The cationic amphiphile 3,4-bis(tetradecyloxy)benzylamine inhibits LPS signaling by competing with endotoxin for CD14 binding

Article abstract of DOI:10.1016/j.bcp.2010.06.019

The identification of the bacterial endotoxin receptors for innate immunity, most notably the Toll-like receptor 4 (TLR4), has sparked great interest in therapeutic manipulation of innate immune system. We have recently developed synthetic molecules that have been shown to inhibit TLR4 activation in vitro and in vivo. Here we present the synthesis and the biological characterization of a new molecule, the cationic amphiphile 3,4-bis(tetradecyloxy)benzylamine, with a structure strictly related to the previously developed TLR4 modulators. This compound is able to inhibit in a dose-dependent manner the LPS-stimulated TLR4 activation in HEK cells. In order to characterize the mechanism of action of this compound, we investigated possible interactions with the extracellular components that bind and shuttle LPS to TLR4, namely LBP, CD14, and MD-2. This compound inhibited LBP/CD14-dependent LPS transfer to MD-2.TLR4, resulting in reduced formation of a (LPS-MD-2-TLR4)₂ complex. This effect was due to inhibition of the transfer of LPS from aggregates in solution to sCD14 with little or no effect on LPS shuttling from LPS/CD14 to MD-2. This compound also inhibited transfer of LPS monomer from full-length CD14 to a truncated, polyhistidine tagged CD14. Taken together, our findings strongly suggest that this compound inhibits LPS-stimulated TLR4 activation by competitively occupying CD14 and thereby reducing the delivery of activating endotoxin to MD-2.TLR4.

Full text of DOI:10.1016/j.bcp.2010.06.019

Biochemical Pharmacology 80 (2010) 2050–2056
Contents lists available at ScienceDirect
Biochemical Pharmacology
journal homepage: www.elsevier.com/locate/biochempharm
The cationic amphiphile 3,4-bis(tetradecyloxy)benzylamine inhibits
LPS signaling by competing with endotoxin for CD14 binding
Matteo Piazza a, Valentina Calabrese a, Chiara Baruffa a, Theresa Gioannini b,c,e
,
Jerrold Weiss b,d, Francesco Peri a,
*
a Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza, 2; 20126 Milano, Italy
b Department of Internal Medicine and The Inflammation Program, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52246, United States
c Department of Biochemistry, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52246, United States
d Department of Microbiology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52246, United States
e Iowa City Veterans’ Administration Medical Center, Iowa City, Iowa 52246, United States
A R T I C L E I N F O
A B S T R A C T
Article history:
The identification of the bacterial endotoxin receptors for innate immunity, most notably the Toll-like
receptor 4 (TLR4), has sparked great interest in therapeutic manipulation of innate immune system. We
have recently developed synthetic molecules that have been shown to inhibit TLR4 activation in vitro
and in vivo. Here we present the synthesis and the biological characterization of a new molecule, the
Received 30 April 2010
Accepted 10 June 2010
Keywords:
TRL4
cationic amphiphile 3,4-bis(tetradecyloxy)benzylamine, with
a structure strictly related to the
previously developed TLR4 modulators. This compound is able to inhibit in a dose-dependent manner
the LPS-stimulated TLR4 activation in HEK cells. In order to characterize the mechanism of action of this
compound, we investigated possible interactions with the extracellular components that bind and
shuttle LPS to TLR4, namely LBP, CD14, and MD-2. This compound inhibited LBP/CD14-dependent LPS
transfer to MD-2.TLR4, resulting in reduced formation of a (LPS-MD-2-TLR4)2 complex. This effect was
due to inhibition of the transfer of LPS from aggregates in solution to sCD14 with little or no effect on LPS
shuttling from LPS/CD14 to MD-2. This compound also inhibited transfer of LPS monomer from full-
length CD14 to a truncated, polyhistidine tagged CD14. Taken together, our findings strongly suggest
that this compound inhibits LPS-stimulated TLR4 activation by competitively occupying CD14 and
thereby reducing the delivery of activating endotoxin to MD-2.TLR4.
CD14
LPS
Endotoxin
Septic shock
Inflammation
ß 2010 Elsevier Inc. All rights reserved.
