for 30 s at 62 °C for
human IFNβ, 58 °C for human IL-1β, 60 °C for human RSAD2, and 60
°C for human β-actin; and 30 s of extension at 72 °C. Primer sequences
were as follows: human IFNβ forward 5′-CAACTTGCTTG-
GATTCCTACAAAG-3′ reverse 5′-GTATTCAAGCCTCCCATT-
CAATTG-3′; human IL-1β forward 5′-AGAATGACCTGAG-
CACCTTC-3′, reverse 5′-GCACATAAGCCTCGTTATCC-3′;
human RSAD2 forward 5′-AGAATACCTGGGCAAGTTGG-3′,
reverse 5′-GTCACAGGAGATAGCGAGAATG-3′; β-actin (forward
5′-AAGATGACCCAGATCATGTTTGAGACC-3′, reverse 5′-AGC-
CAGTCCAGACGCAGGAT-3′) was used as a housekeeping gene.
Gene expression was calculated using the ΔΔCt function and expressed
as fold change compared to not treated cells.
neapolis). The optical density of each well was determined using a
microplate reader set to 450 nm (wavelength correction: 570 nm).
Western Blot Analysis. Immunoblotting of caspase-1 and mature
IL-1β from precipitated supernatant was performed as described.41 For
cell extracts, cells were washed twice in ice-cold PBS and lysed in
radioimmunoprecipitation assay buffer (RIPA) buffer (CST, #9806),
supplemented with protease (Roche, Mannheim, Germany) and
phosphatase inhibitors (CST ##5870). After centrifugation at 13 000
RCF for 30 min at 4 °C, the supernatants were collected as whole cell
lysates. Methanol/chloroform precipitated cell supernatants and cell
lysates were resuspended in the Laemmli buffer, denatured for 5 min at
100 °C, and separated on 10 or 13% polyacrylamide gels. Proteins were
transferred on poly(vinylidene difluoride) (PVDF) filters (Bio-Rad),
blocked in 5% w/v BSA TTBS, and incubated with the primary and
corresponding secondary antibodies indicated below. Proteins were
revealed by chemiluminescence (LiteAblot EXTEND, Euroclone) and
detected using Odyssey Fc LI-COR Imaging System. The PVDF
membrane filters were incubated with the following primary antibodies:
anti-phospho NF-κB (Ser536) (93H1) rabbit mAb (CST #3033;
diluted 1:1000); anti-phospho-p38 MAPK (Thr180/Tyr182) (D3F9)
XP rabbit mAb (CST #4511; diluted 1:1000); anti-phospho-IRF-3
(Ser386) (E7J8G) XP rabbit mAb (CST #37829 diluted 1:1000); anti-
phospho-STAT1 (Tyr701) (58D6) rabbit mAb (CST #9167 diluted
1:1000); anti-IL-1β (3A6) mouse mAb (CST #12242 diluted 1:1000);
anti-cleaved-IL-1β (Asp116) (D3A3Z) rabbit mAb (CST #83186
diluted 1:1000); caspase-1 (D7F10) Rabbit mAb (CST #3866 diluted
1:1000); and anti-β-actin (13E5) rabbit mAb (CST #4970 diluted
1:1000). Secondary antibodies used were anti-rabbit or anti-mouse IgG
and HRP-linked secondary antibody (Cell Signaling #7074 and #7076,
diluted 1:3000). Densitometric analysis was carried out using Image J.
Mice Immunization Experiments. The in vivo protocols were
reviewed by the Queen’s University Animal Welfare and Ethical Review
Body (AWERB), and the work was carried out under an approved UK
Home Office Project License (PPL2807). Chicken ovalbumin (OVA,
Sigma-Aldrich) was resuspended in pyrogen-free Dulbecco’s phos-
phate-buffered saline (DPBS) (Sigma-Life Science) at 5 mg/mL.