1. Introduction
microbial components, lipopolysaccharides (LPS) and lipooligo-
saccharides (LOS) and their bioactive portions, the lipodisaccharide
The ability of an organism to protect itself from microbial
challenge requires the rapid detection of ‘‘pathogen-associated
molecular patterns’’ by a variety of pathogen sensors, in particular
Toll-like receptor 4 (TLR4) that activate the host response by
rapidly triggering pro-inflammatory processes [1–3]. Among
lipid A, commonly defined as endotoxin (E), are potent stimulants
of immune responses [4]. The induction of inflammatory responses
by LPS is achieved by the coordinate and sequential action of four
principal endotoxin-binding proteins: the lipopolysaccharide-
binding protein (LBP), the cluster differentiation antigen (CD14),
the myeloid differentiation protein (MD-2) and Toll-like receptor 4
(TLR4) [5]. LBP interacts with endotoxin-rich bacterial membranes
and purified endotoxin aggregates, catalyzing extraction and
transfer of E monomers to CD14 that in turn transfers endotoxin
to MD-2 and to MD-2-TLR4 heterodimer [6]. The role of CD14
seems to be fundamental when endotoxin has very low
concentration and when TLR4 is stimulated by the smooth LPS
(LPS with the entire O-chain oligosaccharide), while the rough
chemotype of LPS can stimulate efficiently TLR4 even in the
absence of CD14 [7]. Besides being an essential chaperone or
matchmaker assisting the TLR4-MD-2-endotoxin complex forma-
tion, CD14 very likely has an important role in cellular pathways
not necessarily related to the TLR4 signaling. It has been recently
Abbreviations: TLR, Toll-like receptor; LPS, lipopolysaccharide; E, endotoxin; LBP,
lipopolysaccharide-binding protein; CD14, cluster differentiation antigen 14; MD-
2, myeloid differentiation protein; HEK, human embryonic kidney cells; AcOEt,
ethyl acetate; EtP, petroleum ether; TEA, triethylamine; MeOH, methanol; HSA,
human serum albumin; DMSO, dimethyl sulfoxyde; EtOH, ethanol; PBS, phosphate
buffer; DMEM, Dulbecco Modified Eagle Medium; His, histidine; TLR4ECD, TLR4
ectodomain; LOSagg, LOS aggregates; IgG, immuno globulin G; IL-8, interleukin-8;
ELISA, enzyme-linked immunoabsorbent assay; FBS, fetal bovine serum; pNpp,
para-nitrofenil phosphate; MTT, 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylforma-
zan; Boc, tert-butyloxycarbonyl; sAP, secreted alkalyne phosphatase; sCD14,
soluble CD14; tCD14, truncated CD14.
* Corresponding author. Tel.: +39 02 64483453; fax: +39 02 64483565.
E-mail address: francesco.peri@unimib.it (F. Peri).
0006-2952/$ – see front matter ß 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.bcp.2010.06.019

M. Piazza et al. / Biochemical Pharmacology 80 (2010) 2050–2056
2051
discovered that CD14 regulates dendritic cells life cycle after LPS
exposure through a signal pathway based on NFAT activation,
totally independent from the TLR4-activated intracellular pathway
[8]. Moreover, although the role of CD14 in LPS response is well
established, the participation of CD14 to other TLR-dependent
ligands has been less well studied. In particular, it has been shown
that CD14 contributes to innate immune response activated by
other, non-LPS ligands, including many TLR1-TLR2 ligands [9,10].
Some recent studies also suggest that therapies targeting CD14
may also interfere with TLR3 activation by viral nucleic acids, thus
holding out the possibility that these agents may be effective in the
control of viral as well as bacterial diseases in which excess
immune responsiveness damages the host [11]. With the aim to
develop new lipid A mimetics containing a non-natural, non-
hydrolizable glycosidic bond, we serendipitously found that amino
glycolipids and aromatic ammonium salts (Fig. 1) are active in
inhibiting lipid A and LPS-promoted cytokines production in innate
immunity cells such as macrophages or dendritic cells [12].
Molecule 1 and compounds with a similar chemical structure
(Fig. 1) inhibit LPS-induced TLR4 activation on HEK/TLR4 cells and
LPS-induced septic shock in mice [13].
simplified and the biological activity is retained in vitro, or even
increased in vivo [13]. We therefore projected to do the same
functional group change on compound 7, thus obtaining 8. Second,
compounds containing alkyl ammonium groups are generally
toxic, especially in the case of N-methyl ammonium ions present in
compound 7, that are supposed to be methylating agents in vivo.
Compound 8 was therefore designed with the aim to reduce the in
vitro and in vivo toxicity. In this paper we present the synthesis and
the biological characterization of compound 8 in terms of activity
on cells, toxicity and binding to purified receptors.
2. Materials and methods
2.1. Chemistry, synthesis of 3,4-bis(tetradecyloxy)benzylamine
2.1.1. Tert-butyl 3,4-dihydroxybenzylcarbamate
To a solution of 3,4-dihydroxybenzylamine (90 mg, 0.41 mmol)
in dry pyridine (4 mL) at 0 8C, di-tert-butyldicarbonate (88 mg,
0.41 mmol) was added and the mixture was reacted for 4 h at 0 8C.
The solution was concentrated in vacuo and the residue was
dissolved in AcOEt and washed with HCl 1N solution and brine
then dried with anhydrous sodium sulphate and evaporated in
vacuo. The crude product was purified by flash chromatography on
silica gel (EtP/AcOEt 7:3) to afford the tert-butyl 3,4-dihydrox-
ybenzylcarbamate (78 mg, 78%) as a white solid. Rf (AcOEt/EtP
Our structure–activity studies point out that amphiphilic
compounds with a positively charged ammonium or protonatable
nitrogen, such as compounds 1, 2, 5, 6 and 7 are active in blocking
TLR4-mediated endotoxin stimulus, while very similar compounds
lacking the positive charge (molecules 3 and 4) are totally inactive
[13]. These molecules showed in vivo activity as inhibitors of
LPS-induced lethality [13] and are also able to combat other
pathologies caused by TLR4 activation, such as inflammation and
neuropathic pain [14,15]. Interestingly, if the cyclic pyranose
scaffold of the sugar is replaced by an aromatic ring, the biological
activity is retained, and compound 7 (Fig. 1) showed to be highly
potent as anti-sepsis agent [13]. The mechanism of action of
bioactive molecules 1, 2, 5, 6, 7 has been investigated and it was
found that these compounds inhibit TLR4 activation by endotoxin
by competitively occupying CD14 and thereby reducing the
delivery of activating endotoxin to MD-2/TLR4 [16]. In this paper
we present the synthesis and the characterization of the biological
activity of 3,4-bis(tetradecyloxy)benzylamine 8, whose chemical
structure is strictly related to the ammonium salt 7 (Fig. 1). The
rationale to design this compound is based on the following points.