Endotoxins were removed by Pierce High-Capacity Endotoxin
Removal Spin Columns (Thermo Scientific). The endotoxin level of
purified OVA was determined by the Limulus Amebocyte Lysate
(LAL) Gel-clot method (Associates of Cape Cod; East Falmouth, MA)
in the form of single test vials. The samples were assessed at a sensitivity
of 0.125 endotoxin unit (EU)/mL. OVA concentration was determined
by BioRad Protein Assay Dye Reagent (Bio Rad) and bovine serum
albumin (BSA, Sigma-Aldrich) as a reference standard. Six-week-old
female C57BL/6 mice were purchased from Envigo, U.K. For the pilot
toxicity experiment, mice (n = 3 per treatment) were injected
subcutaneously on the flank with 10 μg of adjuvants (FP11, FP18, or
MPLA) suspended in 50 μL of PBS. The mice were monitored and
weighed daily for 7 days. For immunization, C57BL/6 mice (n = 8 per
treatment) were immunized by subcutaneous injection on the flank
with 50 μL of 500 μg of OVA mixed with 10 μg of FP11, FP18, or
MPLA resuspended in PBS. A control group of OVA without the
adjuvant was also included. Mice were given a booster immunization on
the alternative flank 22 days after prime immunization. Serum, obtained
from blood samples drawn from the tail vein, was examined for anti-
OVA antibodies at days 21 and 41. The antibody levels in sera were
measured by ELISA. Wells of polystyrene microplates Nunc Maxisorp
(Thermo Scientific) were coated with 50 μL of OVA at 4 μg/mL
diluted in 50 mM carbonate/bicarbonate buffer, pH 9.6, at 4 °C
overnight. The coating solution was removed, and plates were washed
with 300 μL of PBS/Tween 20 (0.05%). Additional blocking was
achieved by adding 200 μL of blocking buffer (BSA 5%). Plates were
covered and incubated at room temperature for 1 h and then washed
three times with PBS/Tween20. Fifty microliters of serum diluted in
half-strength blocking buffer (from 1:100 to 1:12 800) was added to the
wells and incubated for 90 min at room temperature. After incubation,
plates were washed four times with PBS/Tween20. Affinity purified
horseradish peroxidase-conjugated goat anti-mouse IgG (catalog
number: 170-6516, Bio-Rad, U.K.) diluted to 1:5000 was added to
wells for 1 h at RT. After washing four times with PBS/Tween20, 50 μL
Enzyme-Linked Immunosorbent Assay (ELISA). TNF-α, IL-1β,
IL-6, and IFNβ levels were measured in TDP supernatants and cell
lysates after the indicated timing using the following sensitive enzyme-
linked immunosorbent assays (ELISA) (R&D Systems; #DY210-05,
#DY201-05, #DY200-05, #DY206-05, #DY208-05, #DY814-05 Min-
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of the substrate solution 3,3′,5,5′-tetramethylbenzidine was added per
well and incubated in the dark at room temperature for 10 min. After
sufficient color development, 30 μL of stop solution (2 N H2SO4) was
added and the absorbance of each well was read with a POLARstar
Omega microplate reader (BMG Labtech, Ortenberg, Germany) at 450
nm.
All animal experiments performed in this study were conducted in
compliance with institutional guidelines.
Statistical Information. All experimental results represent the
Guanbo Wang − The Wellcome-Wolfson Institute for
Experimental Medicine, Queen’s University of Belfast, Belfast
BT9 7BL, U.K.
Rebecca Ingram − The Wellcome-Wolfson Institute for
Experimental Medicine, Queen’s University of Belfast, Belfast
BT9 7BL, U.K.
Sonsoles Martin-Santamaria − Department of Structural and
Chemical Biology, Centro de Investigaciones Biologicas
Margarita Salas, 28040 Madrid, Spain; orcid.org/0000-
0002-7679-0155
Grisha Pirianov − Department of Biomedical and Forensic
Sciences, Anglia Ruskin University, Cambridge CB1 1PT, U.K.
Marco De Andrea − CAADCenter for Translational
Research on Autoimmune and Allergic Disease, University of
Eastern Piedmont, 28100 Novara, Italy; Department of Public
Health and Pediatric Sciences, University of Turin, 10126
Turin, Italy
Miguel A. Valvano − The Wellcome-Wolfson Institute for
Experimental Medicine, Queen’s University of Belfast, Belfast
BT9 7BL, U.K.
mean
standard deviation (SD) of at least three independent
experiments unless specified. In real-time polymerase chain reaction
(PCR) and western blot experiments, gene expression and protein
amount were evaluated in relation to the housekeeping gene β-actin.
Gene expression is represented as fold change compared to untreated
cells, and results were evaluated using the one-sample Wilcoxon test.
The western blots shown were representative data from at least two
independent experiments. For ELISA experiments, means were
compared by t-tests (two groups) or one-way ANOVA (three or
more groups). Tukey multiple comparison test following one-way
ANOVA was performed to obtain adjusted P values. For statistical
comparisons of immunization results, the area under the ELISA
titration curves was examined by Brown−Forsythe and Welch one-way
ANOVA tests with an α of 0.05. This study includes no data deposited
in external repository.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00896
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00896.
■
Notes
sı
The authors declare no competing financial interest.
○F.A.F. and A.M. are first co-authors.
Molecular formula strings (CSV)
ACKNOWLEDGMENTS
■
computational methods; docking calculations; synthesis
procedures; all NMR spectra; cytotoxicity of compounds
FP11 and FP18; log P and log S calculations (PDF)
The support from European Union’s Horizon 2020 research and
innovation programme under the Marie Skłodowska-Curie
grant agreement No. 860325 (www.BactiVax.eu), Italian
Ministry grant PRIN 2017, no. 2017E44A9P (BacHounds:
Supramolecular nanostructures for bacteria detection), and the
Spanish Ministry for Science and Innovation (grants CTQ2017-
88353-R and PRE2018-086249) is acknowledged.
AUTHOR INFORMATION
Corresponding Author
Francesco Peri − Department of Biotechnology and Biosciences,
University of Milano-Bicocca, 20126 Milano, Italy;
orcid.org/0000-0002-3417-8224;
■
Email: francesco.peri@unimib.it
ABBREVIATIONS USED
■
Authors
AP-1, activator protein 1; CD14, cluster of differentiation 14;
CSA, camphorsulfonic acid; ELISA, enzyme linked immuno-
sorbent assay; ERK, extracellular regulated kinase; FA, fatty acid;
IFNs, interferons; IFNAR, interferon-α/β receptor; IgG,
immunoglobulin G; IL-, interleukin-; IRF3, interferon regu-
latory factor 3; ISGs, interferon stimulated genes; LPS,
lipopolysaccharide; R-LPS, rough lipopolysaccharide; S-LPS,
smooth lipopolysaccharide; MAPK, mitogen-activated protein
kinase; MD, molecular dynamics; MD-2, myeloid differentiation
factor 2; MPLA, monophosphoryl lipid A; MyD88, myeloid
differentiation factor 88; NF-κB, nuclear factor kappa-light-
chain enhancer of activated B cells; NLRP3, NLR family pyrin
domain containing 3; NLRs, nucleotide-binding oligomeriza-
tion domain (NOD)-like receptors; NT, untreated; OVA,
ovalbumin; PAMPs, pathogen-associated molecular patterns;
PRRs, pattern recognition receptors; RSAD2/Viperin, radical S-
adenosyl methionine domain containing 2; SD, standard
deviation; SEAP, secreted embryonic alkaline phosphatase;
SPR, surface plasmon resonance; STAT1, signal transducer and
activator of transcription; TBAF, tributylammonium fluoride;
TFA, trifluoroacetic acid; TLRs, toll-like receptors; TNFα,
tumor necrosis factor α; TDM, THP-1 derived macrophages;
TRIF, TIR domain-containing adaptor inducing IFN-β
○Fabio A. Facchini − Department of Biotechnology and
Biosciences, University of Milano-Bicocca, 20126 Milano,
Italy; orcid.org/0000-0002-4339-5845
○Alberto Minotti − Department of Biotechnology and
Biosciences, University of Milano-Bicocca, 20126 Milano, Italy
Andrea Luraghi − Department of Biotechnology and
Biosciences, University of Milano-Bicocca, 20126 Milano, Italy
Alessio Romerio − Department of Biotechnology and
Biosciences, University of Milano-Bicocca, 20126 Milano, Italy
Nicole Gotri − Department of Biotechnology and Biosciences,
University of Milano-Bicocca, 20126 Milano, Italy
Alejandra Matamoros-Recio − Department of Structural and
Chemical Biology, Centro de Investigaciones Biologicas
Margarita Salas, 28040 Madrid, Spain; orcid.org/0000-
0003-1563-9408
Andrea Iannucci − Department of Translational Medicine,
University of Eastern Piedmont, 28100 Novara, Italy;
CAADCenter for Translational Research on Autoimmune
and Allergic Disease, University of Eastern Piedmont, 28100
Novara, Italy
Charys Palmer − Department of Biomedical and Forensic
Sciences, Anglia Ruskin University, Cambridge CB1 1PT, U.K.
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NOTE ADDED AFTER ASAP PUBLICATION
■
This paper was published ASAP on August 12, 2021 with several
incorrect abbrevations throughout the paper. The corrected
version was reposted on August 17, 2021.
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J. Med. Chem. 2021, 64, 12261−12272