First, when replacing the alkyl ammonium group of 1 with the
primary amine of 5 in sugar-derived compounds, the synthesis is
8:2): 0.65. 1H NMR (400 MHz, CD3OD):
d = 6.70 (s, 1 H, ArH-2), 6.68
(d, 1 H, ArH-6, J = 9.8 Hz), 6.56 (dd, 1 H, ArH-5, J = 8.0, 1.6 Hz), 4.05
(s, 2 H, –CH2N), 1.43 (s, 9 H, –C(CH3)3). 13C NMR (100 MHz,
CD3OD):
d = 156.08, 149.02, 148.12, 135.14, 122.42, 118.92,
118.26, 47.48, 31.55.
2.1.2. Tert-butyl 3,4-bis(tetradecyloxy)benzylcarbamate
Tert-butyl 3,4-dihydroxybenzylcarbamate (50 mg, 0.21 mmol)
was dissolved in dry DMF (5 mL) and potassium carbonate (60 mg,
0.42 mmol) was added. After 1 h at room temperature, tetra-
decanoyl bromide (174 mg, 0.62 mmol) was added dropwise and
the mixture was reacted at 80 8C overnight. The solvent was
concentrated in vacuo, the residue was dissolved in AcOEt and
washed with water and brine. The organic phase was dried with
anhydrous sodium sulphate and evaporated in vacuo and the crude
product was purified by flash chromatography on silica gel (EtP/
AcOEt 9:1) to afford tert-butyl 3,4-bis(tetradecyloxy)benzylcarba-
mate (110 mg, 83%) as a white solid. Rf (EtP/AcOEt 9:1): 0.40. 1H
[(ig._1)TD$FIG]
NMR (400 MHz, CDCl3):
–CH2N), 3.97 (t, 4 H, CH2
H, –C(CH3)3), 1.25 (m, 44 H, CH2) 0.88 (t, 6 H, CH3, J = 6.0 Hz). 13C
NMR (100 MHz, CD3OD): = 156.08, 149.50, 148.63, 131.79,
d
= 6.79 (m, 3 H, ArH-2,5,6), 4.21 (s, 2 H,
a
, J = 5.6 Hz), 1.79 (m, 4 H, CH2 ), 1.46 (s, 9
b
d
120.08, 114.10, 113.54, 69.62, 69.44, 63.93, 32.15, 29.93, 29.89,
29.86, 29.81, 29.66, 29.59, 29.51, 29.44, 28.62, 26.48, 23.01, 14.35.
2.1.3. 3,4-Bis(tetradecyloxy)benzylamine
Tert-butyl 3,4-bis(tetradecyloxy)benzylcarbamate (750 mg,
1.27 mmol) was dissolved in dry CH2Cl2 under argon atmosphere,
trifluoroacetic acid (3 mL, 39.17 mmol) was added dropwise and
the solution was reacted for 2 h at room temperature. The mixture
was concentrated in vacuo, the residue was dissolved in CH2Cl2 and
washed with NaOH 0.1 M solution then the organic layer was dried
with anhydrous sodium sulphate and evaporated in vacuo to afford
3,4-bis(tetradecyloxy)benzylamine (675 mg, quantitative) as
white powder. Rf (AcOEt/MeOH/TEA 9:1:0.1): 0.42. 1H NMR
(400 MHz, CD3OD):
5,6), 3.97 (m, 4 H, CH2
1.28 (m, 44 H, CH2), 0.88 (t, 6 H, CH3, J = 6.0 Hz). 13C NMR
(100 MHz, CD3OD): = 149.51, 148.21, 136.39, 119.43, 114.27,
d
= 6.95 (s, 1 H, ArH-2), 6.86 (m, 2 H, ArH-
a
), 3.71 (s, 2 H, CH2NH2), 1.76 (m, 4 H, CH2 ),
b
d
113.18, 69.70, 69.45, 46.49, 32.17, 29.95, 29.91, 29.89, 29.68, 29.61,
29.57, 26.29, 22.94, 14.37.
Fig. 1. Cationic amphiphiles derived from
benzylamine (compounds 7 and 8).
D-glucose (compounds 1–6) and from

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M. Piazza et al. / Biochemical Pharmacology 80 (2010) 2050–2056
2.2. Biology: materials
concentration) was added and the solution was incubated for 30 min
at 37 8C. Products of the reactions were analyzed by size exclusion
chromatography (Mr [3H]LOS.CD14ꢂ25 000 Da) and by co-capture to
Ni chelating resin.
Tritiated lipooligosaccharide ([3H]LOS, 25 000 cpm/pmol) was
metabolically labeled and isolated from an acetate auxotroph of
Neisseria meningitidis serogroup B as described. LBP and sCD14
were gifts from Xoma (Berkley, CA) and Amgen Corp. (Thousand
Oaks, CA), respectively. Human serum albumin (HSA) was obtained
as an endotoxin-free, 25% stock solution (Baxter Health Care,
Glendale, CA). Chromatography matrices (Sephacryl HR S200 and
S300) were purchased from GE Healthcare and metal chelation
matrix, HisLink is from Promega. ESF921 medium for High Five
insect cells was purchased from Expressions Systems. In the assays
described below, stocks of molecule 8 were freshly prepared in a
75% DMSO/25% EtOH solution and then further diluted to the
appropriate concentration as needed in PBS, pH 7.4, 0.1% HSA, or
DMEM 0.1% HSA when used for cellular assays.
2.2.4. Size exclusion chromatography
Sephacryl S300 and S200 columns used for determination of
apparent Mr of the [3H]LOS-containing products were calibrated
with the following Mr standards: blue dextran (2 ꢁ 106, V0),
thyroglobulin (650 000), ferritin (440 000), catalase (232 000),
IgG (158 000), HSA (66 000), ovalbumin (44 500), myoglobin
(17 500), vitamin B12 (1200, Vi). The reaction mixture for the
generation of [LOSMD-2TLR4ECD]2 was applied in a volume of
0.9 mL to a Sephacryl HR S300 column (1.6 cm ꢁ 70 cm) pre-
equilibrated in PBS, pH 7.4, 0.1% HSA and eluted in the same buffer
at a flow-rate of 0.5 mL/min at room temperature using AKTA
Purifier or Explorer 100 fast protein liquid chromatography (GE
Healthcare), with the collection of 1 mL fractions. Radioactivity in
collected fractions was analyzed by liquid scintillation spectros-
copy (Beckman LS liquid scintillation counter). Total recovery of
[3H]LOS was ꢄ70% in all experiments. For formation of
[3H]LOSCD14 complexes, the chromatographic analyses were
carried out, using a Sephacryl HR S200 column (1.6 cm ꢁ 30 cm)
with the same experimental conditions.
2.2.1. Preparation of recombinant proteins
Preparative amounts of sMD-2 and tCD14-His6 were generated
from infections of High Five insect cells with baculovirus
containing the cDNA for human MD-2-His6 inserted into pBAC3
or for human tCD14 (amino acids 1–156) inserted into pBAC11 as
described previously [17]. Conditioned medium containing MD-2
was stored at ꢀ80 8C until needed. tCD14-His6 was purified using
Ni FF Sepharose resin (GE Healthcare) with an imidazole gradient;
purified tCD14-His6 was stored at 4 8C [17].
Conditioned medium containing MD-2 associated with TLR4
ectodomain (TLR4ECD) proteins (MD-2/TLR4ECD) was produced in
HEK293T cells as described previously [6]. Expression vectors
containing DNA of interest for production of FLAG-TLR4ECD, amino
acids 24-631, (pFLAG-CMV-TLR4) and MD-2-FLAG-His (pEF-BOS)
have been previously described and characterized [6]. Media
containing secreted proteins were concentrated 10–20-fold using
Millipore Centricon-10 before use. Conditioned medium contain-
ing secreted MD-2/TLR4ECD proteins maintained activity to react
with [3H]LOSsCD14 for at least 6 months when stored at 4 8C.
2.2.5. Evaluation of transfer of [3H]LOS to His6-tagged proteins by co-
capture to metal chelating resin
tCD14-His6 (10
mL for an estimated concentration of 1.6 nM) or
His6-sMD-2 (final concentration of 1.2 nM in the reaction mixture)
was pre-incubated in 0.3 mL PBS, 0.1% HSA in presence or absence of
LBP for 30 min at 37 8C ꢃ different concentrations of synthetic
compounds. [3H]LOSagg or [3H]LOSsCD14 was added followed by
another incubation for 30 min at 37 8C. The reaction mixture was then
incubated with HISLINK resin (20 mL) for 15 min at 25 8C, allowing His-
taggedtCD14and sMD-2tobeadsorbed ontobeads. Theresinwasspun
down for 2 min at 2000 g, supernatant was removed, and the resin was
washed twice with 300 mL PBS, 1% HSA using the same procedure. The
[3H]LOS absorbed onto the beads or recovered in the supernatant was
quantified by liquid scintillation spectroscopy. Capture of [3H]LOS in
the absence of His-tagged proteins was less than 12%.
2.2.2. Preparation of [3H]LOSagg and [3H]LOSsCD14 complex
[3H]LOSagg and [3H]LOSsCD14 complex were prepared as
previously described [6]. Briefly, [3H]LOSagg (Mr > 20 ꢁ 106) were
obtained after hot phenol extraction of [3H]LOS followed by
ethanol precipitation of [3H]LOSagg
,
and ultracentrifugation.
2.2.6. HEK cell activation assay
Monomeric [3H]LOSCD14 complexes (Mr ꢂ 60 000) were prepared
by treatment of [3H]LOSagg for 30 min at 37 8C with substoichio-
metric LBP (molar ratio 200:1 LOS:LBP) and 1–1.5 molar excess
sCD14 followed by gel exclusion chromatography (Sephacryl S200,
1.6 cm ꢁ 70 cm column) in PBS, pH 7.4, 0.03% HSA to isolate
monomeric [3H]LOSsCD14 complex. Radiochemical purity of
[3H]LOSsCD14 was confirmed by S200 chromatography [6,17].
293/hTLR4A-MD2-CD14 cells and parental HEK, as negative
control, have been purchased by Invivogen and cultured as
described [18]. For cell activation assays, 293/hTLR4A-MD2-CD14
or parental HEK cells were grown to confluency in 96-well plates.
Cell monolayers were washed twice with warm PBS and incubated
overnight at 37 8C, 5% CO2, and 95% humidity in 200
mL DMEM
supplemented with molecule 8 (0–10 M), and LPSagg (1 nM, from
m
Sigma-AldrichTM) as stimulus. The final organic solvent (DMSO/
ethanol) concentration per well is less than 0.5%. After the
incubation, plates were centrifuged at 1000 rpm for 5 min and
supernatants were collected. Activation of HEKcells was assessedby
measuring the accumulation of extracellular IL-8 by ELISA according
to supplier protocol (BD Clontech, Inc., Palo Alto, CA).
2.2.3. Preparation of [3H]LOSProtein(s) complexes in the presence of
molecule 8
[3H]LOSMD-2TLR4 complex was generated by a three-step
process. sCD14 (0.8 nM, final concentration) was pre-incubated
with LBP (4 pM, final concentration) ꢃ molecule 8 (0–20 mM) for
30 min at 37 8C in PBS, pH 7.4, 0.1% HSA (0.875 mL). After this
incubation, [3H]LOSagg (up to 5 mL for a final concentration of 0.8 nM)
was added; the mixture was incubated for 30 min at 37 8C and then
supplemented with conditioned HEK293T cell medium containing
preformed MD-2TLR4ECD heterodimer [6] (125 mL for an estimated
final concentration of 0.2 nM) and incubated again for 15 min at
37 8C. Analysis of the reaction products was determined by gel size
exclusion chromatography as described below. To examine the ability
of the molecule to affect LOS-sCD14 transfer, sCD14 (0.8 nM, final
concentration) was pre-incubated with molecule 8 (10 mM in PBS, pH
7.4, 0.1% HSA) for 30 min at 37 8C then [3H]LOS. (0.8 nM, final
2.2.7. HEK-BlueTM assay
HEK-BlueTM-4 cells (HEK-BlueTM LPS Detection Kit, InvivoGen)
were cultured according to manufacturer’s instructions. Briefly,
cells were cultured in DMEM high glucose medium supplemented
with 10% fetal bovine serum (FBS), 2 mM glutamine, 1ꢁ
NormocinTM (InvivoGen), 1ꢁ HEK-BlueTM Selection (InvivoGen).
HEK-BlueTM-4 cells were detached by the use of a cell scraper and
the cell concentration was estimated by using a cell counter. The
cells were diluted in DMEM high glucose medium supplemented
with DMEM high glucose medium supplemented with 10% fetal

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M. Piazza et al. / Biochemical Pharmacology 80 (2010) 2050–2056
2053
Scheme 1. Synthesis of bis(tetradecyloxy)benzylamine 8. Reagents and conditions: (a) Boc2O, dry pyridine, 0 8C, 4 h (78%); (b) tetradecanoyl bromide, K2CO3, dry DMF, 80 8C,
overnight (83%); (c) trifluoroacetic acid, RT, 2 h, quantitative yield.
bovine serum (FBS), 2 mM glutamine, 1ꢁ NormocinTM (InvivoGen),
transcription factors such as NF-kB and AP-1. This reporter gene
allows to monitor the activation of TLR4 signal pathway by
endotoxin.
HEK-293/hTLR4A-MD2-CD14 cells were stimulated with LPS
(Sigma-Aldrich) at a concentration of 1 nM after pre-exposure to
the molecule 8, at four different concentrations ranging from 0.5 to
1ꢁ HEK-BlueTM Selection (InvivoGen) and 200
m
L of cell suspen-
sion (20 000 cells) were added to each well. After O.N. incubation at
37 8C in a CO2 incubator cells reached confluency and supernatant
removed, cell monolayers were washed twice with warm PBS
without Ca2+ and Mg2+ and incubated overnight at 37 8C, 5% CO2,
and 95% humidity in 200
(0–20 M), and LPSagg (1 nM) as stimulus. After the incubation,
plates were centrifuged at 1000 rpm for 5 min and supernatants
were collected. 5 L of each sample was added to 95 L PBS, pH 8,
m
L DMEM supplemented with molecule 8
10 mM, and the production of the cytokine IL-8 was monitored
(Fig. 2A). A concentration-dependent inhibition of IL-8 production
was observed. HEK-BlueTM-4 was treated with LPS after exposition
m
m
m
to four concentrations of molecule 8, from 0.5 to 10 mM (Fig. 2B). In
0.84 mM pNpp for a final concentration of 0.8 mM pNpp. Plates
were incubated for 2 h in the dark at RT then the plate reading was
assessed by using a spectrophotometer at 405 nm. As a control, we
treated the cells with ꢃ LPS (1 nM) alone.
this case too, a dose-dependent inhibition of phosphatase activity
was observed, indicating an inhibition of TLR4 activation. In this
latter case, the inhibitory effect of compound 8 was more evident,
giving a 57% reduction of TLR4 activation and phosphatase activity
when LPS reaches the concentration of 10
mM. To asses if molecule 8
2.2.8. MTT cell viability assay1 (Sigma-AldrichTM
)
could provide a change in IL-8 production in absence of TLR4
receptor complex, we treatedparental HEK cells (that do not express
TLR4) with increasing amount of the synthetic compound. No
change in IL-8 production was observed in this latter case. A viability
assay was performed to verify if reduction in LPS-stimulated IL-8 by
compound8isnotduetoageneralcytotoxiceffect. MTTtest(Fig. 2C)
clearly showed that molecule 8 is not toxic.
Parental HEK cells were seeded at a density of 2 ꢁ 104 cells/mL
in 100
mL medium (DMEM without red phenol) per well in 96-
well-plate. Compound 8 was diluted with PBS and added to the
cells to yield a final concentration of 20 M. 24 h after incubation
at 37 8C, the medium was removed, monolayer washed two times
with warm PBS and 100 L of DMEM without phenol red + 0.05 mg
MTT was added per well. After 2 h incubation at 37 8C and 5% CO2,
formazan crystals were dissolved by adding 100 L of MTT
m
m
m
3.3. Compound 8 inhibits the formation of tritiated
lipooligosaccharide-CD14 ([3H]LOS-CD14) and [3H]LOS-MD-2-TLR4
complexes
solubilization solution (anhydrous isopropanol 0.1N HCl with 10%
Triton X-100). Formazan concentration in the wells was deter-
mined measuring the absorbance at 570 nm. As positive control for
cytotoxicity (C+), we set up triplicate wells containing cells treated
with a compound known to be toxic to the cells.
We then wanted to verify if the inhibition of lipooligosaccharide
(LOS) signal by our compound is due to an interference with the
formation of (LOS-MD-2-TLR4)2 complex. We have previously
shown that both reactions of monomeric LOS-sCD14 with MD-2-
TLR4ECD and of monomeric LOS-MD-2 with TLR4 ectodomain
(TLR4ECD) produced a complex of apparent Mr 190 000 containing
LOS, MD-2 and TLR4ECD that nearly matches the mass of a dimer of
a 1:1:1 complex of LOS-MD-2-TLR4 [6]. We then modified the
protocol of (LOS-MD-2-TLR4)2 complex formation for evidencing a
possible interference of compound 8 in this process [16]. Soluble
CD14 (sCD14) was pre-incubated with LBP in the presence or
3. Results
3.1. Synthesis of bis(tetradecyloxy)benzylamine 8
Bis(tetradecyloxy)benzylamine
8 was synthesized starting
from commercially available 3,4-dihydroxybenzylamine according
to Scheme 1. The amino group was protected as tert-butylox-
ycarbonyl (Boc) derivative using Boc anhydride in pyridine, then
the tert-butyl 3,4-dihydroxybenzylcarbamate was reacted with
tetradecanoyl bromide in the presence of potassium carbonate to
afford the tert-butyl 3,4-bis(tetradecyloxy)benzylcarbamate. The
amine deprotection using trifluoroacetic acid in dichloromethane
gave finally bis(tetradecyloxy)benzylamine 8.
absence of molecule 8 (10
mM), then were added and incubated,
successively, [3H]LOS and the preformed MD-2-TLR4 dimer. Size-
exclusion chromatography by gel-filtration of the reaction mixture
(Fig. 3) showed that the presence of compound 8 inhibited the
formation of the peak corresponding to an Mr of about 190 kDa,
corresponding to the (LOS-MD-2-TLR4)2 complex, while increasing
the earlier eluting peak corresponding to high molecular weight
LOS aggregates.
3.2. Compound 8 inhibits LPS-stimulated TLR4 activation in HEK-293/
hTLR4A-MD2-CD14 and HEK-BlueTM-4 cells
Interestingly, also the peak corresponding to the mass of LOS-
sCD14 complex is reduced when incubation is done in the presence
of compound 8 (Fig. 3).
The activity of compound 8 in modulating the LPS signaling
through the TLR4 receptor was tested in vitro using HEK cells.
Two types of cells were used: the HEK-293/hTLR4A-MD2-CD14
cells stably transfected with TLR4, MD2, and CD14 genes and
HEK-BlueTM-4 cells that, in addition, stably express an optimized
alkaline phosphatase gene engineered to be secreted (sAP),
placed under the control of a promoter inducible by several
3.4. Compound 8 inhibits the endotoxin loading on CD14
While being an interesting preliminary indication, the observed
effect of inhibition of 190 kDa complex formation by molecule 8

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M. Piazza et al. / Biochemical Pharmacology 80 (2010) 2050–2056
Fig. 2. (A) Co-administration of LPS and increasing amount of molecule 8 (0–10
m
M) to 293/hTLR4A-MD2-CD14 cells. Cell activation was measured as extracellular
M) and
M molecule 8, of 0.5% DMSO/EtOH and of a positive
production of IL-8 through an ELISA assay. (B) Measurement of activated NF-
k
B level in HEK-BlueTM cells when increasing amount of molecule 8 (0–10
m
supplemented with LPS as stimulus. (C) MTT assay of parental HEK cells (that do not express TLR4) in the presence of 10
control.
m
Results shown represent the mean SEM of three independent experiments, each in triplicate. The asterisks indicate a significant statistical difference (P < 0.01; ANOVA,
Dunnett’s test) between data in the analyzed group and reference data (LPS alone for panel A and DMSO/EtOH for panel C).
could derive from the interference of this compound at different
levels in the sequence of reactions leading to the formation of the
complex. To gain further information, we divided the process into
two main steps: (1) the formation of dimeric [3H]LOS-CD14
complex and (2) the subsequent transfer of LOS from the complex
with CD14 to MD2. The first step is the LBP-aided formation of LOS-
CD14 from LOS aggregates. We formed the complex by incubating
LOS and CD14 in the presence or in the absence of 8 (10 M). After
incubation, the reaction mixture was analyzed by gel sieving
chromatography using a Sephacryl HR S200 resin and the elution
profile showed a reduction in the formation of the [3H]LOS-CD14
peak (Fig. 4).
m
[(ig._3)TD$FIG]
[i(g._4)TD$FIG]
Fig. 3. Molecule 8 inhibits the formation of the [[3H]LOS-MD-2-TLR4ECD
The incubation mixture containing [3H]-LOSagg
LBP, sCD14 and MD-2-
]2 complex.
Fig. 4. Transfer of [3H]-LOS monomers to sCD14 is inhibited by 10
mM of molecule 8.
,
Transfer of tritiated endotoxin from aggregates to sCD14 and formation of [3H]-LOS-
sCD14 complex is monitored with Sephacryl S200 chromatography. Profiles shown
are representative of three independent experiments.
TLR4ECD ꢃ the synthetic compound (10 mM) was analyzed by gel-sieving analysis
using a Sephacryl S300 as described in Section 2. Profiles shown are representative of
three experiments for each condition (both in presence or absence of molecule 8).

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M. Piazza et al. / Biochemical Pharmacology 80 (2010) 2050–2056
2055
Fig. 5. (A) Dose-dependent inhibition of [3H]-LOS transfer to His-tagged tCD14. This capture assay shows the dependency between molecule 8 concentration and the
inhibition of [3H]-LOS-sCD14 complex formation. (B) Molecule 8 does not inhibit [3H]-LOS transfer from CD14 to MD2. [3H]-LOS–MD-2 complex formation is not inhibited if
MD-2 is pre-incubated with molecule 8 suggesting that the compound could not compete with LOS for MD-2 binding. Data shown are representative of three experiments,
each in duplicate, and are expressed as mean of percent of radioactivity captured in comparison with total radioactivity measured ꢃ SEM. The asterisks indicate a significant
statistical difference (P < 0.01; ANOVA, Dunnett’s test) between data in the analyzed group and reference data (sample without molecule 8).
To further test this hypothesis, we performed an experiment of
direct antagonism between 8 and LOS for CD14 binding. We have
shown that LOS can be rapidly and efficiently transferred from
LOS-CD14 to free CD14 [19]. A capture experiment was carried out
in which [3H]LOS is transferred to His-tagged tCD14 from CD14
complex according to the following equilibrium: [3H]LOS-
CD14 + tCD14 $ CD14 + [3H]LOS-tCD14. Once formed, the
[3H]LOS.tCD14 complex is efficiently captured by Ni++-coated
HISLINK resin and the resulting radioactivity quantified [16].
Unlike previous experiments, in which LOSagg were used, in this
experiment LOS is present in monomeric form associated to CD14
in the [3H]LOS-CD14 complex. A preformed [3H]LOS.CD14 complex
was incubated with His-tagged tCD14 ꢃ molecule 8, the complex
captured with resin (Fig. 5A).
The percents of radioactivity captured in different conditions
are depicted in Fig. 5 showing that 8 inhibits the transfer of
monomeric LOS from CD14 complexes to soluble tagged CD14. A
control capture experiment in which the endotoxin is transferred
from [3H] LOS.CD14 to His-tagged MD-2 confirmed that molecule 8
does not inhibit this reaction (Fig. 5B).
presence or absence of molecule 8, and then incubated with
tritiated LOS in the form of aggregates ([3H]LOSagg) followed by
conditioned medium containing preformed MD-2-TLR4ECD hetero-
dimer. Size-exclusion chromatography of the reaction mixture
showed that, in the absence of molecule 8, virtually all [3H]LOS
aggregates (LOSagg) were converted to later eluting species (i.e.,
smaller [3H]LOS-containing complexes) corresponding to, from the
left, [[3H]LOS-MD-2-TLR4ECD]2 (Mr ꢂ 190 000) and [3H]LOS-sCD14
(Mr ꢂ 60 000). The identity of these complexes was confirmed by
co-capture of [3H]LOS with immobilized monoclonal antibodies to
CD14 [20] or to epitope tags in His6-MD-2 or in Flag-TLR4ECD [6]
and by co-elution with purified, defined [3H]LOSprotein com-
plexes.
In contrast, in the presence of 10
m
M molecule 8, accumulation
was
of both [3H]LOS-sCD14 and [[3H]LOS-MD-2-TLR4ECD 2
]
markedly reduced and most [3H]LOS was recovered in the void
volume presumably as large [3H]LOS aggregates (Fig. 3). The
inhibition of accumulation of [3H]LOSsCD14 suggested a primary
effect of molecule 8 on LBP/sCD14-dependent extraction and
transfer of [3H]LOS monomers from [3H]LOSagg to [3H]LOSsCD14.
To test this hypothesis more directly, we examined the effect of
molecule 8 on LBP/sCD14-dependent conversion of [3H]LOSagg to
[3H]LOS-sCD14. As shown in Fig. 4, molecule 8 caused a clear
reduction in conversion of [3H]LOSagg to [3H]LOS-sCD14, with
decreased accumulation of [3H]LOSsCD14 accompanied by in-
creased recovery of [3H]LOS as high Mr LOS aggregates.
4. Discussion
Compound 8 is active in inhibiting in a dose-dependent way
LPS-stimulated TLR4 activation in HEK cells stably transfected with
TLR4, CD14, and MD-2 genes. After pre-incubation with compound
8, and subsequent treatment with LPS, we observed a reduction in
the production of the IL-8 (Fig. 2A) and, in the case of HEK-BlueTM-4
The above experiments demonstrate an ability of molecule 8 to
inhibit extraction and transfer of [3H]LOS monomers from [3H]LOS
aggregates to CD14. This targeted inhibitory effect could be
explained by a selective effect on extraction of LOS monomers from
aggregates that is needed for generation of [3H]LOSsCD14 or a
selective effect on net transfer of [3H]LOS monomers to sCD14 vs.
sMD-2. To test the latter possibility more directly, we took
advantage of the ability of monomeric E-sCD14 complexes to serve
as a donor of E monomers to either CD14 or MD-2. For this purpose,
we used [3H]LOS-sCD14 (full-length sCD14, no His tag) as [3H]LOS
donor and either His6-tagged truncated sCD14 (tCD14; residues 1–
156) or His6-tagged sMD-2 as [3H]LOS acceptors. Thus, [3H]LOS
transfer from [3H]LOS-sCD14 to tCD14-His6 or to His6-sMD-2
could be readily measured by assay of co-capture of [3H]LOS to
HISLINK resin as described in Section 2.
cells, a dose-dependent inhibition of the NF-kB induced produc-
tion of alkaline phosphatase (Fig. 2B). Compound 8 did not present
detectable cellular toxicity (Fig. 2C). In order to clarify the
mechanism of action at a molecular level, and identify its
molecular target, we tested the effect of molecule 8 on the
sequential extraction and transfer of tritiated lipooligosaccharide
[3H]LOS monomers from [3H]LOSagg to LBP, CD14, and MD-
2TLR4ECD, reactions that are key to efficient delivery of activating
endotoxin to MD-2-TLR4 [18,6].
To do this, we performed an experiment of sequential receptor
incubation in the presence and absence of compound 8, followed
by mass determination of formed complexes by gel sieving
chromatography. sCD14 was pre-incubated with LBP in the

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M. Piazza et al. / Biochemical Pharmacology 80 (2010) 2050–2056
[6] Prohinar P, Re F, Widstrom R, Zhang D, Teghanemt A, Gibson BW, Weiss JP.
Molecule 8 markedly inhibited transfer of [3H]LOS from full-
length sCD14 (no His tag) to tCD14-His6 (Fig. 5A) but did not inhibit
transfer of [3H]LOS to His6-sMD-2 (Fig. 5B).
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Our findings demonstrate that molecule 8 affects this multi-
step pathway in a relatively selective manner, acting mainly to
inhibit transfer to and/or stable occupation of CD14 by lipooligo-
saccharide ([3H]LOS) monomers. Similar inhibitory effects of
molecule 8 were seen when the transfer of [3H]LOS from either
[3H]LOS aggregates or from monomeric [3H]LOSsCD14 was
examined (Figs. 3–5), indicating that an additional effect of
molecule 8 on interactions between LBP and endotoxin-rich
interfaces was unlikely and not necessary for its inhibitory action.
Remarkably, shuttling of LOS monomers from CD14 to MD-2 is
unaffected by molecule 8. The apparently selective targeting of
CD14 by this compounds is very similar to that observed for
compounds 1 and 7 [16]. The functional properties described
suggest that molecule 8 could be considered lead compounds in
the development of new anti-endotoxic agents selectively target-
ing CD14. Increasing evidence is emerging that TLR4 and CD14 are
implicated in other important pathologies, such as neuropathic
pain, related to the microglia TLR4 activation [21], or Alzheimer
disease [22,23]. The broader role of CD14 in other biological
recognition/response pathways may further expand the potential
pharmacological applications of this compound.
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endotoxin from MD-2 to CD14. J Biol Chem 2007;282:36250–6.
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the interaction of endotoxin with lipopolysaccharide-binding protein and
sCD14 and resultant cell activation. J Biol Chem 2002;277:47818–25.
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glial activation: improving the clinical efficacy of opioids by targeting glia.
Trends Pharmacol Sci 2009;30:581–91.
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C, Garcia-Gorostiaga I, Berciano J, Combarros O. Interaction between CD14 and
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Acknowledgement
We acknowledge NIH/NIAID, grant number [1R01AI059372]
‘‘Regulation of MD-2 function and expression’’.
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